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https://openalex.org/W2426707173
https://europepmc.org/articles/pmc4931099?pdf=render
English
null
iStent® Trabecular Microbypass Stent: An Update
Journal of ophthalmology
2,016
cc-by
6,733
Correspondence should be addressed to Michael Waisbourd; mwaisbourd@willseye.org Correspondence should be addressed to Michael Waisbourd; mwaisbourd@willseye.org Received 12 October 2015; Accepted 15 May 2016 Academic Editor: Michele Figus Received 12 October 2015; Accepted 15 May 2016 Received 12 October 2015; Accepted 15 May 2016 Academic Editor: Michele Figus Copyright © 2016 Arthur Fernandes Resende et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Due to the high rates of complications and failure experienced with current glaucoma procedures, there is a continuous search for a safer and more effective glaucoma surgery. A new class of procedures termed minimally invasive glaucoma surgeries (MIGS) aim to fill this void by offering an alternative method of IOP reduction associated with markedly reduced complication rates and shorter recovery times. The iStent, a trabecular microbypass stent, is a MIGS device that has quickly gained popularity. The device allows aqueous humor to directly drain from the anterior chamber into Schlemm’s canal by bypassing an obstructed trabecular meshwork. This review examines publications about the iStent, focusing on the device’s efficacy, safety, and cost when a single iStent or multiple iStents are implanted in combination with cataract surgery or as a solo procedure. Current data suggest that the iStent is a safe and effective tool in the management of mild-to-moderate glaucoma, notable for its limited complications and absence of serious adverse events following implantation. As valuable experience is gained performing ab interno MIGS, increasing familiarity with angle anatomy and iStent placement, and as newer stent designs are developed, there is promise of continual improvement in the surgical management of glaucoma. Hindawi Publishing Corporation Journal of Ophthalmology Volume 2016, Article ID 2731856, 9 pages http://dx.doi.org/10.1155/2016/2731856 Hindawi Publishing Corporation Journal of Ophthalmology Volume 2016, Article ID 2731856, 9 pages http://dx.doi.org/10.1155/2016/2731856 Hindawi Publishing Corporation Journal of Ophthalmology Volume 2016, Article ID 2731856, 9 pages http://dx.doi.org/10.1155/2016/2731856 Review Article iStent„ Trabecular Microbypass Stent: An Update Arthur Fernandes Resende, Neal Sanjay Patel, Michael Waisbourd, and L. Jay Katz Glaucoma Research Center, Wills Eye Hospital, Philadelphia, PA 19107, USA 1. Introduction popularity since first being published by Spiegel et al. in 2007 [7]. Being the smallest US Food and Drug Administration approved device ever implanted in the human body, the iStent is a 1 mm heparin-coated, nonferromagnetic, surgical grade titanium stent with a ridged, snorkel design pictured in Figure 1(a). The device allows aqueous humor to directly drain from the anterior chamber into Schlemm’s canal by bypassing an obstructed trabecular meshwork. Requiring only a short surgical procedure for implantation, the iStent benefits from a relatively fast learning curve; it is inserted ab interno through a clear corneal incision guided by direct gonioscopy (Figure 1(b)). Additionally, the iStent has the potential to be a fiscally favorable alternative to traditional treatments by reducing medication burden in the long term [8]. Herein, we review the literature on the iStent trabecular microbypass stent published between January 2007 and April 2016 in order to better understand its efficacy, safety, cost considerations, and future directions. A PubMed search for “iStent” revealed 44 articles. Each of these full-text articles was reviewed. Secondary searches for “trabecular bypass” and minimally invasive glaucoma surgery or “MIGS” identified Glaucoma is the leading cause of irreversible blindness world- wide, affecting over 65 million people [1]. The primary goal in the treatment of glaucoma is the management of intraocular pressure (IOP), which is traditionally first attempted through use of topical medications or laser therapy [2]. However, when these methods fail, surgery is often required to prevent vision loss. Due to the high rates of complications and failure experienced with current glaucoma procedures (e.g., trabeculectomy and tube shunt implantation) [3], there is a continuous search for a safer and more effective glaucoma surgery. Through the use of novel nonpenetrating and bleb- independent approaches, a new class of procedures termed minimally invasive glaucoma surgeries (MIGS) aim to fill this void by offering an alternative method of IOP reduction associated with markedly reduced complication rates and shorter recovery times compared with traditional glaucoma surgery [4–6].h The iStent (Glaukos, Laguna Hills, CA), a trabecular microbypass stent, is a MIGS device that has quickly gained Journal of Ophthalmology 2 additional relevant articles. Randomized controlled trials (RCT) and relevant case series were included in this review and are listed in Tables 1 and 2. Review articles and cost studies were cited in this paper as well. stent. 2. Outcomes of iStent Implantation with Cataract Surgery Although cataract surgery is known to reduce IOP by itself (by approximately 2 mmHg) [9], combining this procedure with the implantation of an iStent through the same surgical incision can have a greater impact on reducing IOP and medication burden [10–12]. As such, the most popular and well-researched use of the iStent is when its implantation is performed simultaneously with cataract surgery. One of the earliest reports of this combined surgery in 2008 [13] demonstrated that 70% of subjects (𝑛= 33/47) were able to discontinue all previous IOP lowering medications, with a mean IOP reduction of 5.7 ± 3.8 mmHg at 6 months (25.4% reduction, 𝑃< 0.001), a reduction greater than what has been evidenced in previous studies with cataract surgery as a solo procedure [10, 12–15]. The largest prospective, randomized, controlled trial to be performed on this topic as of yet is a multicenter study conducted by the US iStent Study Group [15]. The study enrolled a total of 240 eyes with cataract and OAG which were randomized into 2 groups to receive either phacoemulsifica- tion alone (𝑛= 123) or phacoemulsification combined with a single iStent (𝑛= 117). In 2011 Samuelson et al. [15] pub- lished results from a 12-month follow-up, with the primary efficacy measure defined as an unmedicated IOP ≤21. The iStent group performed significantly better than the group receiving phacoemulsification alone, with 72% reaching the desired outcome of an IOP of <22 mmHg without glaucoma medications in the iStent group compared to 50% in the control group (𝑃< 0.001). A secondary efficacy measure specified as an IOP reduction ≥20% without medication resulted in a similarly significant outcome, demonstrating 18% treatment difference between subjects receiving an iStent group and those receiving phacoemulsification alone (66% versus 48%, 𝑃 = 0.003). While the mean reduction in IOP was similar in both groups at 12 months (as expected due to the study protocol calling for active management of IOP with medication), the iStent group subjects were able to achieve their IOP reduction with significantly fewer medications. The time to first medication was significantly longer in the iStent group, with control subjects taking more ocular hypotensive medications at 1 week compared to iStent group subjects at 1 year. 2. Outcomes of iStent Implantation with Cataract Surgery At 12 months the mean decrease in medications from baseline was larger in the iStent group (1.4 versus 1.0, 𝑃= 0.005) and fewer subjects in the iStent group required IOP lowering medication compared to those in the control group (15% versus 35%, 𝑃 = 0.001). In 2012, Craven et al. [19] reported the data from a 24-month follow-up in the same study subjects. Although a difference in IOP lowering medication use between the 2 groups was no longer statistically significant (the study protocol was not sufficiently powered for a 2-year efficacy evaluation), a difference favoring the iStent group was still observed when considering the same primary efficacy outcome of an unmedicated IOP ≤21 (𝑃= 0.036). A 2013 prospective, uncontrolled, interventional case series by Patel et al. [14, 16] examined the efficacy and outcomes of the combined iStent implantation and cataract surgery in 40 eyes with open-angle glaucoma (OAG). The study concluded that the procedure resulted in a signifi- cant reduction in IOP at 6 months postoperatively, with a mean reduction of 4.4 mmHg from a mean baseline IOP of 21.1 mmHg (20.9% reduction, 𝑃< 0.0001). Dependence on topical IOP lowering medication was also reduced signifi- cantly, with a mean number of medications reduced from 2.3 to 0.6 (𝑃< 0.01). By a 6-month follow-up, 66% percent of patients were medication-free, with further 20% only requiring 1 ocular hypotensive medication; additionally all patients on oral acetazolamide prior to surgery (𝑛= 6) were able to discontinue its use. An identical case series conducted by Arriola-Villalobos et al. [16] in 2012 examined a smaller population (𝑛= 19) but offered the longest follow-up for this combined procedure currently published in literature. At a mean follow-up of 53 months, a significant reduction in mean IOP of 3.16 mmHg was still demonstrated from a baseline of 19.4 mmHg (16.3% reduction, 𝑃= 0.002). While all subjects were using at least 1 IOP lowering medication at baseline, by the end of follow- up 8 subjects (42.1%) still did not require any hypotensive medication. Although limited by being an uncontrolled study with a small sample size, these results suggest that the efficacy of iStent implantation may persist in the long term.hfi The most recent published data on the long-term efficacy of combined cataract and iStent implantation demonstrated outcomes very similar to previous studies. 1. Introduction Although not statistically significant, the microbypass stent combined with phacoemulsification group demon- strated a consistently reduced IOP throughout the entire study period, starting from 17.8 ± 2.7 mmHg at baseline to 16.1±2.0 mmHg at 12 months and finally to 15.9±2.3 mmHg at 48 months. At long-term follow-up after washout, IOP in the group receiving phacoemulsification alone was sig- nificantly greater than that at baseline (20.4 ± 3.2 versus 16.7 ± 3.0 mmHg, 𝑃= 0.002) and a 14.2% difference in IOP compared to the combined group was reported, which was statistically significant (17.5 ± 2.3 mmHg in the combined group versus 20.4±3.2 mmHg in the control group, 𝑃= 0.02) [18].h 2. Outcomes of iStent Implantation with Cataract Surgery In one study with a 3-year follow-up, iStent placement achieved an IOP reduction from 24.1±6.9 mmHg at the baseline to 14.9±2.3 mmHg, with use of glaucoma medications eliminated in 74% of patients [17]. Fea et al. also published long-term results, including a comparison between cataract extraction as a solo procedure and cataract extraction combined with implantation of one A meta-analysis comparing iStent implantation with phacoemulsification versus phacoemulsification alone has been recently published by Malvankar-Mehta et al. [11] in 2015. This meta-analysis reported that while both strategies caused reduction of IOP and the number of medications used in the long term, the combined iStent implantation had significantly better results. Phacoemulsification alone 3 Journal of Ophthalmology 3 Table 1: Summary of iStent randomized controlled trials. Authors (year) TG (𝑛) CG (𝑛) Device Procedure TG mean IOP reduction (%) CG mean IOP reduction (%) TG med. reduction (%)∗ CG med. reduction (%)∗ Follow-up (months) Samuelson et al. [15] (2011) 117 123 iStent Phaco. versus Phaco. + 1 iStent 8.2 5.4 86.7 73.3 12 Craven et al. [19] (2012) 117 123 iStent Phaco. versus Phaco. + 1 iStent 8.1 4.3 80.0 66.7 24 Fern´andez-Barrientos et al. [26] (2010) 17 16 iStent Phaco. versus Phaco. + 2 iStents 27.3 16.5 100 41.7 12 Fea [10] (2010) 12 24 iStent Phaco. versus Phaco. + 1 iStent 17.3 9.2 80.0 31.6 15 Fea et al. [27] (2014) 94 98 iStent inject 2 iStents versus med. 38.4 36.2 N/A N/A 12 CG, control group; IOP, intraocular pressure; med, medication; Phaco., phacoemulsification; TG, treatment group. ∗The numerical value listed under “med. reduction” represents the decrease in mean number of IOP lowering medications used postoperatively. Journal of Ophthalmology Table 2: Summary of iStent case series. Authors (year) 𝑛 Procedure Device Mean IOP reduction (%) Medication reduction (%)∗ Follow-up (months) Spiegel et al. [7]1 (2007) 6 1 iStent iStent 23.9 18.5 12 Buchacra et al. [20]1 (2011) 10 1 iStent iStent 27.3 62.0 12 Ahmed et al. [21]1 (2014) 39 2 iStents + travoprost iStent 46.9 50 18 Voskanyan et al. [31]1 (2014) 99 2 iStents iStent inject 39.7 N/A 12 Arriola-Villalobos et al. [30]1 (2013) 20 Phaco. + 1 iStent or 2 iStents iStent inject 35.7 76.9 12 Spiegel et al. [13]1 (2008) 47 Phaco. + 1 iStent iStent 25.4 66.7 6 Spiegel et al. [12]1 (2009) 47 Phaco. + 1 iStent iStent 21.4 75.0 12 Arriola-Villalobos et al. 2. Outcomes of iStent Implantation with Cataract Surgery [16]1 (2012) 19 Phaco. + 1 iStent iStent 16.3 63.6 60 Patel et al. [14]1 (2013) 44 Phaco. + 1 iStent iStent 20.9 74.3 6 Belovay et al. [22]1 (2012) 53 Phaco. + iStents (2 or 3) iStent 20.2–20.4 64.3–84.6 12 Klamann et al. [32]2 (2015) 35 1 iStent in phakic OAG iStent inject 33–35 N/A3 6 El Wardani et al. [34]2 (2015) 131 Phaco. alone and 1 iStent or 2 iStents iStent N/A4 275 6 IOP, intraocular pressure; 𝑛, number of eyes enrolled; Phaco., phacoemulsification; OAG, open-angle glaucoma. ∗The numerical value listed under “medication reduction” represents the decrease in mean number of IOP lowering medications used postoperatively. 1Prospective studies. 2Retrospective studies. 3No statically significant difference was found at the end of follow-up. 4Percentage of reduction was not available in abstract (epub ahead of printing). 5Number of medications was reduced by 8% in the phacoemulsification alone group, 27% when using one iStent, and 45% when using 2 iStents. 5 Journal of Ophthalmology Snorkel Lumen 1mm Rail Retention arches Self-trephining tip (a) (b) (c) Figure 1: Illustration of the iStent with dimensions and technical specifications (a); intrasurgical view of the trabecular meshwork with a direct gonioscopy lens (b); flipped view of 2 inserted iStents under gonioscopy (c). ((a) and (b), courtesy of Glaukos Corporation; (c), courtesy of Matt Poe, http://www.ophthalmicphotography.info/). Snorkel Lumen 1mm Rail Retention arches Self-trephining tip (a) (c) (b) (b) (a) (c) Figure 1: Illustration of the iStent with dimensions and technical specifications (a); intrasurgical view of the trabecular meshwork with a direct gonioscopy lens (b); flipped view of 2 inserted iStents under gonioscopy (c). ((a) and (b), courtesy of Glaukos Corporation; (c), courtesy of Matt Poe, http://www.ophthalmicphotography.info/). 2.0 mmHg, IOP was reduced to 14.0 ± 2.2 mmHg by 1 month and 13.0 ± 2.4 by 12 months, with reduction of 1 medication. At the 12-month follow-up, all 39 subjects had achieved an IOP ≤18 mmHg and a reduction ≥20% from baseline solely on travoprost. Moreover, 29 patients (74.4%) achieved an IOP reduction ≥40% from baseline. Following a washout leading up to a 13-month follow-up, it was observed that the mean unmedicated IOP decreased from 25.3 ± 1.8 mmHg preoperatively to 17.1 ± 2.2 mmHg, with an IOP reduction of 8.2 mmHg or 32.4%. The results suggest that iStent implantation could serve as a substitute for patients using multiple ocular hypotensive medications. 2. Outcomes of iStent Implantation with Cataract Surgery Results from a 5-year follow-up on the same subjects are pending and may help elucidate long-term effects of solo iStent implantation. resulted in a 4% mean decrease in IOP from baseline; how- ever, addition of an iStent increased this to a 9% reduction; concurrent implantation of 2 iStents additionally improved this to a 27% reduction in IOP. Moreover, combination surgery resulted in a weighted mean reduction in the number of glaucoma medications by 1.33 per patient, compared to 1.01 with phacoemulsification alone. Success of the combined surgery continued into the long term, with the meta-analysis finding a significant reduction of glaucoma medications by 12 months postoperatively, which remained significant until 4 years of follow-up. Despite significant heterogeneity between studies examined in the meta-analysis, the results concluded that the combined iStent with phacoemulsification surgery significantly outperforms phacoemulsification alone in terms of both IOP reduction and medication burden. f One control trial has been conducted examining the potential for increased efficacy with the addition of a third iStent in a solo procedure. Conducted by Belovay et al. in 2012 [22], the study compared the use of 1, 2, or 3 iStents, with 30 patients enrolled in each of the 3 groups. It showed that the group that simultaneously received 3 iStents presented with a mean IOP of 12.9 ± 1.6 mmHg at 6 months postoperatively, compared to a baseline of 24.3 ± 3.7 mmHg, with a reduction of 41%. While this outperformed the single iStent group, which experienced a mean IOP reduction of 31%, it performed similarly to the 2 iStents group which had an identical IOP reduction of 41%. 3. iStent as a Solo Procedure Following a 1-month medication washout period at month 12 for eyes on medication, mean unmedicated IOP at months 12–13 were 14.9 ± 1.9 mmHg, 13.6 ± 2.1 mmHg, and 12.7 ± 2.1 mmHg in the three respective groups. IOP reduction was sustained in each of the groups throughout the 18-month postoperative period, with a greater reduction observed in the multiple-stent groups versus the one-stent group. At 18 months, mean IOP was 15.6 ± 1.5 mmHg in the one-stent group, 13.8 ± 1.3 mmHg in the two-stent group, and 12.1 ± 1.2 mmHg in the three-stent group [24]. While reports of most complications are consistent across all studies, other studies have found a wide range in frequency of complications related to iStent malpositioning. Compared to the iStent Study Group results of a 3% incidence among subjects, a 2010 study by Fern´andez-Barrientos et al. [26] found that 6 of 34 (17.6%) iStents were malpositioned upon follow-up, and results from Fea in 2010 [10] showed that 2 of 12 (16.7%) were malpositioned. It is notable, however, that none of these cases led to any significant adverse events, nor did any require resurgery. It is likely that this variation among reported results is largely due to a lack of specific standardized criteria defining malpositioning, along with the absence of a universal protocol for determining if surgical intervention is necessary. Among studies examined in this review, surgical intervention (e.g., iStent repositioning or removal) or laser procedures were necessitated in 4.5% to 11.3% of study subjects that experienced complication related to iStent malpositioning and obstruction. A recent 2015 meta-analysis assessing solo iStent implan- tation, conducted by Malvankar-Mehta et al. [25], examined 5 studies with a total of 248 subjects for quantitative synthesis. Despite significant heterogeneity between studies, the meta- analysis concluded that a 22% weighted mean IOP reduction from baseline was observed at 18 months after 1 iStent was implanted, 30% weighted mean IOP reduction from baseline was observed at 6 months after 2 iStents were implanted, and 41% weighted mean IOP reduction from baseline was observed at 6 months after 3 iStents were implanted, with a statistically significant reduction found in all 3 groups. 3. iStent as a Solo Procedure Subjects were submitted to washout and divided into groups Journal of Ophthalmology 6 cataract surgery rather than the result of issues with the iStent itself.h to receive either 1 (𝑛 = 38), 2 (𝑛 = 41), or 3 (𝑛 = 40) stents. The same primary efficacy end point of an IOP reduction ≥20% without medication at 12 months compared to baseline unmedicated IOP and secondary end point of IOP ≤18 mmHg without medication at 12 months were used in this study. Additional measures included a proportional analysis of subjects with IOP ≤15 mmHg at 12 months. For all analyses of efficacy, patients could not be using topical ocular medication at 12 months and must not have undergone any additional surgical procedures for glaucoma by month 12. The randomized control trial for the iStent Study Group, reported on by Samuelson et al. [15] and Craven et al. [19], found that, among the 240 eyes enrolled, implantation of the iStent along with cataract surgery did not result in substantial additional risk or adverse events. The increased efficacy demonstrated in the iStent group during the 24-month study was achieved with no compromise in visual outcomes and with a safety profile comparable to cataract surgery alone. The most common early postoperative complications found in the iStent group were related to iStent malpositioning and obstruction (by iris, blood, etc.). Incidence of these events in the iStent Study Group was 3% and 4%, respectively. A variety of options for managing these complications have proven to be safe and clinically viable, ranging from observation while waiting for spontaneous resolution to more aggressive options such as laser therapy, stent repositioning, or stent replacement. In the iStent Study Group, 5 subjects who received an iStent (4.5%) required secondary surgery to fix iStent related complications (3 stent repositionings, 1 stent replacement, and 1 laser iridoplasty). Importantly, there was no evidence of any severe adverse events following iStent implantation. Both the primary and secondary efficacy end points were achieved by 89.2% of one-stent, 90.2% of two-stent, and 92.1% of three-stent subjects. At 12-month follow-up, an IOP ≤15 mmHg without medication was achieved by 64.9% (𝑛= 24, 95% CI: 47.5%–79.8%) of one-stent subjects, 85.4% (𝑛= 35, 95% CI: 70.8%–94.4%) of two-stent subjects, and 92.1% (𝑛 = 35, 95% CI: 78.6%–98.3%) of three-stent subjects. 3. iStent as a Solo Procedure While currently not performed commonly, the implantation of an iStent as a solo procedure has been advocated for by some authors. The earliest studies on this topic were prospective, interventional case series on patients with OAG published by Spiegel et al. in 2007 [7]. The study demon- strated a mean IOP reduction of 23.9% among the 6 patients examined in the study, from a baseline of 20.2 ± 6.3 mmHg to 15.3 ± 3.7 mmHg. Buchacra et al. [20] published results from a similar case series in 2011 which examined 10 patients with secondary OAG (including traumatic, steroid induced, pseudoexfoliative, and pigmentary glaucoma) who under- went single iStent implantation without cataract surgery. Of the 10 patients enrolled in this study, 7 had phakic lenses. The surgery was found to be effective, with 8 patients averaging a 27.3% reduction in IOP after 12 months. Additionally, the solo procedure proved to be very safe, with no complications reported among phakic eyes. A prospective pilot study evaluated the solo implantation of 2 iStents in 39 patients using one topical IOP lowering medication prior to washout. The primary end point was IOP reduction ≥20% without medication compared to baseline unmedicated IOP at 12-month follow-up and a secondary end point of IOP ≤18 mmHg without medication at 12-month follow-up. The primary and secondary efficacy end points were each achieved by 92.3% of subjects (𝑛= 36; 95% CI: 79.1%, 98.4%). Additionally, mean reduction in IOP from baseline was 44%. Most subjects maintained these target IOP thresholds through month 36, with an IOP reduction ≥20% achieved by 86.2% (𝑛= 25; 95% CI: 68.3%, 96.1%) and IOP ≤18 mmHg achieved by 89.7% of patients (𝑛= 26; 95% CI: 72.6%, 97.8%) [23].fi A prospective, nonrandomized study conducted by Ahmed et al. [21] examined the efficacy of 2 iStents implanted simultaneously in a solo procedure (Figure 1(c)). The study enrolled 39 phakic subjects with OAG which were on 2 IOP lowering medications preoperatively. Following a washout of all medications, subjects received iStent implantation surgery and were concurrently started on travoprost topical medication. From a mean baseline prewashout IOP of 22.2 ± Katz et al. also evaluated the efficacy and safety of the implantation of 1 or multiple iStents as a solo procedure. 3. iStent as a Solo Procedure Additionally, a significant reduction in ocular hypotensive medication use was seen after implantation in all 3 groups, with a mean reduction of 1.2 bottles per patient at 18 months after 1 iStent was implanted, 1.45 bottles per patient at 6 months after 2 iStents were implanted, and 1 bottle per patient at 6 months after 3 iStents were implanted. Although data was limited by the fact that only a single study had examined the impact of 3 iStents, results suggested that the IOP decrease correlates positively with the number of iStents injected. Overall, the meta-analysis concluded that iStent implantation as a solo procedure is effective in lowering IOP and reducing dependency on topical glaucoma medications. Hyphema is another complication seen in the early postoperative stage and is usually a consequence of blood reflux from the iStent. It is in fact a sign that indicates patency of the iStent and typically resolves spontaneously within 1 week postoperatively. Studies where hyphema was reported largely did not specify if it was a notable complication or normal reflux as a result of the procedure. Given this, the reported frequency of this complication varies greatly among studies from 2.3% to 70% [14, 20]. One rare complication occurs when an ophthalmologist cannot locate an implanted iStent under gonioscopy post- operatively, with one such example of this seen in a study by Fea et al. [27] published in 2014. Ichhpujani et al. [28] conducted a laboratory study in 2010 testing the efficacy of 3 different imaging technologies in their ability to locate a deliberately misplaced iStent. The results demonstrated that ultrasound biomicroscopy was able to locate the “missing” 5. iStent inject The newer, second generation of the iStent is a smaller version of the original model (Figures 2(a) and 2(b)), called the GTS- 400 iStent inject (Glaukos, Laguna Hills, CA). Developed to reduce IOP in the same safe and effective way, the iStent inject is proposed to have an easier learning curve largely due to the device’s completely different structure compared to the first generation, notably evidenced by the absence of the snorkel (Figures 2(a) and 2(c)). The new device also includes a modified injector that can be simultaneously loaded with 2 stents, an important improvement that allows surgeons to place both stents with a single entry into the eye. Bahler et al. [29] conducted a laboratory study with the new device utilizing human donor eyes, with a method similar to what has already been performed with first-generation iStent. The results demonstrated that addition of a second iStent significantly increased the outflow.i Complication rates with the iStent inject have been found to be comparable to those from the previous model. It is notable that Klamann et al. [32] showed that blood reflux occurred in 91% of the surgeries involving the iStent inject; however, there was no incidence of complicated hemorrhage. il One of the first reports on the iStent inject was published in 2013 by Arriola-Villalobos et al. [30], in which 20 patients underwent combined phacoemulsification and implantation of 2 iStent inject stents as part of a prospective, uncontrolled, interventional case series study. At a 12-month follow-up, the mean washout baseline IOP of 26±3.1 mmHg was decreased by 35.7% to 16.7 ± 2.2 mmHg (9.4 ± 3 mmHg reduction, 𝑃< 0.001). Mean number of glaucoma medications also fell from 1.3 ± 0.6 to 0.3 ± 0.5 (𝑃< 0.001), with 75% patients still completely off medication at 1 year. The study observed no adverse events and concluded that combined cataract surgery with implantation of 2 iStent inject stents seems to be a safe and effective procedure. 4. Complications of iStents A key aspect of the iStent, as a MIGS device, is its favorable safety profile. Clinical trials and case series have consistently reported few to no adverse events following its implantation [10, 15, 19, 26]. Moreover, when complications occur they are often due to issues with the simultaneously performed 7 Journal of Ophthalmology (a) (b) (c) Figure 2: Illustration of the second-generation iStent inject (a); size comparison of the iStent inject (b); schematic illustration of iStent inject placement in trabecular meshwork (c). ((a)–(c), courtesy of Glaukos Corporation). (b) (c) (a) (b) (c) (a) Figure 2: Illustration of the second-generation iStent inject (a); size comparison of the iStent inject (b); schematic illustration of iStent inject placement in trabecular meshwork (c). ((a)–(c), courtesy of Glaukos Corporation). cond-generation iStent inject (a); size comparison of the iStent inject (b); schematic illustration of iStent inject work (c). ((a)–(c), courtesy of Glaukos Corporation). Figure 2: Illustration of the second-generation iStent inject (a); size comparison of the iStent inject (b); schem placement in trabecular meshwork (c). ((a)–(c), courtesy of Glaukos Corporation). iStents with the best reliability, compared to optical coherence tomography and B-scan ultrasonography. Fea et al. [27] conducted a prospective, multicenter, randomized clinical trial in 6 countries, enrolling OAG patients with uncontrolled IOP on 1 medication who either underwent implantation of 2 iStent inject stents or received medical therapy consisting of a fixed combination. Ninety- four patients were enrolled in this study for the iStent group, the majority of whom were phakic (98%) and Caucasian (100%). After 12 months of follow up, 94.7% of the eyes in the iStent group reported an IOP reduction ≥20% without use of any medications. The mean baseline IOP after washout was 25.2 ± 1.4 mmHg, and after 12 months the mean IOP decreased to 13.0 ± 2.3 mmHg. A favorable safety profile was achieved in the iStent group as measured by a stable best corrected visual acuity and cup-to-disk ratio among subjects throughout the study, as well as few adverse events. iStents with the best reliability, compared to optical coherence tomography and B-scan ultrasonography. 7. Conclusion and during 9 years of treatment in the Collaborative Initial Glaucoma Treatment Study,” Ophthalmology, vol. 115, no. 6, pp. 927–933, 2008. The considerable focus and interest of the medical com- munity with MIGS in the last decade demonstrates that ophthalmologists are anxious for advancements in the sur- gical treatment of glaucoma. It is clear that single trabecular microbypass stents do not have an IOP reducing power comparable to more invasive surgeries such as trabeculec- tomy or tube shunt surgery. However, it is important to understand that the iStent is not intended to totally replace these procedures. MIGS, such as the iStent, instead have the potential to be a valuable option for glaucoma surgeons due to their precise indications, consistent efficacy, and ability to increase patient prognosis and quality of life. Additional high quality randomized controlled trials are still needed to confirm the advantages of MIGS over cataract surgery alone [33].hi [3] [3] S. J. Gedde, L. W. Herndon, J. D. Brandt, D. L. Budenz, W. J. Feuer, and J. C. Schiffman, “Surgical complications in the Tube Versus Trabeculectomy study during the first year of follow-up,” American Journal of Ophthalmology, vol. 143, no. 1, pp. 23–31.e2, 2007. [4] H. Saheb and I. I. K. Ahmed, “Micro-invasive glaucoma surgery: current perspectives and future directions,” Current Opinion in Ophthalmology, vol. 23, no. 2, pp. 96–104, 2012. [5] L. M. Brand˜ao and M. C. Grieshaber, “Update on minimally invasive glaucoma surgery (MIGS) and new implants,” Journal of Ophthalmology, vol. 2013, Article ID 705915, 12 pages, 2013. [6] G. M. Richter and A. L. Coleman, “Minimally invasive glau- coma surgery: current status and future prospects,” Journal of Clinical Ophthalmology, vol. 10, pp. 189–206, 2016. [7] D. Spiegel, W. Wetzel, D. S. Haffner, and R. A. Hill, “Initial clinical experience with the trabecular micro-bypass stent in patients with glaucoma,” Advances in Therapy, vol. 24, no. 1, pp. 161–170, 2007. The favorable safety profile consistently demonstrated across studies is one of the key features of the iStent, as the potential for serious adverse events can be a significant deterrent for patients and physicians when considering sur- gical interventions during the initial and moderate stages of glaucoma. New devices are continually being developed and improved, and current MIGS devices are likely only the beginning of a new era in glaucoma management. 6. Cost Considerations There is a lack of studies considering the cost effectiveness of the iStent; there exists only 1 published study to date on this topic [8]. The study, based in Canada, demonstrated that implantation of 2 iStents could possibly reduce the costs of the glaucoma treatment in comparison to the use of topical medications. Over a 6-year period, potential savings were estimated to be CA$1272 when comparing the iStent to a drug regimen using 2 medications and CA$2124 when compared to 3 medications. The study was limited by solely consid- ering the placement of 2 simultaneous iStents, a relatively uncommon practice that is not approved in some countries (e.g., United States). Further limitations arose from difficulty predicting the effective duration at which iStents can continue to control IOP due to the lack of randomized controlled trials with long-term follow-up times. It is also important to be aware of variation in cost of medications, surgery fees, and many other variables like the cost of the device itself. The financial viability of the iStent device will play a crucial role for patients and doctors when deciding to implement this technology, making it critical for more studies to be conducted on this topic. f In 2014, Voskanyan et al. [31] presented results from a prospective, multicenter, open-label study on the implan- tation of 2 iStent inject stents as a solo procedure in 99 phakic and pseudophakic subjects with OAG. At a 12-month follow-up, mean baseline washout IOP values decreased by 10.2 mmHg (39.7% reduction) from 26.3 ± 3.5 to 15.7 ± 3.7 mmHg. Additionally, 66% of subjects had an IOP ≤18 mmHg without medication, and 81% achieved an IOP ≤18 mmHg with either 1 or no medications. Medication burden also improved in 86.9% of subjects, with 15.2% expe- riencing a reduction of 1 medication and 71.7% discontinuing use of 2 or more medications postoperatively. Journal of Ophthalmology 8 7. Conclusion As valuable experience is gained performing ab interno MIGS, increasing familiarity with angle anatomy and iStent placement, and as newer stent designs are developed, there is promise of continual improvement in the surgical management of glaucoma. [8] Y. Iordanous, J. S. Kent, C. M. L. Hutnik, and M. S. Malvankar- Mehta, “Projected cost comparison of trabectome, istent, and endoscopic cyclophotocoagulation versus glaucoma medica- tion in the ontario health insurance plan,” Journal of Glaucoma, vol. 23, no. 2, pp. e112–e118, 2014. [9] S. L. Mansberger, M. O. Gordon, H. Jampel et al., “Reduction in intraocular pressure after cataract extraction: the ocular hypertension treatment study,” Ophthalmology, vol. 119, no. 9, pp. 1826–1831, 2012. [10] A. M. Fea, “Phacoemulsification versus phacoemulsification with micro-bypass stent implantation in primary open-angle glaucoma: randomized double-masked clinical trial,” Journal of Cataract and Refractive Surgery, vol. 36, no. 3, pp. 407–412, 2010. Competing Interests [11] M. S. Malvankar-Mehta, Y. Iordanous, Y. N. Chen et al., “iStent with phacoemulsification versus phacoemulsification alone for patients with glaucoma and cataract: a meta-analysis,” PLoS ONE, vol. 10, no. 7, Article ID e0131770, 2015. Dr. L. Jay Katz receives research support from Allergan, Aerie Pharm, Bausch & Lomb, Mati Therapeutics, Innfocus Inc., and Diopsys Inc. Dr. Katz is a consultant and/or on the advisory board of Allergan, Alcon, Glaukos, Aerie Pharm, Bausch & Lomb, Sucampo, Inotek, Sensimed AG, Alimera Sciences, ForSight Vision, Mati Therapeutics, and Ocular Therapeutix. Dr. Katz is also speaker for Allergan, Alcon, Merck, and Lumenis. Dr. Katz is a stock shareholder for Glaukos, Mati Therapeutics, and Aerie Pharm and is a medical monitor for Glaukos. All others coauthors have no commercial relationship to declare. [12] D. Spiegel, W. Wetzel, T. Neuhann et al., “Coexistent primary open-angle glaucoma and cataract: interim analysis of a tra- becular micro-bypass stent and concurrent cataract surgery,” European Journal of Ophthalmology, vol. 19, no. 3, pp. 393–399, 2009. [13] D. Spiegel, J. Garc´ıa-Feijo´o, J. Garc´ıa-S´anchez, and H. Lamielle, “Coexistent primary open-angle glaucoma and cataract: prelim- inary analysis of treatment by cataract surgery and the iStent trabecular micro-bypass stent,” Advances in Therapy, vol. 25, no. 5, pp. 453–464, 2008. Acknowledgments [14] I. Patel, T. A. de Klerk, and L. Au, “Manchester iStent study: early results from a prospective UK case series,” Clinical and Experimental Ophthalmology, vol. 41, no. 7, pp. 648–652, 2013. This study was funded by the Glaucoma Service Foundation to Prevent Blindness, Philadelphia, PA. [15] T. W. Samuelson, L. J. Katz, J. M. Wells, Y.-J. Duh, and J. E. Giamporcaro, “Randomized evaluation of the trabecular micro-bypass stent with phacoemulsification in patients with glaucoma and cataract,” Ophthalmology, vol. 118, no. 3, pp. 459– 467, 2011. This study was funded by the Glaucoma Service Foundation to Prevent Blindness, Philadelphia, PA. References [1] Y.-C. Tham, X. Li, T. Y. Wong, H. A. Quigley, T. Aung, and C.- Y. Cheng, “Global prevalence of glaucoma and projections of glaucoma burden through 2040: a systematic review and meta- analysis,” Ophthalmology, vol. 121, no. 11, pp. 2081–2090, 2014. [16] P. Arriola-Villalobos, J. M. Mart´ınez-de-la-Casa, D. D´ıaz-Valle, C. Fern´andez-P´erez, J. Garc´ıa-S´anchez, and J. Garc´ıa-Feijo´o, “Combined iStent trabecular micro-bypass stent implantation and phacoemulsification for coexistent open-angle glaucoma and cataract: a long-term study,” British Journal of Ophthalmol- ogy, vol. 96, no. 5, pp. 645–649, 2012. [2] D. C. Musch, B. W. Gillespie, L. M. Niziol, L. F. Cashwell, and P. R. Lichter, “Collaborative Initial Glaucoma Treatment Study G. Factors associated with intraocular pressure before Journal of Ophthalmology 9 [17] T. H. Neuhann, “Trabecular micro-bypass stent implantation during small-incision cataract surgery for open-angle glaucoma or ocular hypertension: long-term results,” Journal of Cataract & Refractive Surgery, vol. 41, no. 12, pp. 2664–2671, 2015. [31] L. Voskanyan, J. Garc´ıa-Feijo´o, J. I. Belda, A. Fea, A. J¨unemann, and C. Baudouin, “Prospective, unmasked evaluation of the iStent Inject system for open-angle glaucoma: synergy trial,” Advances in Therapy, vol. 31, no. 2, pp. 189–201, 2014. [18] A. M. Fea, G. Consolandi, M. Zola et al., “Micro-bypass implantation for primary open-angle glaucoma combined with phacoemulsification: 4-year follow-up,” Journal of Ophthalmol- ogy, vol. 2015, Article ID 795357, 4 pages, 2015. [32] M. K. J. Klamann, J. Gonnermann, M. Pahlitzsch et al., “iStent inject in phakic open angle glaucoma,” Graefe’s Archive for Clinical and Experimental Ophthalmology, vol. 253, no. 6, pp. 941–947, 2015. [33] M. L. Zhang, P. Hirunyachote, and H. Jampel, “Combined surgery versus cataract surgery alone for eyes with cataract and glaucoma,” The Cochrane Database of Systematic Reviews, vol. 7, Article ID CD008671, 2015. [19] E. R. Craven, L. J. Katz, J. M. Wells, and J. E. Giamporcaro, “Cataract surgery with trabecular micro-bypass stent implan- tation in patients with mild-to-moderate open-angle glaucoma and cataract: two-year follow-up,” Journal of Cataract and Refractive Surgery, vol. 38, no. 8, pp. 1339–1345, 2012. [34] M. El Wardani, C. Bergin, F. Achache, and E. Sharkawi, “Evaluating the trabecular micro-bypass stent combined with phacoemulsification compared to phacoemulsification alone,” Klinische Monatsbl¨atter f¨ur Augenheilkunde, vol. 232, no. 4, pp. 442–445, 2015. [20] O. Buchacra, S. Duch, E. Milla, and O. Stirbu, “One-year analysis of the istent trabecular microbypass in secondary glaucoma,” Clinical Ophthalmology, vol. 5, no. 1, pp. 321–326, 2011. [21] I. I. K. References Ahmed, L. J. Katz, D. F. Chang et al., “Prospective evaluation of microinvasive glaucoma surgery with trabecular microbypass stents and prostaglandin in open-angle glaucoma,” Journal of Cataract and Refractive Surgery, vol. 40, no. 8, pp. 1295–1300, 2014. [22] G. W. Belovay, A. Naqi, B. J. Chan, M. Rateb, and I. I. K. Ahmed, “Using multiple trabecular micro-bypass stents in cataract patients to treat open-angle glaucoma,” Journal of Cataract and Refractive Surgery, vol. 38, no. 11, pp. 1911–1917, 2012. [23] E. D. Donnenfeld, K. D. Solomon, L. Voskanyan et al., “A prospective 3-year follow-up trial of implantation of two tra- becular microbypass stents in open-angle glaucoma,” Clinical Ophthalmology, vol. 9, pp. 2057–2065, 2015. [24] L. J. Katz, C. Erb, G. A. Carceller et al., “Prospective, randomized study of one, two, or three trabecular bypass stents in open- angle glaucoma subjects on topical hypotensive medication,” Clinical Ophthalmology, vol. 9, pp. 2313–2320, 2015. [25] M. S. Malvankar-Mehta, Y. N. Chen, Y. Iordanous, W. W. Wang, J. Costella, and C. M. Hutnik, “iStent as a solo procedure for glaucoma patients: a systematic review and meta-analysis,” PLoS ONE, vol. 10, no. 5, Article ID e0128146, 2015. [26] Y. Fern´andez-Barrientos, J. Garc´ıa-Feijoo, J. M. Mart´ınez-de- la-Casa, L. E. Pablo, C. Fernandez-Perez, and J. G. Sanchez, “Fluorophotometric study of the effect of the glaukos trabecular microbypass stent on aqueous humor dynamics,” Investigative Ophthalmology and Visual Science, vol. 51, no. 7, pp. 3327–3332, 2010. [27] A. M. Fea, J. I. Belda, M. Rekas et al., “Prospective unmasked randomized evaluation of the iStent inject versus two ocular hypotensive agents in patients with primary open-angle glau- coma,” Clinical Ophthalmology, vol. 8, pp. 875–882, 2014. [28] P. Ichhpujani, L. J. Katz, R. Gille, and E. Affel, “Imaging modalities for localization of an iStent,” Ophthalmic Surgery Lasers and Imaging, vol. 41, no. 6, pp. 660–663, 2010. [29] C. K. Bahler, C. R. Hann, T. Fjield, D. Haffner, H. Heitzmann, and M. P. Fautsch, “Second-generation trabecular meshwork bypass stent (iStent inject) increases outflow facility in cultured human anterior segments,” American Journal of Ophthalmology, vol. 153, no. 6, pp. 1206–1213, 2012. [30] P. Arriola-Villalobos, J. M. Mart´ınez-de-la-Casa, D. D´ıaz-Valle et al., “Mid-term evaluation of the new Glaukos iStent with pha- coemulsification in coexistent open-angle glaucoma or ocular hypertension and cataract,” British Journal of Ophthalmology, vol. 97, no. 10, pp. 1250–1255, 2013.
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Nonsense-Mediated mRNA Decay Immunity Can Help Identify Human Polycistronic Transcripts
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Abstract Funding: The authors have no support or funding to report. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E il d it @ t il Competing Interests: The authors have declared that no competing interests exist. * E il d it @ t il Competing Interests: The authors have declared that no competing interests exist. * E-mail: dorits@mta.ac.il Nonsense-Mediated mRNA Decay Immunity Can Help Identify Human Polycistronic Transcripts Guy Shahaf, Dorit Shweiki* Bioinformatics Program, School of Computer Science, The Academic College of Tel Aviv-Yaffo, Tel Aviv, Israel Citation: Shahaf G, Shweiki D (2014) Nonsense-Mediated mRNA Decay Immunity Can Help Identify Human Polycistronic Transcripts. PLoS ONE 9(3): e91535. doi:10.1371/journal.pone.0091535 Copyright:  2014 Shahaf, Shweiki. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Eukaryotic polycistronic transcription units are rare and only a few examples are known, mostly being the outcome of serendipitous discovery. We claim that nonsense-mediated mRNA decay (NMD) immune structure is a common characteristic of polycistronic transcripts, and that this immunity is an emergent property derived from all functional CDSs. The human RefSeq transcriptome was computationally screened for transcripts capable of eliciting NMD, and which contain an additional ORF(s) potentially capable of rescuing the transcript from NMD. Transcripts were further analyzed implementing domain-based strategies in order to estimate the potential of the candidate ORF to encode a functional protein. Consequently, we predict the existence of forty nine novel polycistronic transcripts. Experimental verification was carried out utilizing two different types of analyses. First, five Gene Expression Omnibus (GEO) datasets from published NMD-inhibition studies were used, aiming to explore whether a given mRNA is indeed insensitive to NMD. All known bicistronic transcripts and eleven out of the twelve predicted genes that were analyzed, displayed NMD insensitivity using various NMD inhibitors. For three genes, a mixed expression pattern was observed presenting both NMD sensitivity and insensitivity in different cell types. Second, we used published global translation initiation sequencing data from HEK293 cells to verify the existence of translation initiation sites in our predicted polycistronic genes. In five of our genes, the predicted rescuing uORFs are indeed identified as translation initiation sites, and in two additional genes, one of two predicted rescuing uORF is verified. These results validate our computational analysis and reinforce the possibility that NMD-immune architecture is a parameter by which polycistronic genes can be identified. Moreover, we present evidence for NMD-mediated regulation controlling the production of one or more proteins encoded in the polycistronic transcript. Citation: Shahaf G, Shweiki D (2014) Nonsense-Mediated mRNA Decay Immunity Can Help Identify Human Polycistronic Transcripts. PLoS ONE 9(3): e91535. doi:10.1371/journal.pone.0091535 eiki D (2014) Nonsense-Mediated mRNA Decay Immunity Can Help Identify Human Polycistronic Transcripts. PLoS ONE 9(3): e91535 0091535 Editor: Christophe Antoniewski, CNRS UMR7622 & University Paris 6 Pierre-et-Marie-Curie, France Received November 21, 2012; Accepted February 13, 2014; Published March 12, 2014 Received November 21, 2012; Accepted February 13, 2014; Published March 12, 2014 Copyright:  2014 Shahaf, Shweiki. This is an open-access article distributed under the terms of the Creative Commons Att unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Known human polycistronic transcript architecture Our main hypothesis was that polycistronic transcripts share a distinctive NMD-immune architecture, leading to the production of stable mRNA, maximally available for translation. We thus assessed the architecture of the known human polycistronic and bicistronic transcripts (Figure 1). In two bicistronic genes (LASS1- GDF1, and SNURF-SNRPN), NMD immunity is hypothesized to be contributed by both CDSs (LASS1-GDF1, all exon junctions are covered by the two CDSs; SNURF CDS ends 45 nucleotides upstream to the exon junction). In the MFRP-C1QTNF5 gene, three ORFs are responsible for NMD-immune architecture, yet only two of them are documented to encode the known proteins (the first encodes MFRP and the last - C1QTNF5). The MFRP- C1QTNF gene was found to be strongly expressed in human medulla oblongata [3]. Thus, we speculated that, either the middle ORF is also translated, contributing to the removal of the EJCs, or that EJC removal results from an upstream ribosome-dependent spatial effect, occurring during translation reinitiation of the C1QTNF5 CDS (with its AUG positioned only 44 nucleotides downstream to the exon junction; see discussion section). Finally, the MTPN stop codon in the MTPN-LUZP6 transcripts is positioned in the terminal exon solely responsible for transcript NMD-immunity, while LUZP6 is encoded by a cryptic ORF positioned in the 39 UTR region, which uses IRES and a non- AUG translation initiation codon [5]. Hence, in the four known human bicistronic examples, NMD-immune architecture is demonstrated. Yet, given the very few documented genes available, more evidence is necessary. Turning a polycistronic transcript into NMD-immune following the pioneer round of translation requires the removal of all EJCs subsequent to the translation of the functional CDS. In this manuscript we raise the hypothesis that human (and most likely mammalian) functional polycistronic transcripts share a distinctive NMD-immune architecture, which is an emergent property of all functional CDSs. Further, we argue that the definition of potentially NMD-eliciting transcripts (failing to fulfill the "55 nucleotide rule") ought to include the 5’ UTR of the molecule, as occurs in many upstream open reading frame (uORF) containing transcripts. In non-polycistronic transcripts. uORFs, which are found in almost 50% of human genes, are mainly characterized by their negative regulatory effect [24,25,26,27]. In other words, the upstream CDS in polycistronic transcripts and the regulatory uORF differ by their NMD-induction potential. Introduction EJCs, positioned ,20–24 nucleotides upstream to the exon-exon junction, are detached and removed. It was demonstrated that translating ribosomes are responsible for the removal of the EJCs positioned within the coding region, during the pioneer round of translation [9,10,11]. Un-removed EJCs in prematurely transla- tion-terminated transcripts trigger NMD degradation. By and large, PTCs elicit NMD if positioned more than 55 nucleotides upstream to the terminal exon-exon junction, known as the ‘‘55 nucleotide rule’’. Stop codons positioned downstream to this site (in the penultimate or the terminal exon) fail to elicit NMD and are considered NMD immune [7,12]. The vast majority of eukaryotic genes are considered mono- cistronic with a single transcription unit encoding for a single protein (alternatively-spliced variants included). Polycistronic transcription units (no trans-splicing involved; i.e., "eukaryotic operon") are rare in eukaryotes and specifically in mammals, and therefore little is known on how they differ from the monocistronic ones. Genomically organized polycistronic units are known in several organisms (e.g., nematodes, Arabidopsis thaliana) yet those are trans-spliced and each monocistronic unit is translated indepen- dently [1]. Further, episodic occurrences of eukaryotic bicistronic transcripts, which do not undergo trans-splicing are documented (including STNA-STNB in Drosophila; GK-GPR in tomato and mammalian GDF-1-LASS1, SNRPN-SNURF, MTPN-LUZP6 and MFRP- C1QTNF5) [1,2,3,4,5]. Seven polypeptides constitute the mammalian NMD core mechanism: up-frameshift protein 1 (UPF1), UPF2, UPF3 (comprised isoforms UPF3 and UPF3X) SMG1, SMG5, SMG6 and SMG7. UPF1 is the most conserved, essential protein, with RNA-dependent ATPase and 59-39 helicase activities [13,14]. UPF1 was shown to directly interact with both cap-binding- protein CBP80 and translation termination factors eRF1 and/or eRF3, thus likely linking NMD and translation termination activities [15,16]. In the event of premature termination, UPF1 and SMG1 interact with EJC-associated UPF2 and UPF3X. Consequent to UPF1/SMG1- EJC interaction, SMG1-mediated UPF1 phosphorylation occurs, triggering translational repression and NMD induced degradation [17,18]. Until recently the Newly synthesized mRNAs are subjected to a pioneer round of translation in which premature termination codon (PTC) contain- ing transcripts are identified and degraded in various degrees of efficiency via the Nonsense-mediated mRNA decay (NMD) mechanism [6,7]. In mammals, NMD onset is primarily associated with the identification of un-removed exon-junction protein complexes (EJCs) in PTC-containing transcripts [8]. During the pioneer round event, previously deposited splicing-dependent March 2014 | Volume 9 | Issue 3 | e91535 1 PLOS ONE | www.plosone.org Polycistronic mRNAs Share NMD-Immune Architecture Figure 1. Known human polycistronic transcripts architecture. Introduction Exon junctions highlighted in bold, uncovered exon junction coordinates are indicated in bold; annotated CDS in turquoise; ORF in purple; CDS, ORF and transcript coordinates are indicated. doi:10.1371/journal.pone.0091535.g001 Figure 1. Known human polycistronic transcripts architecture. Exon junctions highlighted in bold, uncovered exon junction coordinates are indicated in bold; annotated CDS in turquoise; ORF in purple; CDS, ORF and transcript coordinates are indicated. doi:10.1371/journal.pone.0091535.g001 functional protein. Polycistronic (mainly bicistronic) transcript prediction is presented and discussed. common belief was that NMD is restricted to the pioneer round of translation and only to mRNAs which are associated with cap- binding-protein CBP80-CBP20 complex. Following the removal of the EJCs and the CBP80-CBP20 complex and its replacement by eIF4E, the transcript therefore becomes NMD immune, free to undergo multiple translation cycles [14,19,20,21]. Recently, however, several lines of evidences indicated that NMD may also occur on eIF4E-bound transcripts, which are already being translated [22,23]. common belief was that NMD is restricted to the pioneer round of translation and only to mRNAs which are associated with cap- binding-protein CBP80-CBP20 complex. Following the removal of the EJCs and the CBP80-CBP20 complex and its replacement by eIF4E, the transcript therefore becomes NMD immune, free to undergo multiple translation cycles [14,19,20,21]. Recently, however, several lines of evidences indicated that NMD may also occur on eIF4E-bound transcripts, which are already being translated [22,23]. Novel polycistronic transcript prediction in the 3’ UTR of penultimate or upstream NMD-eliciting transcripts analyzed for IRES elements with no positive results. (ii) No significant similarity between the candidate ORF sequence and the annotated CDS (or CDSs of alternatively spliced isoforms of the same gene; lower than 50% similarity). A high degree of sequence similarity was assumed to indicate gene rearrangement rather than the existence of a functional ORF. (iii) The potential ORF encoded protein shares a significant similarity to other proteins in the protein database or contains functional domains according to InterProScan analysis (or both – see Methods). In addition, candidate polycistronic transcripts were screened for transcript architecture conservation in other organisms, utilizing BLAST analysis to GenBank databases. p p g p Based on the architecture of the known bicistronic transcripts, we devised a strategy for the identification of novel polycistronic genes. Polycistronic transcript search was limited to potentially NMD-eliciting transcripts with an annotated stop codon posi- tioned in the penultimate or upstream exon. Transcripts in which the annotated stop codon is positioned in the terminal exon (similar to the MTPN-LUZP6 gene) were excluded from this study due to the following reasons: (i) the vast majority of the known mammalian bicistronic genes share an NMD-immune architecture contributed by all functional CDSs; (ii) no other criteria were indicative enough: our preliminary results show that ORF coding potential alone is insufficient to distinguish functional ORFs from non-functional ones (data not shown). Furthermore, comparative genomics per se seem to be inadequate based on the lack of evolutionary conservation in the known bicistronic genes. Out of the 93 potential rescuing ORFs, 53 (39 transcripts) were discarded due to high homology between the rescuing ORF and the annotated CDS. The remaining ORFs were further analyzed according to the criteria elaborated above. Eight candidate bicistronic transcripts (6 genes) were identified, out of which two were discarded because the predicted protein was identified to contain only a signal peptide sequence, with no other known protein domains (See Methods section). From the remaining six transcripts, three novel (2 genes) and three known bicistronic transcripts (SNRPN, MFRP and LASS1; GI’s: 29540557, 223633880 and 110349723, respectively) were identified (Table 1, only novel candidates are presented). In all, 30035 Refseq records were analyzed for potentially failing to fulfill the ‘‘55 nucleotides rule’’ and eliciting NMD, as detailed in the Methods section. Of these, 113 transcripts contained an annotated stop codon positioned 55 nucleotides or more upstream to the terminal exon-exon junction. Novel polycistronic transcript prediction in the 3’ UTR of penultimate or upstream NMD-eliciting transcripts Those were further analyzed for the existence of ORFs which are potentially capable of turning the transcript from NMD-eliciting into NMD- immune. Ninety three potential rescuing ORFs were identified in 68 transcripts. Annotated ATG exon position: implications for polycistronic transcripts prediction The existence of a rescuing ORF overlapping an exon junction is far from sufficient in order to identify a polycistronic transcript. We therefore assessed potential functional ORFs based on the following criteria: (i) Existence of a translation initiation sequence. Two potential elements - Kozak-like sequence and internal ribosome entry sites (IRESs) in the 5’ end can be considered. We avoided relying on IRES identification as a search criterion because its presence in cellular mRNAs is still debated [28,29]. Over 85 reported cellular IRES-containing transcripts share long 5’ UTRs, multiple uAUGs and a similar GC content, yet a considerable amount of genes fit this profile with no evidences for IRES existence [29,30]. Furthermore, IRESs are characterized structurally, with no known consensus sequence and therefore in silico identification is problematic, and most studies focus on empirical data validation not on novel IRES prediction [30,31,32]. Indeed, when screening the known bicistronic transcripts for IRES sequences, utilizing UTRScan and IRSS, no IRES elements were identified (data not shown) [33,34]. Still, all polycistronic candidates reported in this manuscript were computationally Limiting the search for functional ORFs to the 3’ UTR of the mRNA seems arbitrary. One CDS may indeed be more dominant over the other in terms of its expression level, yet it is not necessarily the first in the polycistronic transcript (e.g., SNURF- SNRPN). Similar to the strategy undertaken in the former stage, we needed to distinguish transcripts which contain a regulatory uORF from polycistronic ones in which the upstream CDS is still unknown. The upstream CDSs in polycistronic transcripts and regulatory uORFs differ first and foremost by their NMD- induction potential. Thus we performed a preliminary analysis aiming to identify potentially NMD-eliciting transcripts based on mRNA 5’ screening. We analyzed the distribution of the annotated ATG exon position in human RefSeq transcripts and evaluated how many of them are potentially NMD-eliciting (unless a rescuing ORF will be revealed). NMD degradation induction relies on EJCs that remain after the pioneer round of translation. Since no known sequence-based parameters are available to indicate whether translation re-initiation will occur in sequential Table 1. Novel bicistronic transcript candidates followed 39 UTR analysis of penultimate or upstream NMD-eliciting transcripts. Known human polycistronic transcript architecture A computational-based approach was utilized to survey monocistronic and polycistronic transcript architecture and to predict the existence of novel polycistronic transcripts in the human transcriptome. We screened the human RefSeq dataset for potentially NMD-eliciting transcripts, according to the classic definition and our modified one. Further, we aimed to isolate those transcripts containing ORFs capable of "rescuing" the mRNA from its NMD-eliciting destiny, i.e., overlapping the exon junctions or positioned in their proximity (as detailed in the Methods section). We then applied domain-based strategies (see below) to predict the potential of the candidate ORF to encode a March 2014 | Volume 9 | Issue 3 | e91535 PLOS ONE | www.plosone.org 2 Polycistronic mRNAs Share NMD-Immune Architecture Novel polycistronic transcript prediction in the 3’ UTR of penultimate or upstream NMD-eliciting transcripts 59 UTR-based novel polycistronic transcript prediction NMD insensitivity of polycistronic transcripts. mRNA expression datasets from Gene Identification by NMD inhibition (GINI) experiments, in which mRNA levels are compared in the presence and absence of NMD- inhibitors (emetine, caffeine and NMD-specific siRNA inhibitors) were utilized to identify polycis- tronic transcript NMD sensitivity. Five datasets representing a variety of cell types were downloaded from the Gene Expression Omnibus database (GEO) and analyzed. Tables 4 and 5 summarize the NMD sensitivity status of the known bicistronic (Table 4) and polycistronic predicted genes (Table 5; detailed information in Tables S2A and S2B) found in the different experiments. Overall, the known bicistronic genes display consid- erable, stable expression in the different cell types analyzed (Table 4, Table S2A). Fourteen of the predicted genes fulfilled our primary criterion, i.e. genes which all their documented transcripts seem polycistronic (see Methods section). Out of these, twelve are represented in the various datasets that were used for validation (C20orf203, ERVFRD-1, FRRS1, HMGB1, LOC401052, NMD insensitivity of polycistronic transcripts. mRNA expression datasets from Gene Identification by NMD inhibition (GINI) experiments, in which mRNA levels are compared in the presence and absence of NMD- inhibitors (emetine, caffeine and NMD-specific siRNA inhibitors) were utilized to identify polycis- tronic transcript NMD sensitivity. Five datasets representing a variety of cell types were downloaded from the Gene Expression Omnibus database (GEO) and analyzed. Tables 4 and 5 summarize the NMD sensitivity status of the known bicistronic (Table 4) and polycistronic predicted genes (Table 5; detailed information in Tables S2A and S2B) found in the different experiments. Overall, the known bicistronic genes display consid- erable, stable expression in the different cell types analyzed (Table 4, Table S2A). Fourteen of the predicted genes fulfilled our primary criterion, i.e. genes which all their documented transcripts seem polycistronic (see Methods section). Out of these, twelve are represented in the various datasets that were used for validation (C20orf203, ERVFRD-1, FRRS1, HMGB1, LOC401052, After dividing the transcriptome into groups according to the annotated ATG position and the existence of rescuing uORFs, we turned to predict the 5’ UTR-related novel polycistronic transcript potential. A total of 4130 transcripts (13.8% of Refseq transcrip- tome) constitute the dataset from which we aimed to differentiate transcripts with regulatory uORFs from those with functional upstream CDSs. Annotated ATG exon position: implications for polycistronic transcripts prediction In 29 of these 81 transcripts, InterProScan analysis identified only a signal peptide sequence and/or transmembrane regions, and they were therefore discarded (See Methods section). The remaining 52 transcripts are considered polycistronic candidates, among which three are known transcripts (SNURF-SNRPN, LUZP6 and GDF1; GIs: 29540556, 190886450 and 110349791, respectively). An addition- al three undergo an unusual transcription pattern: leptin receptor (LEPR, GI: 310923183), which is reported to share the same promoter and the first two exons with the leptin receptor overlapping transcript (LEPROT) gene [36]; The IGF 2 read- through product (GI: 183603938); And the GPR75- ASB3 gene (G protein-coupled receptor 75-ankyrin repeat and SOCS box containing 3; GI: 188528701) read-through product [37] (Table 3, a detailed description in Table S1). ORFs, our approach is applicable only for those cases in which the uORF/CDS and the annotated ATG are positioned in different exons and therefore at least one remaining EJC potentially exists. Transcripts for which the first exon contains the 59 UTR and the annotated ATG, as well as potentially encoding ORF, were not included in our study as they require experimental evaluation of re-initiation and NMD-eliciting potential. We found that only 59% of the annotated ATGs are positioned in the first exon of the transcript and the rest are positioned in the second or downstream exons (Table 2). ORFs, our approach is applicable only for those cases in which the uORF/CDS and the annotated ATG are positioned in different exons and therefore at least one remaining EJC potentially exists. Transcripts for which the first exon contains the 59 UTR and the annotated ATG, as well as potentially encoding ORF, were not included in our study as they require experimental evaluation of re-initiation and NMD-eliciting potential. We found that only 59% of the annotated ATGs are positioned in the first exon of the transcript and the rest are positioned in the second or downstream exons (Table 2). Transcripts in which the annotated ATG is positioned in the second or downstream exons were analyzed for 59 UTR ORF existence (12320 records; 41% of the Refseq transcriptome). Of these, 6118 transcripts (20.3% of total Refseq transcripts) contain no ORF in their 59 UTR, i.e., the ribosomal 43S pre-initiation complex is assumed to scan the mRNA until the annotated ATG is reached (detaching pre-deposited EJCs on its way) [19,20,21]. These transcripts were therefore not considered as candidates for 59 UTR-related NMD-induction. Annotated ATG exon position: implications for polycistronic transcripts prediction Gene Symbol Gene Name Transcript GI Predicted functional ORF position Kozak Sequence InterProScan BlastP C20orf203 Chromosome 20 open reading frame 203 292658848 1876..2109 CTTACTATGT signal peptide 1–19; PTHR12138, family-not- named domain 11–49 No NAT15 N-acetyltransferase 15 (GCN5-related, putative) 134254454 1165..1716 CAGAGCATGC signal peptide 1–23; 95% identity with hypothetical protein LOC100609520 [Pan troglodytes] 183 a.a.; 86% identity with hypothetical protein LOC100443079 [Pongo abelii] 223 a.a. 134254439 1115..1666 doi:10.1371/journal.pone.0091535.t001 PLOS ONE | www.plosone.org 3 March 2014 | Volume 9 | Issue 3 | e91535 Table 1. Novel bicistronic transcript candidates followed 39 UTR analysis of penultimate or upstream NMD-eliciting transcripts. Gene Symbol Gene Name Transcript GI Predicted functional ORF position Kozak Sequence InterProScan BlastP C20orf203 Chromosome 20 open reading frame 203 292658848 1876..2109 CTTACTATGT signal peptide 1–19; PTHR12138, family-not- named domain 11–49 No NAT15 N-acetyltransferase 15 (GCN5-related, putative) 134254454 1165..1716 CAGAGCATGC signal peptide 1–23; 95% identity with hypothetical protein LOC100609520 [Pan troglodytes] 183 a.a.; 86% identity with hypothetical protein LOC100443079 [Pongo abelii] 223 a.a. 134254439 1115..1666 doi:10.1371/journal.pone.0091535.t001 PLOS ONE | www.plosone.org 3 March 2014 | Volume 9 | Issue 3 | e91535 Table 1. Novel bicistronic transcript candidates followed 39 UTR analysis of penultimate or upstream N March 2014 | Volume 9 | Issue 3 | e91535 3 Polycistronic mRNAs Share NMD-Immune Architecture Table 2. Annotated ATG exon position in human RefSeq transcripts. Table 2. Annotated ATG exon position in human RefSeq transcripts. Exon no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 No. of Transcripts 17715 8908 2369 687 217 77 35 15 6 1 1 1 1 2 % of Total 59.0 29.7 7.9 2.3 0.72 0.26 0.12 0.050 0.020 0.003 0.003 0.003 0.003 0.007 doi:10.1371/journal.pone.0091535.t002 relatively small size of human uORFs (average length 51.5 nucleotides) [35]. ORF encoding potential and Kozak sequence recognition were carried out as described above and in Methods. Out of the 4130 candidate transcripts screened, 335 were identified to contain ORFs larger than 99 nucleotides with no significant similarity between the candidate ORF sequence and the annotated CDS. Of these, 81 transcripts: (i) contain a Kozak- like sequence in proximity to the candidate AUG and (ii) the potential ORF-encoded protein shares a significant similarity to other proteins in the protein database and/or contains functional domains according to InterProScan analysis. Novel polycistronic transcript validation We hypothesized that human polycistronic mRNAs share a unique configuration, in which functional CDSs are mutually organizes in an NMD-immune structure. This architecture was demonstrated in four known bicistronic genes (LASS1-GDF1, SNURF-SNRPN, MFRP-C1QTNF5 and MTPN-LUZP6), and was further used to predict the existence of 49 novel polycistronic transcripts. In order to validate our predictions, we screened the literature and databases for known cases of NMD inhibition and transcription initiation site detection. At this point we concluded the following: (i) a considerable portion of RefSeq transcripts contain two or more ORFs, are in NMD-immune architecture, and therefore have the potential to function as polycistronic mRNAs; (ii) NMD-eliciting potential in the human transcriptome is likely higher then so far evaluated, due to 59 UTR-related NMD-eliciting architecture (2063 records; 6.9% of total Refseq transcripts). Annotated ATG exon position: implications for polycistronic transcripts prediction Another 4130 transcripts (13.8% of total Refseq transcripts), contain one or more ORFs in their 59 UTR, yet are expected to display NMD-immunity due to their architecture which theoretically ensures removal of all EJCs according to the ‘‘55-nucleotide rule’’, implemented on the 59 UTR. Finally, 2063 records (6.9% of total Refseq transcripts), although they contain an ORF in their 59 UTR, are predicted to have an NMD-eliciting architecture, since not all EJCs are expected to be removed after the pioneer round of translation, even if re-initiation occurs at the annotated ATG. 59 UTR-based novel polycistronic transcript prediction Two working assumptions guided this stage: (i) the first ATG identified by the 43S pre-initiation complex can be positioned in the second and downstream exon, and all EJCs deposited upstream to it are removed. Therefore no full exon-junctions coverage is required, and instead we screened for exon-junction coverage between the end of the first ORF identified and the annotated ATG. (ii) potential ORFs were analyzed only if the ORF was larger than 99 nucleotides. This cutoff value was set based on the size range of known polycistronic encoded proteins (59 to 580 amino acids, LUZP6 and MFRP, respectively) and the March 2014 | Volume 9 | Issue 3 | e91535 PLOS ONE | www.plosone.org 4 Polycistronic mRNAs Share NMD-Immune Architecture Table 3. Novel human polycistronic transcript candidates followed 5’ UTR analysis. LOC442578, MGC119295, STAG3L3, TXNDC6, UTP14C, ZNF117 and ZNF841), mostly displaying NMD insensitivity (Table 5, Table S2B). ZNF841 exhibits NMD sensitivity in the sole experiment that monitored this gene, in mononuclear leukocytes taken from both healthy and prostate cancer patients (GSE24204). 59 UTR-based novel polycistronic transcript prediction Three more genes display partial NMD sensitivity: GeneID Gene Symbol Gene Name 80823 BHLHB9 basic helix-loop-helix domain containing, class B, 9 6046 BRD2 bromodomain containing 2 84798 C19orf48 chromosome 19 open reading frame 48 9139 CBFA2T2 core-binding factor, runt domain, alpha subunit 2; translocated to, 2 966 CD59 CD59 molecule, complement regulatory protein 9425 CDYL chromodomain protein, Y-like 56616 DIABLO diablo, IAP-binding mitochondrial protein 405754 ERVFRD-1 endogenous retrovirus group FRD, member 1 57579 FAM135A family with sequence similarity 135, member A 391059 FRRS1 ferric-chelate reductase 1 2657 GDF1 growth differentiation factor 1 81491 GPR63 G protein-coupled receptor 63 10936 GPR75 G protein-coupled receptor 75 3146 HMGB1 high mobility group box 1 3481 IGF2 insulin-like growth factor 2 (somatomedin A) 3781 KCNN2 potassium intermediate/small conductance calcium-activated channel, subfamily N, member 2 3953 LEPR leptin receptor 401052 LOC401052 hypothetical LOC401052 767558 LUZP6 leucine zipper protein 6 8195 MKKS McKusick-Kaufman syndrome 318 NUDT2 nudix (nucleoside diphosphate linked moiety X)-type motif 2 5569 PKIA protein kinase (cAMP-dependent, catalytic) inhibitor alpha 11272 PRR4 proline rich 4 (lacrimal) 80758 PRR7 proline rich 7 5724 PTAFR platelet-activating factor receptor 494115 RBMXL1 RNA binding motif protein, X-linked-like 1 5265 SERPINA1 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 6579 SLCO1A2 solute carrier organic anion transporter family, member 1A2 6638 SNRPN small nuclear ribonucleoprotein polypeptide N 441273 SPDYE2 speedy homolog E2 (Xenopus laevis) 1E+08 SPDYE2L WBSCR19-like protein 3 442578 STAG3L3 stromal antigen 3-like 3 51807 TUBA8 tubulin, alpha 8 347736 TXNDC6 thioredoxin domain containing 6 9724 UTP14C UTP14, U3 small nucleolar ribonucleoprotein, homolog C (yeast) 9189 ZBED1 zinc finger, BED-type containing 1 9189 ZBED1 zinc finger, BED-type containing 1 51351 ZNF117 zinc finger protein 117 8187 ZNF239 zinc finger protein 239 339324 ZNF260 zinc finger protein 260 353274 ZNF445 zinc finger protein 445 55769 ZNF83 zinc finger protein 83 162962 ZNF836 zinc finger protein 836 284371 ZNF841 zinc finger protein 841 Novel polycistronic transcript candidates are presented (alphabetically sorted by gene symbol). Documented genes highlighted in bold. doi:10.1371/journal.pone.0091535.t003 LOC442578, MGC119295, STAG3L3, TXNDC6, UTP14C, ZNF117 and ZNF841), mostly displaying NMD insensitivity (Table 5, Table S2B). ZNF841 exhibits NMD sensitivity in the sole experiment that monitored this gene, in mononuclear leukocytes taken from both healthy and prostate cancer patients (GSE24204). LOC442578, MGC119295, STAG3L3, TXNDC6, UTP14C, ZNF117 and ZNF841), mostly displaying NMD insensitivity (Table 5, Table S2B). ZNF841 exhibits NMD sensitivity in the 59 UTR-based novel polycistronic transcript prediction Three more genes display partial NMD sensitivity: March 2014 | Volume 9 | Issue 3 | e91535 PLOS ONE | www.plosone.org March 2014 | Volume 9 | Issue 3 | e91535 March 2014 | Volume 9 | Issue 3 | e91535 5 Polycistronic mRNAs Share NMD-Immune Architecture Table 4. NMD sensitivity status of human bicistronic genes in published NMD-inhibition experiments. Table 4. NMD sensitivity status of human bicistronic genes in published NMD-inhibition experiments. Table 4. NMD sensitivity status of human bicistronic genes in published NMD-inhibition experiments. GEO Dataset/ Cell type Citation NMD -Inhibition method Gene symbol ProbeID Type of transcripts identified NMD Sensitivity GSE1703 Hela Cells Mendell, JT. et al, Nat Genet. 36, 1073 - 1078 (2004); PMID:15448691 RENT1-siRNA GDF1-LASS1 887_at bicistronic transcripts (NM_0212673; NM_001492) NMD insensitive 888_s_at monicistronic variant (NM_198207) NMD insensitive SNRPN-SNURF 34842_at both monicistronic and bicistronic variants NMD insensitive GSE16170 Hela Cells Choe, J. et al EMBO Rep 11(5): 380-386 (2010); PMID: 20395958 Ago2 siRNA; UPF1 and Ago2 siRNA. SNRPN-SNURF ILMN_1656537 both monicistronic and bicistronic variants NMD insensitive MTPN-LUZP6 ILMN_2180682 bicistronic transcript (NM_145808) NMD insensitive GSE20491 Clear cell renal cell carcinoma Duns, G. et al, Cancer Res 70(11):4287–4291 (2010). PMID:20501857 Emetine or caffeine inhibition SNRPN-SNURF ILMN_1660000 bicistronic transcript (NM_005678) NMD insensitive MTPN-LUZP6 ILMN_218068, ILMN_1791478 bicistronic transcript (NM_145808) NMD insensitive GSE24204 Prostate cancer Mattila, H., University of Tampere. Finland (unpublished). Emetine inhibition GDF1-LASS1 25143 two bicistronic transcripts (NM_0212673; NM_001492) NMD insensitive MFRP-C1QTNF5 37231, 20996 bicistronic transcripts (NM_031433; NM_015645) NMD insensitive MTPN-LUZP6 4388, 23064, 41236 bicistronic transcript (NM_145808) NMD insensitive GSE29788 Head and neck cell lines Sharma. S., et al, Mol Cancer Ther. 10(9): 1751–1759, (2011). PMID: 21764905 Emetine inhibition SNRPN-SNURF 201522_x_at, 206042_x_at both monicistronic and bicistronic variants NMD insensitive doi:10.1371/journal.pone.0091535.t004 doi:10.1371/journal.pone.0091535.t004 The UTP14C gene displays a mixed expression pattern in different cell types, with NMD sensitivity in one experiment and insensitivity in three others. The FRRS1 gene exhibits NMD insensitivity in one experiment (GSE16170) using two treatments (Ago2 siRNA - targets CBP80/20-bound mRNAs and thus considered a regulator of NMD and UPF1 and Ago2 siRNA), and NMD sensitivity in another experiment, in mononuclear leukocytes of prostate cancer patients but not of healthy patients (GSE24204). Finally, the HMGB1 gene displays a mixed expression pattern, indicating NMD sensitivity in one out of 4 experiments. Yet in the same experiment, one HMGB1 probe produces an NMD sensitivity pattern while the other probe indicates NMD insensitivity (GSE29788). 59 UTR-based novel polycistronic transcript prediction The probe which is responsible for the NMD sensitivity pattern identifies also an additional antisense transcript (AF83771), for which we have no CDS annotation, and thus may be an artifact. It may be argued that NMD inhibition is not be fully achieved in GINI experiments and thus lead to somewhat distorted results, yet several observa- tions oppose this notion, including: different methods of NMD inhibition were undertaken in the various experiments analyzed; the mixed results of NMD sensitivity and insensitivity, which was limited to a narrow number of our genes and not for the entire array; and significant, high levels of expression displayed for only some of our genes. All these characteristics do not fit a general NMD-inhibition failure pattern. as some transcripts may show detectable levels of seemingly stable mRNA even though NMD degradation does occur. A more direct verification is therefore desired, reinforcing our validation results. We therefore crosschecked our predicted transcript list with a dataset of experimentally verified TISs, produced by utilizing global translation initiation sequencing (GTI-seq) in the transcrip- tome of human embryonic cell line (HEK293) [38]. Out of the 49 polycistronic transcripts predicted in this study, 9 transcripts (5 genes) are listed in the Lee et al. TIS dataset, in the exact position and with the same ORF size as predicted in our study (Table 6). Additionally, two more transcripts, in which two uORFs are expected to rescue the transcript from NMD, are partially listed in this dataset with only one TIS verified. Discussion The function of NMD in quality control surveillance and as a gene expression regulatory apparatus is well documented. Diverse events contribute to PTC occurrence, with a key role for alternative splicing, nonsense mutations and SNP-related events [39,40,41,42]. The regulatory aspect of NMD was demonstrated in several physiological settings including the regulation of selenoprotein mRNAs, splicing-factor gene expression, physiolog- ically-related classes of transcripts in Hela cells and others [14,43]. Additionally, a role for an exon-truncated class of alternative polyadenylation as an NMD-rescue regulatory mechanism was previously suggested by us [44]. Translation initiation site (TIS) detection in predicted polycistronic transcripts. NMD is not 100% efficient [6,7]. Consequently, NMD insensitivity validation per se is not sufficient, March 2014 | Volume 9 | Issue 3 | e91535 PLOS ONE | www.plosone.org 6 Polycistronic mRNAs Share NMD-Immune Architecture Table 5. NMD sensitivity status of human polycistronic predicted genes in published NMD-inhibition experiments. Table 5. NMD sensitivity status of human polycistronic predicted genes in published NMD-inhibition experiments. GEO Dataset/ Cell type Citation NMD -Inhibition method Gene symbol ProbeID Type of transcripts identified NMD Sensitivity GSE1703 Hela Cells Mendell, JT. et al, Nat Genet. 36, 1073–1078 (2004); PMID:15448691 RENT1-siRNA ZNF117 36783_f_at NM_015852. NMD insensitive UTP14C 39405_at UTP14C (chr13) and UTP14 (chrX) genes. NMD insensitive GSE16170 Hela Cells Choe, J. et al EMBO Rep 11(5): 380–386 (2010); PMID: 20395958 Ago2 siRNA; UPF1 and Ago2 siRNA. HMGB1 ILMN_2231242 NM_002128 NMD insensitive Both with Ago2 siRNA and UPF1 + Ago2 siRNAs. UTP14C ILMN_1686645 NM_021645 FRRS1 ILMN_2214734 NM_001013660 LOC401052 ILMN_1791423 NM_001008737 MGC119295 ILMN_2144654 NM_001031618 LOC442578 ILMN_1791375 NM_001013739 GSE20491 Clear cell renal cell carcinoma Duns, G. et al, Cancer Res 70(11):4287–4291 (2010). PMID:20501857 Emetine or caffeine inhibition HMGB1 ILMN_223124; ILMN_1791466 NM_002128 NMD insensitive. (both emetine and caffeine; in 10 cell lines) UTP14C ILMN_1686645 NM_021645 NMD insensitive (both emetine and caffeine; in 10 cell lines) GSE24204 Prostate cancer Mattila, H., University of Tampere. Finland (unpublished). Healthy and cancerous cells Emetine inhibition C20orf203 27463 AK091025 NMD insensitive HMGB1 27795, 2170, 7063, 8395 NM_002128 NMD insensitive UTP14C 32662 NM_021645 NMD sensitive ZNF841 39976 NM_001136499 NMD sensitive TXNDC6 7699, 11753, 4719 NM_178130 NMD insensitive FRRS1 31823 NM_001013660 NMD insensitive in healthy cells; NMD sensitive in cancer cells LOC401052 13485 NM_001008737 NMD insensitive ERVFRD-1 14886 NM_207582 NMD insensitive STAG3L3 3563 NM_001013739 NMD insensitive GSE29788 Head and neck cell lines Sharma. S., et al, Mol Cancer Ther. Discussion Finally, we succeeded to find 9 predicted polycistronic transcripts (5 genes) in a translation initiation sites dataset, with the exact position and ORF size as predicted in our study (and 2 additional partial matches, for a total of 7 genes) [38]. No overlap between these gene lists of validated polycistronic transcripts was found. Yet, it is expected that different cell types will exhibit different patterns of polycistronic gene regulation, thus, one does not exclude the other. Altogether, 16 novel polycistronic genes are experimentally validated. It was shown that following translation of a uORF and the release of the 60S subunit, the 40S subunit may remain on the mRNA and resume scanning for as far as 600 nucleotides, without re-initiating translation [54]. Consequently, theoretically, EJCs may be removed from downstream exon junctions in the absence of near-by translation re-initiation. Undoubtedly, this observation is of importance for searching for an NMD-immune architecture, yet the lack of additional knowledge on the conditions affecting 40S scanning and EJC removal prevents us from implementing this knowledge in our study. Almost half of the human genes have uORFs in their 59 UTRs, capable of reducing protein expression by 30 to 80% [24,53,55,56,57]; and though these findings are well documented and acknowledged, they are not assimilated into the vast majority of studies screening and evaluating the NMD fraction of the transcriptome in different contexts. In this study we challenged the classical definition of the "55 nucleotide rule", arguing that it should be "stretched" to the 5’ UTR of the transcripts. Namely, we claim that the search for polycistronic-related functional ORFs should take place both in the 3’ and 5’ UTRs, upstream and downstream to the annotated CDS. Subsequently, we estimate the fraction size of NMD-eliciting transcripts in human Refseq transcriptome to be approximately 7.3%, significantly larger in comparison to its size (0.4%) when analyzing only the 3’ UTR. Moreover, we further argue that while evaluating the potential for NMD, both sides of the exon-exon junction (upstream and downstream) ought to be equally considered. EJCs deposited 20– 24 nucleotides upstream to the exon junction are being pushed away and removed by the ribosome. The ribosome’s spatial dimensions dictate a downstream EJC displacement even if the stop codon is positioned 50 to 55 nucleotides upstream to the exon junction. Discussion in Lee et al Table S1 3953 LEPR NM_001003680 (310923183) 74..184 111 22 9113 NM_002303 (310923184) 23 9114 NM_001003679 (310923185) 24 9112 8195 MKKS NM_018848 (25914751) 261..452 192 28 19993 9189 ZBED1 NM_004729 (57165426) 43..165 123 45 22240 NM_001171136 (283806700) 43..168 126 46 22242 80823 BHLHB9 NM_030639 (216547631) 101..211 111 4 19752 NM_001142528 (216547671) 101..226 126 5 19742 494115 RBMXL1 NM_001162536 (242247050) 378..548 171 35 10363 84798 C19orf48 NM_199249 (40548381) [139..243] 337..378 42 7 21003 339324 ZNF260 NM_001166036 (260436927) 201..299 [390..485] 99 49 8477 doi:10.1371/journal.pone.0091535.t006 Table 6. Human polycistronic transcripts found in Lee et al TIS dataset: Novel polycistronic transcripts candidates that were found in Lee et al TIS dataset with exact match both in ORF start position and length; missing rescuing ORF in brackets. Table 6. Human polycistronic transcripts found in Lee et al TIS dataset: Novel polycistronic transcripts candidates that were found in Lee et al TIS dataset with exact match both in ORF start position and length; missing rescuing ORF in brackets. doi:10.1371/journal.pone.0091535.t006 polycistronic transcripts with an NMD-immune architecture, permitting both mRNA stability and a regulatory mean to control the expression of all or some of the functional CDSs. Bioinformatics-based analysis, indicating the existence of known protein domains in the predicted functional-ORFs encoded proteins or their similarity to known proteins, supports our assumption regarding the encoded proteins’ potential to be produced and active. mRNA expression datasets from GINI experiments were utilized for experimental verification, aiming to explore whether the allegedly polycistronic transcripts are insensitive to NMD. All the known bicistronic transcripts and an additional eleven genes from our predicted gene list displayed NMD insensitivity for various NMD inhibitors, including emetine, caffeine and UPF1 and Ago2 siRNAs (other genes in our list were not sampled in those GINI experiments). The gene ZNF481, which was studied in a single experiment, displayed an NMD sensitive profile. An additional three of the eleven genes displayed a mixed expression pattern of both NMD sensitivity and insensitivity in different cell types, possibly manifesting time- dependent and process-dependent translation re-initiation regula- tion. Furthermore, some of the genes displayed significantly high levels of expression which do not fit the expression pattern of inefficient NMD. Hence, these results reinforce our view that an NMD-immune architecture is likely to play a role in polycistronic transcript expression regulation. Discussion 10(9): 1751–1759,(2011) PMID: 21764905 Emetine inhibition HMGB1 200679_x_at; 200680_x_at NM_002128 NMD insensitive 214938_x_at NM_002128 and AF283771 - anti -sense transcript NMD sensitive UTP14C 203614_at NM_021645 NMD insensitive ZNF117 207117_at; 207605_x_at NM_015852 NMD insensitive doi:10.1371/journal.pone.0091535.t005 doi:10.1371/journal.pone.0091535.t005 indicate the transcript’s destiny, but may rather hint on regulatory stratification. The NMD mechanism is not 100% efficient. Up to 25% of the PTC-containing transcripts escape NMD degradation [6,7]. An efficient re-initiation site within a short distance of a nonsense mutation and in the same exon, was demonstrated to elicit NMD rescue and immunity of the transcripts [45,46]. At times, escape was shown to be associated with the regulatory mechanism responsible for the introduction of the PTC (e.g. ApoB48 and thrombopoietin translation control) [47,48]. Overall, 5.8% protein isoforms of the SWISSPROT database are derived from PTC containing transcripts, indicating its regulatory effect [49]. Hence, the existence of a PTC in a given transcript does not necessarily The novelty of our work is in suggesting a yet undescribed connection between NMD and polycistronic gene architecture. In this study we hypothesized that human polycistronic mRNAs (and most likely mammalian ones in general) share a unique configuration, in which functional CDSs are mutually organized in an NMD-immune architecture. Indeed, we detected NMD- immune transcript architecture in the known human bicistronic genes. Further, we analyzed the human Refseq transcriptome, predicting the existence of 49 novel polycistronic transcripts. March 2014 | Volume 9 | Issue 3 | e91535 7 PLOS ONE | www.plosone.org March 2014 | Volume 9 | Issue 3 | e91535 Polycistronic mRNAs Share NMD-Immune Architecture Table 6. Human polycistronic transcripts found in Lee et al TIS dataset: Novel polycistronic transcripts candidates that were found in Lee et al TIS dataset with exact match both in ORF start position and length; missing rescuing ORF in brackets. GeneID Gene Symbol RefSeq Accession (GI) Predicted ORF ORF size Line No. in Table S1 Line No. NMD-candidate identification The remaining 33871 records were analyzed for their potential to elicit NMD. Stop codon and exon-intron partitioning of the mRNA molecule was retrieved based on the Refseq annotation. The annotated stop codon was identified as PTC if the 39 most nucleotide of the stop codon is positioned more than 55 nucleotides upstream of the terminal exon–exon junction. Transcripts in which the stop codon is positioned in the terminal exon or in the 55 nucleotides preceding the terminal exon junction were considered as NMD-immune. 113 records were identified to potentially elicit NMD. ORF identification in 3’ UTRs "Rescuing ORFs", capable of turning NMD-eliciting transcripts into NMD-immune one, were searched screening the mRNA sequence in the three reading-frames (0, +1, +2) from 5’ to 3’ direction. In order for an ORF to be defined as a rescuing one, all exon-exon junctions, downstream to the annotated stop codon, are to be covered according to the "55 nucleotide rule". Namely, the ORF should cover the entire sequence length within the range of 55 nucleotides upstream to the first exon-exon junction-down- stream to the annotated stop codon, and up to at least 55 nucleotides upstream to the terminal junction (or further downstream). For NMD-eliciting transcripts harboring an anno- tated stop codon in the penultimate exon in a distance .55 nucleotides, a minimal rescuing ORF should start and end in the penultimate exon in a distance smaller than 55 nucleotides upstream to the terminal exon. If more than one ORF was Candidate functional ORFs were predicted based on whether the potential ORF encoded protein shares a significant similarity to other proteins in the protein database. Candidate ORFs were translated to potential protein sequences and were analyzed for protein similarity utilizing BLASTP (standalone version) with default parameters against the non-redundant protein sequences (nr) database [61]. A positive ratio (number of positive hits/ORF length) . 0.5 or align length (alignment length/ORF length) . 0.8 was used to identify significant hits. Discussion There is no evidence for the translation of the protein encoded in the central ORF. We think that it is possible that the last ORF is responsible for removal of the EJCs during translation, due to ribosomal spatial hindrance, even though its ATG is positioned 44 nucleotides downstream to the exon junction. Although the MFRP-C1QTNF5 gene potentially supports our view, the distance range downstream to the exon junction ought to be examined and established experimentally before being implemented in computational studies. required in order to turn NMD-eliciting transcript into NMD- immune, they were considered as one unit. Out of 113 NMD-eliciting transcripts, 68 NMD-immune transcripts contained one or more rescuing ORFs (a total of 93 ORFs). At this stage, all rescuing ORFs were considered, including ORFs which overlap one another in different or identical reading- frames. No overlap was allowed between the annotated CDS and rescuing ORFs. Human transcriptome dataset 35157 Human Refseq mRNAs records were downloaded from the RefSeq database (NCBI, Release dated 18/12/2010; http:// www.ncbi.nlm.nih.gov/RefSeq/) [59]. 1286 records were discard- ed due to unavailable information on their exon positions or joint CDS in their annotation. Records with annotated stop codon coordinates positioned after the last exon were discarded for 3’ analysis (14 records). ORF identification in 5’ UTRs Functional uORFs were searched for in the 59 UTRs of RefSeq records for which the annotated CDS start codon is in the second or later exon. An ORF screen was carried out in the three reading- frames (0, +1, +2) from 5’ to 3’. Records which followed our hypothesized polycistronic architecture, namely, exon-junctions coverage between the end of the first ORF identified and the annotated ATG and potential ORFs larger than 99 nucleotides, were further analyzed. Out of 4130 transcripts containing ORFs in their 5’ UTR with an NMD-immune architecture, 335 transcripts were identified to contain ORFs larger than 99 nucleotides and with no significant similarity between the candidate ORF sequence and the annotated CDS, and were further evaluated for polycistronic and bicistronic candidacy. We assume that many functional-ORF encoded proteins are expressed in low levels or to a limited period of time, thus their detection is challenging. Moreover, we believe that seemingly NMD-eliciting, polycistronic transcripts are underrepresented in the mammalian genome annotation, partially due to the tendency to suppress NMD candidate mRNAs from nucleic databases (e.g., RefSeq database policy). NMD-eliciting transcripts seem to include remarkable regulatory features, waiting to be further studied. We therefore hope that our hypothesis will be further verified by experimentalists. Methods Initiation of translation by eukaryotic ribosomes is optimal at the ACCATGG consensus sequence [60]. Yet a purine in position 23 (relative to the A nucleotide in the ATG) followed by ATG at positions +1-+3 is sufficient for efficient translation initiation, thus this minimal sequence was identified as Kozak positive in this study. Initiation of translation by eukaryotic ribosomes is optimal at the ACCATGG consensus sequence [60]. Yet a purine in position Blast analysis BLASTN analysis was carried in order to rule out candidate ORF encoded proteins with significant similarity to the annotated protein of the transcript. ORF sequences were analyzed against the human RefSeq mRNA dataset utilizing BLASTN standalone application with default parameters [61]. No significant similarity between the candidate ORF sequence and the annotated CDS (or CDSs of alternatively spliced isoforms of the same gene) was allowed. A high degree of sequence similarity was hypothesized to indicate a 39 UTR ORF, which is the result of a gene rearrangement event rather than the existence of a functional ORF. Parsing of the BLASTN results was based on a threshold of e-value greater than 1.00E-06 and 50% or more coverage between the CDS from transcripts with the same gene ID and the candidate ORF. Discussion Based on this fact, we argue that in the event of translation re-initiation, EJC removal is likely to occur even if the ORF starts in close proximity downstream to the exon-exon junction, further changing NMD fraction size as estimated computationally. Indeed, yeast ribosome footprint experiments indicated the protection of 28 nucleotides upstream and down- stream to the ATG (212 to +15 nucleotides) [58]. Taking into account EJC size and the re-initiation-related sequence being scanned by the ribosome, a region larger than 12 nucleotides is The rationale for novel polycistronic transcript prediction was based on distinguishing functional CDSs from regulatory ORFs by their potential to elicit NMD and to encode a functional protein. Many studies addressed the question of parameters affecting translation re-initiation following a uORF, including uORF size, length and lack of secondary structure of the intercistronic spacing sequences and the usage of conserved uATG [19,25,26,50,51,52,53]. Yet no sequence-based information is clear enough to pinpoint whether translation re-initiation will occur. Thus, and although many additional parameters may play a role, and other polycis- tronic models are likely to exist, no sequence-based models, other than PTC occurrence seem usable. Our work therefore pro- vides one possible scenario arguing for the existence of cellular March 2014 | Volume 9 | Issue 3 | e91535 PLOS ONE | www.plosone.org 8 Polycistronic mRNAs Share NMD-Immune Architecture likely to be covered. Possibly, this claim is demonstrated in the known human bicistronic gene MFRP-C1QTNF5, whose tran- script contains 3 ORFs contributing to the NMD-immune architecture of the transcript. Yet the gene is recognized by its first and last encoded proteins. There is no evidence for the translation of the protein encoded in the central ORF. We think that it is possible that the last ORF is responsible for removal of the EJCs during translation, due to ribosomal spatial hindrance, even though its ATG is positioned 44 nucleotides downstream to the exon junction. Although the MFRP-C1QTNF5 gene potentially supports our view, the distance range downstream to the exon junction ought to be examined and established experimentally before being implemented in computational studies. likely to be covered. Possibly, this claim is demonstrated in the known human bicistronic gene MFRP-C1QTNF5, whose tran- script contains 3 ORFs contributing to the NMD-immune architecture of the transcript. Yet the gene is recognized by its first and last encoded proteins. References 17. Isken O, Kim YK, Hosoda N, Mayeur GL, Hershey JW, et al. (2008) Upf1 phosphorylation triggers translational repression during nonsense-mediated mRNA decay. Cell 133: 314–327. 1. Blumenthal T (2004) Operons in eukaryotes. Brief Funct Genomic Proteomic 3: 199–211. 2. Gray TA, Saitoh S, Nicholls RD (1999) An imprinted, mammalian bicistronic transcript encodes two independent proteins. Proc Natl Acad Sci U S A 96: 5616–5621. 18. Ohnishi T, Yamashita A, Kashima I, Schell T, Anders KR, et al. (2003) Phosphorylation of hUPF1 induces formation of mRNA surveillance complexes containing hSMG-5 and hSMG-7. Mol Cell 12: 1187–1200. 3. Katoh M (2001) Molecular cloning and characterization of MFRP, a novel gene encoding a membrane-type Frizzled-related protein. Biochem Biophys Res Commun 282: 116–123. 19. Jackson RJ, Hellen CU, Pestova TV (2010) The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol 11: 113–127. 4. Lee SJ (1991) Expression of growth/differentiation factor 1 in the nervous system: conservation of a bicistronic structure. Proc Natl Acad Sci U S A 88: 4250–4254. 20. Kozak M (1989) The scanning model for translation: an update. J Cell Biol 108: 229–241. 5. Xiong Z, Liu E, Yan Y, Silver RT, Yang F, et al. (2006) An unconventional antigen translated by a novel internal ribosome entry site elicits antitumor humoral immune reactions. J Immunol 177: 4907–4916. 21. Sonenberg N, Hinnebusch AG (2009) Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell 136: 731–745. 22. Durand S, Lykke-Andersen J (2013) Nonsense-mediated mRNA decay occurs during eIF4F-dependent translation in human cells. Nature structural & molecular biology 20: 702–709. 6. Anczukow O, Ware MD, Buisson M, Zetoune AB, Stoppa-Lyonnet D, et al. (2008) Does the nonsense-mediated mRNA decay mechanism prevent the synthesis of truncated BRCA1, CHK2, and p53 proteins? Human mutation 29: 65–73. gy 23. Rufener SC, Muhlemann O (2013) eIF4E-bound mRNPs are substrates for nonsense-mediated mRNA decay in mammalian cells. Nature structural & molecular biology 20: 710–717. 7. Isken O, Maquat LE (2007) Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function. Genes & development 21: 1833–1856. 24. Calvo SE, Pagliarini DJ, Mootha VK (2009) Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans. Proc Natl Acad Sci U S A 106: 7507–7512. 8. Maquat LE (2005) Nonsense-mediated mRNA decay in mammals. J Cell Sci 118: 1773–1776. 25. Supporting Information Table S1 Novel polycistronic transcript candidates followed 5’ UTR analysis. (XLSX) Table S2 Table S2A - Known human bicistronic genes in published GINI experiments. Table S2B - Predicted polycistronic genes in published GINI experiments. (PDF) References Churbanov A, Rogozin IB, Babenko VN, Ali H, Koonin EV (2005) Evolutionary conservation suggests a regulatory function of AUG triplets in 5’-UTRs of eukaryotic genes. Nucleic Acids Res 33: 5512–5520. 9. Dostie J, Dreyfuss G (2002) Translation is required to remove Y14 from mRNAs in the cytoplasm. Current biology : CB 12: 1060–1067. 10. Lejeune F, Ishigaki Y, Li X, Maquat LE (2002) The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling. The EMBO journal 21: 3536–3545. y g 26. Kozak M (2001) Constraints on reinitiation of translation in mammals. Nucleic Acids Res 29: 5226–5232. dynamics of mRNP remodeling. The EMBO journal 21: 3536–354 11. Sato H, Maquat LE (2009) Remodeling of the pioneer translation initiation complex involves translation and the karyopherin importin beta. Genes & development 23: 2537–2550. 27. 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Nat Rev Genet 9: 699–712. 15. Hosoda N, Kim YK, Lejeune F, Maquat LE (2005) CBP80 promotes interaction of Upf1 with Upf2 during nonsense-mediated mRNA decay in mammalian cells. Nat Struct Mol Biol 12: 893–901. 31. Filbin ME, Kieft JS (2009) Toward a structural understanding of IRES RNA function. Curr Opin Struct Biol 19: 267–276. 16. Kashima I, Yamashita A, Izumi N, Kataoka N, Morishita R, et al. (2006) Binding of a novel SMG-1-Upf1-eRF1-eRF3 complex (SURF) to the exon junction complex triggers Upf1 phosphorylation and nonsense-mediated mRNA decay. Genes Dev 20: 355–367. 32. Polycistronic transcripts NMD-immunity validation Polycistronic transcripts NMD-immunity validation Five GINI experiments datasets (GSE1703, GSE16170, GSE20491, GSE24204 and GSE29788) in which mRNA levels are compared in the presence and absence of NMD- inhibitors of different sorts (i.e., the chemicals emetine and caffeine and NMD- specific siRNA inhibition; see Tables 4 and 5 for experiment treatment details), were downloaded from the GEO database [64]. Experiments were selected based on data enabling reanalysis, and at least partial overlap between the known and predicted bicistronic genes and probes represented in the array. mRNA expression results were used only if the probe/s available in the array identified the only gene of question and not additional gene family members. Further, since the vast majority of human genes undergo alternative splicing, we obviously preferred relying on probes which distinguish between monocistronic and polycistronic Author Contributions Conceived and designed the experiments: DS. Performed the experiments: GS. Analyzed the data: GS DS. Wrote the paper: DS. Conceived and designed the experiments: DS. Performed the experiments: GS. Analyzed the data: GS DS. Wrote the paper: DS. Functional ORF encoded protein characterization utilizing InterProScan Further, candidate functional ORFs were predicted based on whether the potential ORF contains functional domains according to InterProScan analysis. The ORF-encoded proteins were March 2014 | Volume 9 | Issue 3 | e91535 PLOS ONE | www.plosone.org 9 Polycistronic mRNAs Share NMD-Immune Architecture screened for known protein domains utilizing InerProScan Web Services (http://www.ebi.ac.uk/Tools/InterProScan/) [62]. transcripts of the same gene, when such probes were available. For the predicted genes, we limited ourselves to genes which all their documented transcripts (according to RefSeq annotation), are polycistronic according to our prediction (one or more). If threshold expression levels are defined in the dataset annotation (published manuscripts included), those were taken under consid- eration, and otherwise all available results were included. In order to assess whether gene expression levels significantly differ between NMD-inhibition treatment and control cells, we implemented heteroscedastic two tailed T-Test analysis. For those experiments which specifically set a cut-off value defining the threshold of NMD sensitivity, we used the later (as indicated in Tables 4 and 5 and in Tables S2A and S2B). The signal peptide domain and the transmembrane domain were considered insufficient to predict a functional protein. Although the subcellular localization of proteins is widely studied with many tools, its prediction is not always accurate. Further, non-classical secretion pathways exist, assisting in the secretion of signal-peptide free proteins, contributing to the uncertainty of predicting subcellular localization [63]. Thus, identifying polycis- tronic transcript existence based on a signal peptide or transmembrane domain existence seems less reliable, and addi- tional indications were required. Polycistronic mRNAs Share NMD-Immune Architecture Resch AM, Ogurtsov AY, Rogozin IB, Shabalina SA, Koonin EV (2009) Evolution of alternative and constitutive regions of mammalian 5’UTRs. BMC Genomics 10: 162. 40. Lewis BP, Green RE, Brenner SE (2003) Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. Proc Natl Acad Sci U S A 100: 189-192. 57. Suzuki Y, Ishihara D, Sasaki M, Nakagawa H, Hata H, et al. (2000) Statistical analysis of the 5’ untranslated region of human mRNA using "Oligo-Capped" cDNA libraries. Genomics 64: 286–297. 41. Mort M, Ivanov D, Cooper DN, Chuzhanova NA (2008) A meta-analysis of nonsense mutations causing human genetic disease. Human mutation 29: 1037– 1047. 58. Ingolia NT, Ghaemmaghami S, Newman JR, Weissman JS (2009) Genome- wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324: 218–223. 42. Han A, Kim WY, Park SM (2007) SNP2NMD: a database of human single nucleotide polymorphisms causing nonsense-mediated mRNA decay. Bioinfor- matics 23: 397–399. 59. Pruitt KD, Tatusova T, Maglott DR (2007) NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 35: D61–65. 43. Mendell JT, Sharifi NA, Meyers JL, Martinez-Murillo F, Dietz HC (2004) Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise. Nat Genet 36: 1073–1078. 60. Kozak M (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44: 283–292. 44. Gilat R, Shweiki D (2007) A novel function for alternative polyadenylation as a rescue pathway from NMD surveillance. Biochemical and biophysical research communications 353: 487–492. y y 61. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. Journal of molecular biology 215: 403–410. 45. Buisson M, Anczukow O, Zetoune AB, Ware MD, Mazoyer S (2006) The 185delAG mutation (c.68_69delAG) in the BRCA1 gene triggers translation reinitiation at a downstream AUG codon. Human mutation 27: 1024–1029. 62. Zdobnov EM, Apweiler R (2001) InterProScan—an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17: 847–848. 63. Imai K, Nakai K (2010) Prediction of subcellular locations of proteins: where to proceed? Proteomics 10: 3970–3983. 46. Zhang J, Maquat LE (1997) Evidence that translation reinitiation abrogates nonsense-mediated mRNA decay in mammalian cells. EMBO J 16: 826–833. 64. Barrett T, Troup DB, Wilhite SE, Ledoux P, Rudnev D, et al. Polycistronic mRNAs Share NMD-Immune Architecture Polycistronic mRNAs Share NMD-Immune Architecture untranslated regions of eukaryotic mRNAs. Update 2002. Nucleic Acids Res 30: 335–340. 48. Stockklausner C, Breit S, Neu-Yilik G, Echner N, Hentze MW, et al. (2006) The uORF-containing thrombopoietin mRNA escapes nonsense-mediated decay (NMD). Nucleic acids research 34: 2355–2363. 34. Wu TY, Hsieh CC, Hong JJ, Chen CY, Tsai YS (2009) IRSS: a web-based tool for automatic layout and analysis of IRES secondary structure prediction and searching system in silico. BMC Bioinformatics 10: 160. ( ) 49. Hillman RT, Green RE, Brenner SE (2004) An unappreciated role for RNA surveillance. Genome biology 5: R8. g y 35. Iacono M, Mignone F, Pesole G (2005) uAUG and uORFs in human and rodent 5’untranslated mRNAs. Gene 349: 97–105. 50. Hinnebusch AG (1997) Translational regulation of yeast GCN4. A window on factors that control initiator-trna binding to the ribosome. J Biol Chem 272: 21661–21664. 36. Bailleul B, Akerblom I, Strosberg AD (1997) The leptin receptor promoter controls expression of a second distinct protein. Nucleic acids research 25: 2752– 2758. 51. Poyry TA, Kaminski A, Jackson RJ (2004) What determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame? Genes Dev 18: 62–75. 37. Prakash T, Sharma VK, Adati N, Ozawa R, Kumar N, et al. (2010) Expression of conjoined genes: another mechanism for gene regulation in eukaryotes. PloS one 5: e13284. 52. Hinnebusch AG (2006) eIF3: a versatile scaffold for translation initiation complexes. Trends Biochem Sci 31: 553–562. 38. Lee S, Liu B, Huang SX, Shen B, Qian SB (2012) Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution. Proceedings of the National Academy of Sciences of the United States of America 109: E2424– 2432. 53. Morris DR, Geballe AP (2000) Upstream open reading frames as regulators of mRNA translation. Mol Cell Biol 20: 8635–8642. 54. Miller PF, Hinnebusch AG (1989) Sequences that surround the stop codons of upstream open reading frames in GCN4 mRNA determine their distinct functions in translational control. Genes Dev 3: 1217–1225. 39. Green RE, Lewis BP, Hillman RT, Blanchette M, Lareau LF, et al. (2003) Widespread predicted nonsense-mediated mRNA decay of alternatively-spliced transcripts of human normal and disease genes. Bioinformatics 19 Suppl 1: i118– 121. 55. Kozak M (1991) Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem 266: 19867–19870. 56. References Gilbert WV (2010) Alternative ways to think about cellular internal ribosome entry. J Biol Chem 285: 29033–29038. 33. Pesole G, Liuni S, Grillo G, Licciulli F, Mignone F, et al. (2002) UTRdb and UTRsite: specialized databases of sequences and functional elements of 5’ and 3’ March 2014 | Volume 9 | Issue 3 | e91535 PLOS ONE | www.plosone.org 10 Polycistronic mRNAs Share NMD-Immune Architecture Polycistronic mRNAs Share NMD-Immune Architecture (2009) NCBI GEO: archive for high-throughput functional genomic data. Nucleic acids research 37: D885–890. 47. Chester A, Somasekaram A, Tzimina M, Jarmuz A, Gisbourne J, et al. (2003) The apolipoprotein B mRNA editing complex performs a multifunctional cycle and suppresses nonsense-mediated decay. The EMBO journal 22: 3971–3982. March 2014 | Volume 9 | Issue 3 | e91535 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 11
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The large proper-time expansion of Yang-Mills plasma as a resurgent transseries
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Published for SISSA by Springer Received: November 6, 2018 Accepted: January 11, 2019 Published: February 13, 2019 Received: November 6, 2018 Accepted: January 11, 2019 Published: February 13, 2019 The large proper-time expansion of Yang-Mills plasma as a resurgent transseries Inˆes Aniceto,a,b Jakub Jankowski,c Ben Meiringd and Micha l Spali´nskie,f aInstitute of Physics, Jagiellonian University, ul. Lojasiewicza 11, 30-348 Krak´ow, Poland bMathematical Sciences, University of Southampton, Highfield, Southampton SO17 1BJ, U.K. cFaculty of Physics, University of Warsaw, ul. Pasteura 5, 02-093 Warsaw, Poland dRudolf Peierls Centre for Theoretical Physics, University of Oxford, Parks Rd, Oxford OX1 3PJ, U.K. ePhysics Department, University of Bia lystok, PL-15-245 Bia lystok, Poland fNational Center for Nuclear Research, PL-00-681 Warsaw, Poland E-mail: I.Aniceto@soton.ac.uk, Jakub.Jankowski@fuw.edu.pl, Ben.Meiring@physics.ox.ac.uk, M.Spalinski@uwb.edu.pl Abstract: We show that the late-time expansion of the energy density of N = 4 super- symmetric Yang-Mills plasma at infinite coupling undergoing Bjorken flow takes the form of a multi-parameter transseries. Using the AdS/CFT correspondence we find a gravity solution which supplements the well known large proper-time expansion by exponentially- suppressed sectors corresponding to quasinormal modes of the AdS black-brane. The full solution also requires the presence of further sectors which have a natural interpretation as couplings between these modes. The exponentially-suppressed sectors represent nonhydro- dynamic contributions to the energy density of the plasma. We use resurgence techniques on the resulting transseries to show that all the information encoded in the nonhydrody- namic sectors can be recovered from the original hydrodynamic gradient expansion. The large proper-time expansion of Yang-Mills plasma as a resurgent transseries The large proper-time expansion of Yang-Mills plasma as a resurgent transseries JHEP02(2019)073 Inˆes Aniceto,a,b Jakub Jankowski,c Ben Meiringd and Micha l Spali´nskie,f aInstitute of Physics, Jagiellonian University, ul. Lojasiewicza 11, 30-348 Krak´ow, Poland bMathematical Sciences, University of Southampton, Highfield, Southampton SO17 1BJ, U.K. cFaculty of Physics, University of Warsaw, ul. Pasteura 5, 02-093 Warsaw, Poland dRudolf Peierls Centre for Theoretical Physics, University of Oxford, Parks Rd, Oxford OX1 3PJ, U.K. ePhysics Department, University of Bia lystok, PL-15-245 Bia lystok, Poland fNational Center for Nuclear Research, PL-00-681 Warsaw, Poland E-mail: I.Aniceto@soton.ac.uk, Jakub.Jankowski@fuw.edu.pl, Ben.Meiring@physics.ox.ac.uk, M.Spalinski@uwb.edu.pl Inˆes Aniceto,a,b Jakub Jankowski,c Ben Meiringd and Micha l Spali´nsk aInstitute of Physics, Jagiellonian University, ul. Lojasiewicza 11, 30-348 Krak´ow, Poland bMathematical Sciences, University of Southampton, Highfield, Southampton SO17 1BJ, U.K. cFaculty of Physics, University of Warsaw, ul. Pasteura 5, 02-093 Warsaw, Poland dRudolf Peierls Centre for Theoretical Physics, University of Oxford, Parks Rd, Oxford OX1 3PJ, U.K. ePhysics Department, University of Bia lystok, PL-15-245 Bia lystok, Poland fNational Center for Nuclear Research, PL-00-681 Warsaw, Poland E-mail: I.Aniceto@soton.ac.uk, Jakub.Jankowski@fuw.edu.pl, Ben.Meiring@physics.ox.ac.uk, M.Spalinski@uwb.edu.pl Inˆes Aniceto,a,b Jakub Jankowski,c Ben Meiringd and Micha l Spali´nskie,f Abstract: We show that the late-time expansion of the energy density of N = 4 super- symmetric Yang-Mills plasma at infinite coupling undergoing Bjorken flow takes the form of a multi-parameter transseries. Using the AdS/CFT correspondence we find a gravity solution which supplements the well known large proper-time expansion by exponentially- suppressed sectors corresponding to quasinormal modes of the AdS black-brane. The full solution also requires the presence of further sectors which have a natural interpretation as couplings between these modes. The exponentially-suppressed sectors represent nonhydro- dynamic contributions to the energy density of the plasma. We use resurgence techniques on the resulting transseries to show that all the information encoded in the nonhydrody- namic sectors can be recovered from the original hydrodynamic gradient expansion. Keywords: AdS-CFT Correspondence, Quark-Gluon Plasma, Supersymmetric Gauge Theory ArXiv ePrint: 1810.07130 Open Access, c⃝The Authors. Article funded by SCOAP3. The large proper-time expansion of Yang-Mills plasma as a resurgent transseries https://doi.org/10.1007/JHEP02(2019)073 Contents 1 Introduction 1 2 The bulk gravity solution 5 2.1 Hydrodynamic and nonhydrodynamic sectors 7 2.2 Series solution and numerical implementation 8 2.3 Energy density of the dual theory 9 3 The resurgent transseries of the energy density 11 3.1 The singularities in the Borel plane 11 3.2 A Borel analysis of large-order relations 16 4 Resurgence of QNMs in the hydrodynamic expansion 19 4.1 Behaviour of the hydrodynamic series at the leading Borel singularity 19 4.2 Sub-leading exponentially suppressed behaviour of the hydrodynamic series 21 5 Nonhydrodynamic sectors and effects of QNM coupling 22 5.1 Resurgence of different fundamental sectors 22 5.2 Mixed sectors 24 6 Outlook 25 A Leading singularities in the Borel plane 27 B Large-order predictions from Borel plane residues 27 B.1 Behaviour of an asymptotic series at the first Borel singularity 28 B.2 Behaviour of an asymptotic series at its second Borel singularity 31 JHEP02(2019)073 1 Introduction The discovery of quark-gluon plasma (QGP) at RHIC and the ongoing studies of its prop- erties both there and at the LHC have lead to a burst of activity related to the theoretical description of this new state of matter. Tiny drops of QGP created in these heavy-ion collision experiments appear initially in highly non-equilibrium states. Nevertheless, after a short time this system reaches a state amenable to an effective description formulated in the language of hydrodynamics, which continues up until the medium hadronizes as the local effective temperature drops below the confinement scale. Recent activity aimed at understanding the equilibration of QGP has lead to significant progress concerning the foundational aspects of relativistic hydrodynamics (for reviews see e.g. refs. [1, 2]), such – 1 – as the regime of applicability of hydrodynamics [3–7], the role and meaning of the hy- drodynamic gradient expansion [8] and attractor behaviour far from equilibrium [9–11]. The difficulties of treating real-time evolution far from equilibrium in QCD have provided strong motivation to look for other systems where such an analysis can be more tractable. Since many of the fundamental questions concern relativistic hydrodynamics itself rather than its specific application to QCD, the study of related model systems has proved both fruitful and practical. An important framework where many of these ideas were developed is N = 4 su- persymmetric Yang-Mills theory (SYM), where the AdS/CFT correspondence provides an effective method of carrying out ab-initio calculations of highly non-equilibrium phenom- ena in a strongly-coupled quantum system by relating non-trivial observables at infinite coupling to solvable problems in classical Einstein gravity [12, 13]. While supersymmetric Yang-Mills theory is very different from QCD, these differences are less pronounced at finite temperature and some of the results obtained using AdS/CFT appear to give a qual- itatively useful picture even when interpreted in the context of QGP. Most importantly, since this approach has allowed for reliable calculations of equilibration, it has provided a priceless theoretical laboratory where the emergence of a hydrodynamic behaviour could be explored. JHEP02(2019)073 A kinematic situation of phenomenological interest is that of boost-invariant longitu- dinal expansion — Bjorken flow, which mimics the behaviour of matter during the early stages of an ultra-relativistic heavy ion collision [14]. 1 Introduction Here, under the assumption of con- formality, symmetry constraints impose that the expectation value of the 3+1 dimensional energy-momentum tensor can be expressed in terms of the energy density E as a function of a single parameter, the proper time τ elapsed since the collision event. This leads to an amazingly simple model, yet one which preserves much of the essential complexity of the original problem, making it possible to build a detailed picture of the transition from a highly non-equilibrium initial state to hydrodynamics. A key approximation method which makes analytic calculations possible on the gravity side of the duality is the large proper-time expansion [15]. The bulk geometry obtained in this way implies that the energy density of N = 4 SYM at late times takes the form E (u) ≡Φ0 (u) = u−2 +∞ X k=0 ε(0) k u−k , (1.1) (1.1) where u = τ 2/3, with τ the proper time. Following ref. [8], here and in the following we have suppressed a dimensionful parameter, effectively choosing to measure the proper-time in units set by the scale of the energy density. This expansion is directly related to the hydrodynamic gradient expansion of Bjorken flow, which is the expansion in powers of τE1/4. In fact, given the coefficients of the late proper-time expansion one can calculate those of the gradient expansion and vice-versa — in other words, these two expansions contain the same information. With this understanding we will refer to eq. (1.1) as the hydrodynamic expansion. (0) The dimensionless expansion coefficients ε(0) k are calculable from the gravity solution. The leading 240 coefficients were computed in ref. [8], and further 140 were obtained in – 2 – ref. [16]. The series appearing in eq. (1.1) is asymptotic, and it is natural to ask how to best interpret it. Already in ref. [8] it was observed that the analytic continuation of the Borel transform of this series has branch-point singularities at locations corresponding to frequencies of the least-damped nonhydrodynamic quasinormal modes of the AdS black-brane. This connection was subsequently studied at the level of hydrodynamics, where series expansions such as eq. (1.1) can be generated as asymptotic solutions to MIS-type differential equations such as those introduced in refs. [17, 18]. 1 Introduction These studies [9, 19] lead to the realisation that the proper setting for the hydrodynamic gradient expansion is in fact a transseries [20, 21], which systematically incorporates the contributions from nonhydrodynamic modes. These contributions take the form of terms which are exponentially suppressed at large times, but become significant at earlier times, where they encode the initial state data.1 JHEP02(2019)073 In this paper we present the first calculations which directly reveal the transseries structure at the level of the microscopic theory. This is done by finding a generalisation of the late proper-time expansion guided by the form of asymptotic solutions of the hydrody- namic model studied in ref. [19]. However, in contrast to this hydrodynamic model which features just two conjugate nonhydrodynamic modes, in the case of N = 4 SYM there is an infinite number of nonhydrodynamic modes, naturally ordered by the rate of exponen- tial suppression following from the complex values of the black-brane quasinormal mode (QNM) spectrum. Each such mode introduces a separate sector — a separate asymptotic series weighted by an independent exponential involving the QNM frequencies. The bulk solution will be described in detail in section 2. Here we will present the ensuing form of the energy density of N = 4 SYM. In order to describe this structure explicitly let us introduce some notation which we will employ throughout this paper. With each pair of nonhydrodynamic QNMs (charac- terised by a pair of complex frequencies Ak, Ak with k > 0) we associate a pair of unit vectors ek, ek, whose components vanish apart from a one in slots 2k −1, 2k respectively. These unit vectors form a basis of an infinite-dimensional semi-lattice space N∞ 0 . Each QNM is labelled by a vector n which is equal to one of these basis vectors and introduces an asymptotic expansion of the form, Φn (u) = u−βn +∞ X m=0 ε(n) m u−m , (1.2) (1.2) with some characteristic exponent βn, and expansion coefficients ε(n) m , with the under- standing that the leading coefficient ε(n) 0 ̸= 0. One can view this series as a hydrodynamic “dressing” of the individual QNMs. The contribution of each pair of QNMs comes with a pair of complex normalisation constants, the transseries parameters, which will be denoted by σk, σ¯k. These numbers can be interpreted as integration constants — they contain information about the initial state. 1It is worth noting that different asymptotic analysis of QNM frequencies of black holes can be found in the context of stability of black holes and their infinitely damped QNMs, see e.g. [22–24]. 1 Introduction We find that the energy density at large proper time τ (or, equivalently, for large u) has – 3 – an expansion as a transseries of the form: an expansion as a transseries of the form: E (u, σ) = X n∈N∞ 0 σn e−n·A u Φn (u) . (1.3) E (u, σ) = X n∈N∞ 0 σn e−n·A u Φn (u) . (1.3) (1.3) where n n where where where σn ≡σn1 1 σn1 1 σn2 2 σn2 2 · · · (1.4) σn ≡σn1 1 σn1 1 σn2 2 σn2 2 · · · (1. (1.4) The exponential weights appearing in eq. (1.3) are expressed in terms of the vector of QNM frequencies2 JHEP02(2019)073 A = A1, A1, A2, A2, · · ·  , (1.5) (1.5) n = (n1, n1, n2, n2, · · · ) (1.6) (1.6) with non-negative integer entries. Note that the vector of QNM frequencies in eq. (1.5) determines the form of eq. (1.4), which encodes the initial data of the boundary theory through the values of the transseries parameters. with non-negative integer entries. Note that the vector of QNM frequencies in eq. (1.5) determines the form of eq. (1.4), which encodes the initial data of the boundary theory through the values of the transseries parameters. The collections of contributions to eq. (1.3) with n equal to one of the basis vectors ek, ek will be referred to as fundamental sectors — each such sector corresponds to a specific QNM. Note however that the sum appearing in eq. (1.3) also includes terms with vectors n corresponding to linear combinations of the basis vectors with non-negative coefficients. These contributions will be referred to as mixed sectors. They can be thought of as a reflection of QNM coupling. We will be referring to all of these as nonhydrodynamic sectors. The contribution to eq. (1.3) corresponding to n = 0 (with β0 = 2) is the original hydrodynamic expansion seen in eq. (1.1). This type of (resurgent) transseries structure appears often in studies of asymptotic expansions, and the expansion coefficients appearing in different sectors are known to be related by intricate consistency conditions, the so-called large-order relations, thoroughly described by ´Ecalle’s theory of resurgence [25] (see also e.g. the review [21] and references therein). These remarkable relations in principle allow us to extract the full content of the nonhydrodynamic sectors from the original hydrodynamic series. 2The Ak, Ak and the QNM frequencies are simply related by a proportionality constant (see section 4). 1 Introduction In practice, one can use them as a consistency check of the nonhydrodynamic sectors directly determined from the bulk solution. Such relations provide conclusive evidence that the transseries solution, supplemented by the relevant initial conditions, captured the full non-perturbative behaviour of the fluid. These large-order relations have successfully been used to predict novel non-perturbative physics [26–32] as well as in consistency checks of perturbative and non-perturbative sectors appearing in transseries solutions [19, 33–43]. Our findings are reminiscent of what is known about such series in the context of coupling-constant perturbative expansions in quantum mechanics, where these non- perturbative, exponentially suppressed sectors correspond to specific instanton solutions and are usually referred to as instanton sectors. It is well known that one needs more than 2The Ak, Ak and the QNM frequencies are simply related by a proportionality constant (see section 4). – 4 – just the instanton sectors as non-perturbative corrections to perturbative calculations of en- ergy levels in quantum-mechanical models — one also needs multi-instanton sectors, which correspond to nonlinear effects arising from the quantisation condition (see e.g. refs. [44, 45] and refs. within [21]). In the case at hand, the analogue of multi-instanton sectors are the mixed nonhydrodynamic sectors described above. Their content corresponds to going be- yond the linearised approximation which gives rise to the QNM spectrum. From the point of view of resurgence, these additional sectors are unavoidable and are constrained by the aforementioned large-order relations. Verifying that they are satisfied gives us a very high level of confidence in the consistency of the transseries ansatz in the present context. In section 2 we construct a transseries solution for the gravitational dual to the Bjorken expansion of N = 4 SYM plasma by supplementing the large proper-time expansion with exponentially suppressed contributions, and in subsection 2.3 we extract the coefficients of the dual gauge theory for several transseries sectors of eq. (1.3) to high orders. The asymp- totic expansions of these sectors show similarities with those arising in the context of the hydrodynamic model of ref. [18], whose asymptotic behaviour was investigated in ref. [19]. In particular, the large-order behaviour of the coefficients in these series involve various factorial and exponential growths with complex parameters (related to the exponential weights and characteristic exponents), which make it impossible to apply standard tools used in many recent studies of asymptotic series. 1 Introduction The study of the large-order behaviour of these coefficients can be found in section 3, where we introduce a novel method for reli- ably extracting resurgent information from an asymptotic series whose leading large-order relations depend on multiple complex factorial growths. The numerical checks showing the necessity of the full transseries solution eq. (1.3) to describe the energy density of the N = 4 SYM plasma can be then found in sections 4 (for evidence of inclusion of fundamental sectors) and 5 (for the inclusion of mixed sectors). JHEP02(2019)073 This work offers a full non-perturbative picture of the late time expansion of the N = 4 SYM plasma’s energy density, paving the way to use these new techniques in more general situations. In section 6 we summarise our main results and discuss future applications of our work. 2 The bulk gravity solution The late proper-time expansion for Bjorken flow in N = 4 SYM was studied for the first time analytically in [15] (see also refs. [46–48]) and calculated numerically to high orders in refs. [8, 16]. We adopt the Eddington-Finkelstein (EF) coordinate system which implements the symmetries of Bjorken flow with the following ansatz [3, 49–51] ds2 = −H(r, τ)dτ 2 + 2drdτ + S(r, τ)2  e−2B(r,τ)dy2 + eB(r,τ)dx2 ⊥  . (2.1) (2.1) Here r is the holographic radial co-ordinate with 0 ≤r ≤∞and (τ, y, x1, x2) reduce to proper time, rapidity and transverse spatial dimensions on the r →∞conformal boundary. Here r is the holographic radial co-ordinate with 0 ≤r ≤∞and (τ, y, x1, x2) reduce to proper time, rapidity and transverse spatial dimensions on the r →∞conformal boundary. – 5 – Einstein’s equations can be written in the compact form [51],3 Einstein’s equations can be written in the compact form [51],3 S′′ = −1 2S B′2 , (2.2) S ˙S′ = 2S2 −2 ˙SS′ , (2.3) S ˙B′ = −3 2  ˙SB′ + ˙BS′ , (2.4) H′′ = −3 ˙BB′ −4 + 12 ˙SS′ S2 , (2.5) ¨S = 1 2  ˙SH′ −˙B2S  , (2.6) JHEP02(2019)073 where f′ = ∂rf and ˙f = ∂τ + 1 2H(r, τ)∂r  f. Through the re-definitions where f′ = ∂rf and ˙f = ∂τ + 1 2H(r, τ)∂r  f. Through the re-definitions where f′ = ∂rf and ˙f = ∂τ + 1 2H(r, τ)∂r  f. Through the re-definitions H(r, τ) = r2h(r, τ) (2.7) B(r, τ) = 1 3  log  r2 (rτ + 1)2  + d(r, τ) −1 2b(r, τ)  (2.8) S(r, τ) = r2/3(1 + rτ)1/3 exp 1 3d(r, τ)  (2.9) H(r, τ) = r2h(r, τ) (2.7) B(r, τ) = 1 3  log  r2 (rτ + 1)2  + d(r, τ) −1 2b(r, τ)  (2.8) S(r, τ) = r2/3(1 + rτ)1/3 exp 1 3d(r, τ)  (2.9) (2.7) (2.9) we can express eq. (2.1) in a form convenient for the large-τ expansion [50] ds2 = −r2hdτ 2 + 2dτdr + (rτ + 1)2ebdy2 + r2e−1 2 b+ddx2 ⊥. (2.10) (2.10) For eq. 3Einstein’s equations with cosmological constant Λ = −6 are given by Rµν + 4gµν = 0. 2 The bulk gravity solution (2.10) to reduce to a flat, boost-invariant solution at the boundary we enforce the conditions to reduce to a flat, boost-invariant solution at the boundary we enforce the (2.11) lim r→∞h(r, τ) = 1 , lim r→∞b(r, τ) = 0 , lim r→∞d(r, τ) = 0 . (2.11) For ideal Bjorken flow, the local effective temperature of the plasma at the 4-dimensional Minkowski space boundary should behave as T ∼τ −1 3 at late times. We therefore fix s = 1 rτ −1 3 so that in the late time limit the naive location of the horizon in the s co-ordinate will remain finite. Further, we note that gradient corrections should go like 1 τT ∼τ −2 3 , so we fix u = τ 2 3 and expand the metric functions in inverse powers of u. Note also that 0 < s < 1, u > 0, and that late times correspond to large u. Having in mind the hydrodynamic expansion as well as presence of the transient modes we consider a transseries ansatz for the metric functions of the form h(r, τ) = X n∈N∞ 0 Ωn(u) ∞ X i=0 u−ih(n) i (s) , (2.12) b(r, τ) = X n∈N∞ 0 Ωn(u) ∞ X i=0 u−ib(n) i (s) , (2.13) d(r, τ) = X n∈N∞ 0 Ωn(u) ∞ X i=0 u−id(n) i (s) , (2.14) (2.12) (2.13) (2.14) 3Einstein’s equations with cosmological constant Λ = −6 are given by Rµν + 4gµν = 0. 3Einstein’s equations with cosmological constant Λ = −6 are given by Rµν + 4gµν = 0. – 6 – where Ωn(u) ≡u−n·α e−n·A u . (2.15) Ωn(u) ≡u−n·α e−n·A u . Ωn(u) ≡u−n·α e−n·A u . (2.15) Here (as already described in the introduction) n = (n1, n1, n2, n2, · · · ) ∈N∞ 0 is an in- finite dimensional vector of non-negative, integer components which will be used to de- fine the space of solutions. We will use the field equations to determine the quantities A = A1, A1, A2, A2, · · ·  and α which correspond to particular nonhydrodynamic sec- tors.4 We will denote the hydrodynamic sector by 0 and define special unit vectors ek which have all zero entries except the 2k −1 entry which will be set equal to 1. Similarly, ek is defined to have only the 2k entry non-zero (and equal to 1). 2 The bulk gravity solution These are defined so that the scalar products with A select the corresponding elements, in the sense that ek·A = Ak, and ek · A = Ak. JHEP02(2019)073 After substituting our ansatz into eqs. (2.2) to (2.6), the linear independence of Ωn(u) will imply an infinite hierarchy of equations. These equations can again be expanded in inverse powers of u to find linear ODEs for the functions h(n) i , b(n) i and d(n) i . We use residual gauge invariance to set the radial co-ordinate r to keep the horizon fixed at s = 1 at every order. This will amount to choosing h(n) i (s = 1) = 0 for all i. By imposing regularity in the bulk and flatness at the boundary for the metric functions we are able to solve for functions f(n) i at each order. 4Note that we will assume that each component of A is not a rational multiple of another component. 5In the discussion that follows one can exchange ek for ek where appropriate. 5In the discussion that follows one can exchange ek for ek where appropriate. 2.1 Hydrodynamic and nonhydrodynamic sectors The case of n = 0 will be referred to as the hydrodynamic sector. Any power of Ωn̸=0(u) that enters into the equations of motion will remain linearly independent from terms pro- portional to Ω0(u). Therefore h(0) i , b(0) i and d(0) i can be found independent of the solutions to any other sector. The zero-th order solution (in our chosen gauge) that preserves flatness and bulk regularity will be given by a boosted black-brane written as [52], d(0) 0 (s) = 0 , h(0) 0 (s) = 1 −s4 , b(0) 0 (s) = 0 . (2.16) (2.16) The cases of n = ek and n = ek will be called the fundamental nonhydrodynamic sectors.5 Equations linear in Ωek(u) can depend on the hydrodynamic sector and solutions within its own nonhydrodynamic sector. The zero-th order solution in each case is given by, The cases of n = ek and n = ek will be called the fundamental nonhydrodynamic sectors.5 Equations linear in Ωek(u) can depend on the hydrodynamic sector and solutions within its own nonhydrodynamic sector. The zero-th order solution in each case is given by, d(ek) 0 (s) = 0 , h(ek) 0 (s) = 0 , b(ek) 0 (s) = Zek(s) , (2.17) (2.17) where Zek(s) satisfies where Zek(s) satisfies s(1 −s4)∂2 s −(3 + s4 −2 i ek · ω s)∂s −3 i ek · ω  Zek(s) = 0 , (2.18) (2.18) and where we have defined ω = −2 i 3 A to put eq. (2.18) into the standard form of the Quasinormal Mode (QNM) equation given in infalling EF co-ordinates [53, 54]. Imposing flatness and regularity in Zek(s) turns eq. (2.18) into an eigenvalue problem with an infinite number of solutions ω. We take the eigenvalues ω with negative real part to correspond – 7 – to the unit vector ek, and those with positive real part to correspond to ek, with index k ordering them naturally by the negative imaginary part of each eigenvalue. The infinite dimensional vectors ek and ek can now be understood as spanning the space of QNM frequencies. Each eigenfunction Zek(s) is determined up to an arbitrary integration constant asso- ciated with the choice of Zek(s = 1). This overall normalisation we will later identify as the transseries parameter. 2.1 Hydrodynamic and nonhydrodynamic sectors This freedom comes from the fact that (along with imposing flatness at the boundary) we only require Zek(s) to be regular in the bulk, allowing a family of solutions which satisfy this condition. We can interpret each associated sector as an in- dependent nonhydrodynamic excitation. The vector α is fixed through a similar eigenvalue problem at order i = 1. Our numerically computed values of α and A satisfy α = A 6 with high precision.6 The cases of n ̸= ek and n ̸= ek will be called the mixed sectors. Once we have fixed ω in eq. (2.18), if we replaced ek by a general vector n, then this equation would have no non-trivial solutions obeying the chosen boundary conditions for n ̸= ek. All func- tions h(n) i , b(n) i and d(n) i for n ̸= ek, ek and i ≥0 will be fully determined by solutions in the hydrodynamic and the fundamental nonhydrodynamic sectors, and so will contain no free integration constants. In this way these sectors can be viewed as a cascade of interactions of the fundamental nonhydrodynamic modes rather than independent solutions. JHEP02(2019)073 2.2 Series solution and numerical implementation (2.6) can be written as a constraint at s = 1, In our chosen gauge, eq. (2.6) can be written as a constraint at s = 1, d(n) i (1) = J(n) i , (2.26) (2.26) with J(n) i a number determined by lower order solutions. Eq. (2.5) is redundant, being implied by the other four equations. The implementation of the numerical methods used in this paper are a continuation of the techniques used in ref. [8]. To integrate equations (2.20) to (2.22) we use Chebyshev spectral methods [55] with 400 grid points over the s ∈[0, 1] radial co-ordinate with a minimum of 240 digits of numerical precision.7 JHEP02(2019)073 As mentioned before, in our gauge we have chosen the warp factor h(r, t) to vanish at the naive location of the horizon (s = 1) which results in the boundary condition that h(n) i (s = 1) = 0. Imposing the constraint given by eq. (2.26), and standard flatness and regularity conditions (that all functions d(n) i , h(n) i and b(n) i vanish at s = 0 and are regular at s = 1) fully determines the system for i ≥1 for the hydrodynamic sector, i ≥2 for the fundamental nonhydrodynamic sectors, and i ≥0 for the mixed sectors. A non-obvious implementation is that of i = 1 for the fundamental nonhydrodynamic sectors, where the parameter α must be tuned so as to allow a solution for b(ek) 1 which vanishes at s = 0. For α = A 6 this condition is satisfied for any finite value of b(ek) 1 (s = 1) at the horizon. For i ≥1, the value of b(ek) i at the horizon (s = 1) must be chosen so as to fix b(ek) i+1 (s = 0) = 0. This was employed in our code via a shooting method. 2.2 Series solution and numerical implementation Subsequent equations of motion for i ≥1 where n = 0, ek or ek, (and for i ≥0 where n ̸= ek and n ̸= ek) can be found by directly substituting eqs. (2.12) to (2.14) into eqs. (2.2) to (2.4). They can be written respectively as Ld nd(n) i = jd,n i , (2.20) Lh nh(n) i = jh,n i , (2.21) Lb nb(n) i = jb,n i , (2.22) (2.20) (2.21) (2.22) where jd,n i , jh,n i and jb,n i are source terms, which are sequentially given in terms of solutions at lower orders in i, and solutions at the same order which are determined when solving the equations in the order (2.20) to (2.22). The linear operators above have relatively simple forms, where jd,n i , jh,n i and jb,n i are source terms, which are sequentially given in terms of solutions at lower orders in i, and solutions at the same order which are determined when solving the equations in the order (2.20) to (2.22). The linear operators above have relatively simple forms, Ld n = ∂2 s , (2.23) Lh n = s∂s −4 , (2.24) Lb n = s(1 −s4)∂2 s −(3 + s4 −2i n · ω s)∂s −3i n · ω . (2.25) (2.23) (2.23) (2.24) (2.25) Lh n = s∂s −4 , Lb n = s(1 −s4)∂2 s −(3 + s4 −2i n · ω s)∂s −3i n · ω . (2.25) (2.25) 6A heuristic argument for this value is suggested by refs. [8, 52]. At late times Ωek(u) is expected take the form of a decaying mode with frequency ωk in a slowly evolving plasma of effective temperature T ∼E1/4 given by, 6A heuristic argument for this value is suggested by refs. [8, 52]. At late times Ωek(u) is expected take the form of a decaying mode with frequency ωk in a slowly evolving plasma of effective temperature T ∼E1/4 given by, Ωek(u) ∼exp  i πωk Z dτ T  = exp  i 3 2ωk  u −1 6 log u + O(u−1)  . (2.19) (2.19) Equating Ak = 3 i 2 ωk for every k, we can identify α = A 6 . Equating Ak = 3 i 2 ωk for every k, we can identify α = A 6 . – 8 – In our chosen gauge, eq. 2.3 Energy density of the dual theory It was shown in [15] that imposing the symmetries of conformal Bjorken flow and the conservation of energy and momentum leads to the following form of the energy-momentum tensor: Tµν = diag E, τ 2pL, pT , pT  µν , (2.27) = 3N2 c 8π2 diag  f(τ), −τ 3f′(τ) −τ 2f(τ), f(τ) + 1 2τf′(τ), f(τ) + 1 2τf′(τ)  µν . (2.27) The particular energy-momentum tensor expectation value in the state of the boundary theory which is dual to our bulk geometry can be evaluated via holographic renormal- ization [56], which determines the function f(τ) appearing in eq. (2.27) in terms of the expansion The particular energy-momentum tensor expectation value in the state of the boundary theory which is dual to our bulk geometry can be evaluated via holographic renormal- ization [56], which determines the function f(τ) appearing in eq. (2.27) in terms of the expansion H(r, τ) = r2  1 −f(τ) r4 + . . .  . (2.28) (2.28) Re-expressing our solution in terms of u and s variables we can write, Re-expressing our solution in terms of u and s variables we can write, f(τ) = X n∈N∞ 0 σn Ωn(u) u−2 X i=0 f(n) i u−i ! (2.29) (2.29) 7In each case the computation could be done on a typical laptop to high orders within several hours. – 9 – where the coefficients f(n) i can be read from our gravity solution as f(n) i = −1 4! d4 ds4 h(n) i (s = 0) . (2.30) (2.30) The factor of σn (see eq. (1.4)) is introduced into eq. (2.29) so that the dependence on the choice of initial condition is explicit in the energy density. For generic n, some number of the first coefficients f(n) i in the sum (2.30) will be zero. For convenience we define ε(n) 0 to be (up to normalisation) the first non-zero coefficient of f(n) i (with ε(n) i , i > 0 given by the subsequent coefficients), and absorb the shift of the index into the definition of the characteristic exponent, which will be given by βn. One then arrives at the final formula for the energy density in the form of a transseries, given in eq. 2.3 Energy density of the dual theory (1.3), which we repeat here for convenience, JHEP02(2019)073 E (u, σ) = X n∈N∞ 0 σn e−n·A u Φn (u) , (2.31) (2.31) with with Φn (u) = u−βn +∞ X k=0 ε(n) k u−k . (2.32) (2.32) These coefficients ε(n) k have been included with this submission for the sectors Φn with n = 0, e1, e2, 2e1, (e1 + e1), along with their corresponding exponential weights Ai and characteristic exponents βn. For the hydrodynamic sector Φ0 coefficients for the hydrody- namic expansion were taken from [16]. The normalisations for the hydrodynamic series Φ0 and each of the fundamental sectors Φek, Φek ( associated to each kth QNM frequency) are not fixed, and have been chosen such that ε(0) 0 = π−4, ε(ek) 0 = 1, ε(ek) 0 = 1, k ∈N . (2.33) (2.33) With this choice of normalisation, the mixed sectors have no freedom in their respective coefficients. The list of sectors we have included are as follows: • The first 250 coefficients of the fundamental sector Φe1, and βe1 = −A1 6 + 3 with A1 = i 3 2 ω1, where ω1 ≈3.1195 −i 2.7467 is the lowest nonhydro QNM frequency. • The first 250 coefficients of the fundamental sector Φe1, and βe1 = −A1 6 + 3 wi • The first 200 coefficients of the fundamental sector Φe2, and βe2 = −A2 6 +3 with A2 = i 3 2 ω2 where ω2 ≈5.1695 −i 4.7636 is the second lowest nonhydro QNM frequency. • The first 200 coefficients of the fundamental sector Φe2, and βe2 = −A2 6 +3 with A2 = i 3 2 ω2 where ω2 ≈5.1695 −i 4.7636 is the second lowest nonhydro QNM frequency. • The first 100 coefficients of the mixed sector Φ2e1, with β2e1 = −2 A1 6 + 4 = 2βe1 −2. • The first 100 coefficients of the mixed sector Φ2e1, with β2e1 = −2 A1 6 + 4 = 2βe1 −2. • The first 100 coefficients of the mixed sector Φe1+e1, with βe1+e1 = −A1 6 −A1 6 + 4 = βe1 + βe1 −2. • The first 100 coefficients of the mixed sector Φe1+e1, with βe1+e1 = −A1 6 −A1 6 + 4 = βe1 + βe1 −2. Note that the fundamental sectors Φe1, Φe2 are just complex conjugate of the sectors Φe1 and Φe2, respectively. 8Curiously, the characteristic exponent appears to follow the pattern βn = −n·A 6 + n · 1 + β0, with 1 = (1, 1, 1, · · · ). 2.3 Energy density of the dual theory Furthermore, direct calculation shows that Φ2e1 is complex conjugate of Φ2e1. Their respective characteristic exponents βn are also related by complex conjugation (as are their exponential weights).8 – 10 – 3 The resurgent transseries of the energy density The coefficients appearing in the different sectors of the transseries for the energy den- sity (1.3) calculated in the previous section can be seen to grow factorially at large-order. Thus, each sector is formally defined as an asymptotic series. It is well known that one can remove the factorial growth by performing a Borel transform in each of the asymptotic sectors Φn, which is the first step to the summation of these series. To actually perform such summation one needs to know the singularity structure of the analytic continuation of the Borel transform. In the case at hand this large-order behaviour has an oscillatory component which prevents the application of standard techniques used to extract informa- tion from it. Some methods of approaching this problem were presented in ref. [19], but we have developed a new, powerful approach explained in detail in appendix B, which we will apply below. JHEP02(2019)073 9This analytic continuation is performed by means of diagonal Pad´e approximants. 3.1 The singularities in the Borel plane In general terms, the Borel transform corresponds to mapping a divergent series in the variable u ≫1 to a series in the Borel variable ξ via the map u−α 7→ξα−1 Γ(α) . The series Φn (see eq. (1.2)) is mapped to B [Φn] (ξ) = ξβn−1 +∞ X k=0 ε(n) k Γ(k + βn) ξk . (3.1) (3.1) This series has a finite radius of convergence and we can analyse the singularity structure of its analytic continuation,9 which is necessary if we eventually wish to carry out the Borel summation procedure. Figure 1 shows the poles of the analytic continuation of the Borel transform for the leading asymptotic sectors of our transseries: the hydrodynamic sector Φ0 (top plot), the fundamental sectors Φe1, Φe2 (middle plots) and the mixed sectors Φ2e1 and Φe1+e1 (bottom plots). The condensation of poles is taken to be indicative of a branch point singularity. We can directly check there that the positions of the inferred branch points are determined by the different exponential weights appearing in our transseries solution (1.3). We can see the appearance of the different fundamental sectors (shown in the figure as filled circles) as well as mixed sectors (shown as filled purple diamonds). However, not all the mixed sectors appearing in our transseries (1.3) will contribute to the singularity structure of a given asymptotic sector. Applying resurgence techniques to our transseries, one can predict all the branch point singularities for the Borel transform of each sector. The procedure is thoroughly explained in section 5 of [21] (for multi- parameter transseries). The full list of the branch point singularities for each sector can be found in appendix A, and these were accordingly marked in figure 1 in the form of circles (fundamental) or diamonds (mixed). Analysing the figure further, we note that there are several predicted branch points without any singular behaviour (grey dots). 3.1 The singularities in the Borel plane This is due to the fact that we are only taking a finite and limited number of terms from the original – 11 – ● ● ● ● ● ● ◆ ◆ ◆ ◆ ◆ ◆ × × × × × × × ×     - - -     ● ● ● ● ● ● ◆ ◆ ◆ ◆ ◆ ◆ × × × × × ×     - - -     ● ● ● ● ● ● ◆ ◆ ◆ × × × × × × × × ×     - - -     ● ● ● ● ● ● ● ◆ ◆ ◆ ◆ ◆ ◆ × × × × ×     - - -     ● ● ● ● ● ● ● ◆ ◆ ◆ ◆ ◆ ◆ ◆ ◆ × × ×     - - -     Figure 1. The singularity structure (grey dots) in the complex ξ-plane (Borel plane) associated to the Borel transforms of sectors Φn, with n = 0, e1, e2, 2e1, e1 + e1. This singularity structure is obtained via the poles of the respective Borel-Pad´e approximant (of order N) BPN [Φn]. The Borel- Pad´e approximants used were: BP189 [Φ0], BP135 [Φe1], BP129 [Φe2], BP47 [Φ2e1] and BP47 [Φe1+e1]. The Borel planes have been shifted to be represented on a common axis, with the expansion points shown by a hollow circle and the hydrodynamic expansion located at the origin (in dark yellow). The predicted branch points for each sector (listed in appendix A) have been superimposed on each Borel plane and are divided into fundamental (solid circles) and mixed sectors (purple diamonds). The fundamental sectors include: A1 and A1 (blue), A2 and A2 (red), A3 and A3 (green). Any branch points associated to sectors of the transseries which do not give origin to branch points are shown as orange crosses. 3.1 The singularities in the Borel plane ● ● ● ● ● ● ◆ ◆ ◆ × × × × × × × × ×     - - -     ● ● ● ● ● ● ● ◆ ◆ ◆ ◆ ◆ ◆ ◆ ◆ × × ×     - - -     ● ● ● ● ● ● ◆ ◆ ◆ ◆ ◆ ◆ × × × × × ×     - - -     JHEP02(2019)073 ● ● ● ● ● ● ● ◆ ◆ ◆ ◆ ◆ ◆ × × × × ×     - - -     Figure 1. The singularity structure (grey dots) in the complex ξ-plane (Borel plane) associated to the Borel transforms of sectors Φn, with n = 0, e1, e2, 2e1, e1 + e1. This singularity structure is obtained via the poles of the respective Borel-Pad´e approximant (of order N) BPN [Φn]. The Borel- Pad´e approximants used were: BP189 [Φ0], BP135 [Φe1], BP129 [Φe2], BP47 [Φ2e1] and BP47 [Φe1+e1]. The Borel planes have been shifted to be represented on a common axis, with the expansion points shown by a hollow circle and the hydrodynamic expansion located at the origin (in dark yellow). The predicted branch points for each sector (listed in appendix A) have been superimposed on each Borel plane and are divided into fundamental (solid circles) and mixed sectors (purple diamonds). The fundamental sectors include: A1 and A1 (blue), A2 and A2 (red), A3 and A3 (green). Any branch points associated to sectors of the transseries which do not give origin to branch points are shown as orange crosses. series and approximating the analytic continuation of the Borel transform by means of Pad´e approximants. Depending on the number of terms available the effectiveness with which we can detect branch point singularities in the Borel plane diminishes with distance as one moves away from the origin (or the empty circle in the shifted plots of figure 1). – 12 – The Borel plane singularities shown in figure 1 have an unexpected feature, particularly evident in the middle left plot (the Borel plane singularities of sector Φe1): the hydrody- namic series has a cut associated to it, at the origin of the plot. 3.1 The singularities in the Borel plane This is very surprising: typically, in a resurgent transseries, the perturbative sector has no transseries parameter associated with it (such as the σi appearing in (1.3)). The appearance of the cut associated to the hydrodynamic series suggests otherwise. The resolution of this puzzle is very simple: the energy density as a function of proper time contains in fact a dimensionful parameter, which we have set to a convenient value (following [8]). This parameter is a vestige of the initial conditions and as such it leads to a branch-point singularity and a cut in the Borel plane. We have checked that there are no such cuts if instead of the energy density we consider the pressure anisotropy (defined by A = pT −pL ϵ/3 , see also eq. (2.27)) as a function of τE1/4 whose hydrodynamic gradient expansion is known to be universal at late times, and so does not involve any integration constants.10 JHEP02(2019)073 Each of the branch cuts appearing in the analytically continued Borel transforms con- tains all the information pertaining to the sector associated with it. Naturally, one can expect this information to be encoded in the asymptotic behaviour of the Borel transform of the hydrodynamic sector, B [Φ0], as shown on the top plot of figure 1. In other words, the large-order (factorially divergent) behaviour of the coefficients of this series will encode information about all of the fundamental sectors and mixed sectors. This can be most efficiently and systematically studied through the tools of resurgence, which when applied to our transseries solution (1.3) give rise to the so-called large-order relations, which ex- press precise relations between the coefficients of the different transseries sectors. These large-order relations allow us to very accurately check the consistency of the transseries ansatz (1.3), including the presence of mixed sectors reflecting the non-linear QNM cou- pling. A comprehensive explanation of how to determine these relations can be found in [21] (sections 2 and 5). 3.1 The singularities in the Borel plane Taking as an example the hydrodynamic series, the large-order behaviour of its coeffi- cients, implied by the assumption that our transseries is resurgent, is schematically given by: ε(0) k ≃ k≫1 −S0→e1 2πi Γ(k + β0 −βe1) A k+β0−βe1 1 χ0→e1 −S0→e1 2πi Γ(k + β0 −βe1) A1 k+β0−βe1 χ0→e1+ (3.2) −S0→e2 2πi Γ(k + β0 −βe2) A k+β0−βe2 2 χ0→e2 −S0→e2 2πi Γ(k + β0 −βe2) A2 k+β0−βe2 χ0→e2+ (3.3) −S0→2e1 2πi Γ(k + β0 −β2e1) (2A1)n+β0−β2e1 χ0→2e1 −S0→2e1 2πi Γ(k + β0 −β2e1) 2A1 k+β0−β2e1 χ0→2e1+ (3.4) (3.2) (3.2) (3.3) 10From the independence of the pressure anisotropy on initial conditions we can apply resurgence tech- niques to determine the exact behaviour of the branch cut appearing at the origin of the middle left plot of figure 1: the asymptotic series associated to it is related but not exactly equal to the hydrodynamic series. To do a full resurgent analysis of the transseries describing the energy density, including the cut at the origin, one would have to recover the dependence on this dimensionful parameter. For the purposes of this work we will keep this parameter fixed, thus obtaining the solution in eq. (1.3). – 13 – Each line above corresponds to different exponentially suppressed contributions (top line: leading; middle line: first exponentially suppressed; third line: second exponentially sup- pressed). The proportionality constants S0→n are the so-called Borel residues, which are well known functions of the Stokes constants associated with the problem (see [21] for the exact relations).11 Naturally, if a particular sector Φn does not appear as a singular branch point in the Borel plane of B [Φ0], then it won’t contribute to the large-order behaviour of ε(0) k , because the corresponding Borel residue vanishes, S0→n = 0. The expansions χ0→n(k), which depend solely on the coefficients of the sector Φn and the leading power of the hydrodynamic expansion β0, are asymptotic expansions in k ≫1 and are generally defined as JHEP02(2019)073 χm→n(k) ≃ +∞ X h=0 Γ(k + βm −βn −h) Γ(k + βm −βn) ((n −m) · A)h ε(n) h . (3.5) (3.5) Expanding the above expression at large-order we thus obtain an asymptotic series, e.g. χ0→e1(k) ≃ε(e1) 0 + A1 ε(e1) 1 k + (A1)2 ε(e1) 2 −A1 (β0 −βe1 −1) ε(e1) 1 k2 + · · · . 3.1 The singularities in the Borel plane (3.6) (3.6) Recalling that the coefficients of the hydrodynamic series are all real, from these large- order relations we can predict certain conjugation properties of the Borel residues. Con- sider, for instance, the leading contributions to the large-order behaviour (3.2): these come from the complex conjugate sectors Φe1 and Φe1, whose contributions are of the same order. To sum these contributions into a real result, we need to have S0→e1 = −S0→e1. A question now arises: is it possible to retrieve information concerning the sub-leading, exponentially suppressed contributions, such as (3.3) and (3.4)? To reach these exponen- tially smaller terms, we need to subtract the first line (i.e. eq. (3.3)) from the asymptotic behaviour of the original hydro series coefficients ε(0) k . However, as mentioned before, χ0→n(k) is itself an asymptotic series and we need to perform a resummation of it for each value of k needed to carry out the subtraction. We do so via the so-called ´Ecalle-Borel-Pad´e procedure, wherein we determine its Borel transform, analytically continue it via a Pad´e approximant BPN [χ0→n], and then perform the inverse transform12 to obtain the summed result S0+χ0→n(k): S0+χ0→n(k) ≡ Z eiϵ ∞ 0 dξ e−k ξ BPN [χ0→n] (ξ), (3.7) (3.7) 11For our purposes it is accurate enough to say that every Stokes constant is equal to (minus) the Borel residue of the hydro sector to a fundamental sector, given by Sei = −S0→ei and Sei = −S0→ei. For a more general definition given by alien calculus see [21]. 11For our purposes it is accurate enough to say that every Stokes constant is equal to (minus) the Borel residue of the hydro sector to a fundamental sector, given by Sei = −S0→ei and Sei = −S0→ei. For a more general definition given by alien calculus see [21]. 12The Borel transform is equivalent to applying an inverse Laplace transform to each term of a series. Thus the inverse transform will be a Laplace transform applied to the Borel-Pad´e approximant, with integration contour over the real axis. Given that this Pad´e approximant turns out to have poles along the real axis, to avoid them we need to shift the integration contour slightly above this axis: this is called a lateral resummation. 3.1 The singularities in the Borel plane – 14 – ● ● ● ● ● ● ◆ ◆ ◆ ◆ ◆ ◆ × × × × × × × ×     - - -     Figure 2. Poles of the Borel-Pad´e approximant BP90 [δ1Φ0]. Comparing the figure above to the top plot of figure 1 we can see that the singularity structure of the leading sector can be consistently removed from the coefficients of Φ0, and the information of the higher sectors can be recovered. The slight asymmetry in the figure above is due to the particular choice of lateral resummation where we have integrated along the real line from above. ● ● ● ● ● ● ◆ ◆ ◆ ◆ ◆ ◆ × × × × × × × ×     - - -     JHEP02(2019)073 Figure 2. Poles of the Borel-Pad´e approximant BP90 [δ1Φ0]. Comparing the figure above to the top plot of figure 1 we can see that the singularity structure of the leading sector can be consistently removed from the coefficients of Φ0, and the information of the higher sectors can be recovered. The slight asymmetry in the figure above is due to the particular choice of lateral resummation where we have integrated along the real line from above. where we assumed ϵ > 0 small, and k ≥1 (we cannot use this procedure to determine the summation at k = 0). We can then define the subtracted coefficients as13 δ1ε(0) k ≡ε(0) k + S0→e1 2πi Γ(k + β0 −βe1) A k+β0−βe1 1 S0+χ0→e1 + S0→e1 2πi Γ(k + β0 −βe1) A1 k+β0−βe1 S0+χ0→e1 . (3 (3.8) ( ) The subtracted series defined by these coefficients δ1Φ0(u) ≡u−β0 P+∞ k=1 δ1ε(0) k u−k is again asymptotic, and the singularity structure in the respective Borel plane can be seen in figure 2. As before, in blue are shown the singular points associated to the actions A1, A1, while in red we can see the singular branch points associated to the actions A2, A2. Comparing to the top plot of figure 1, it is evident that indeed the singularities associated to s = A1, A1 have been subtracted, and now the leading singularities are at s = A2, A2. 13The numerical lateral summation S0+χ0→e1 used a diagonal Pad´e approximant of order N = 96. 14This can be traced to the fact that we had to choose a particular lateral resummation to subtract the leading results. Because this summation prescription is the same for complex conjugate sectors, we obtain non-trivial imaginary contributions. 3.1 The singularities in the Borel plane One can now numerically check that the large-order behaviour of the subtracted co- efficients δ1ε(0) k is just given by the second (leading) and third lines (sub-leading) (3.3) and (3.4). From explicitly examining the numerical coefficients, one can notice that Re δ1ε(0) k ∼ Γ(k) |A2|k while Im δ1ε(0) k ∼ Γ(k) |2A1|k , which means we only need the real part of this summation procedure to analyse the contributions from the fundamental sectors Φe2, Φe2, appearing in (3.3). Given that the sectors Φe2 and Φe2 are complex conjugates, with βe2 = βe2, then we again expect S0→e2 = −S0→e2. This conjugacy relation between Borel residues should extend to every fundamental sector: S0→ek = −S0→ek . (3.9) (3.9) The resurgent properties of our transseries also impose several constraints on Stokes constants and consequently on the Borel residues. Of particular interest to our analysis is the expected constraint S0→n ek = (−1)n+1 (S0→ek)n (see [21] for more details). Given the conjugacy relations just mentioned between Borel residues of conjugate fundamental – 15 – sectors, we obtain sectors, we obtain S0→2e1 = −(S0→e1)2 = −(S0→e1)2 = S0→2e1. (3.10) (3.10) From direct gravity calculations we know that Φ2e1 and Φ2e1 are complex conjugates of each other. We could then worry that the large-order contribution coming from the third line (3.4) is not real, as one could have expected. However, the resummations from the pre- vious lines (3.2) and (3.3) will give a non trivial imaginary contribution of the same order.14 The above discussion was framed in the context of the hydrodynamic sector, but an analogous large-order analysis can be done for any sector of the transseries. The upshot of this analysis is that despite the appearance of an infinite number of fundamental QNM sectors one can really analyse each contribution individually due to the hierarchy of expo- nential damping. We have thus verified that our original transseries ansatz is consistent at this level. In the following section we will develop the techniques necessary for a quanti- tative and systematic testing of the resurgence relations between coefficients appearing in different sectors. JHEP02(2019)073 3.2 A Borel analysis of large-order relations In many cases one can directly use the large-order relations to determine the coefficients of exponentially suppressed sectors from the values of the hydrodynamic gradient series using numerical acceleration methods such as Richardson transforms [19, 37]. However, in the case of interest here the oscillatory nature of our problem, due to the presence of complex conjugate sectors with complex characteristic exponents βn, complicates the task exceedingly. This type of issue has already been encountered in the analysis of large-order behaviour of the hydrodynamic model of [18] which was studied in ref. [19]. The equations considered there were designed specifically to model the leading QNMs of N = 4 SYM and they served as a testing ground for the analysis undertaken here. Due to the lack of suitable techniques ref. [19] tested the large-order relations using values of the expansion coefficients appearing in the various sectors. The methods used there started from the large-order relations such as eq. (3.2), and performed the resummation of the expansions χ0→e1, χ0→e1 (using eq. (3.7)). This way one obtains ε(0) k ≃−S0→e1 2πi Γ(k + β0 −βe1) A k+β0−βe1 1 S0+χ0→e1 −S0→e1 2πi Γ(k + β0 −βe1) A1 k+β0−βe1 S0+χ0→e1 + · · · , (3.11) ε(0) k ≃−S0→e1 2πi Γ(k + β0 −βe1) A k+β0−βe1 1 S0+χ0→e1 −S0→e1 2πi Γ(k + β0 −βe1) A1 k+β0−βe1 S0+χ0→e1 + · · · , (3.11) as the leading contributions to the large-order behaviour. In the above equation everything is known except the Borel residues S0→e1, S0→e1, which we know to be related by eq. (3.9) due to the reality condition which our solution satisfies. We can therefore solve for the modulus and argument of the Borel residue S0→e1. While the result formally depends on k, the large-order relations imply that it should saturate at large values of k, which can – 16 –         - - -                  Figure 3. The plots show the convergence of the modulus and argument of Stokes constant (or rather, the appropriate Borel residue). The quantities plotted are k-dependent, but the large-order relations imply that for large enough values of k they should saturate, approaching the values of the modulus and argument of the Stokes constant (for details see ref. [19]). 3.2 A Borel analysis of large-order relations We see that this happens already at moderate values of k. At k = 250 the variation is of order 10−54.         - - -                  JHEP02(2019)073 Figure 3. The plots show the convergence of the modulus and argument of Stokes constant (or rather, the appropriate Borel residue). The quantities plotted are k-dependent, but the large-order relations imply that for large enough values of k they should saturate, approaching the values of the modulus and argument of the Stokes constant (for details see ref. [19]). We see that this happens already at moderate values of k. At k = 250 the variation is of order 10−54. easily be verified, leading to an accurate estimate of the Borel residue. The result of such a procedure — implementing precisely the techniques of ref. [19] but now using the series calculated for N = 4 SYM leads to the results shown in figure 3. This provides further support for our approach. While the arguments presented so far provide strong evidence that we are dealing with a resurgent transseries, we would still like to extract values of the coefficients appearing in the exponentially suppressed transseries sectors from the hydrodynamic gradient expansion alone. The difficulties discussed in the previous paragraph preclude the use of standard techniques, but nevertheless, an equivalent analysis can be carried out at the level of the respective Borel transforms. This new method is presented in some detail in appendix B, but we briefly summarise it below for use in the following sections. ( ) We consider the hydrodynamic series, Φ0(u), whose coefficients ε(0) k have the asymp- totic large-order behaviour shown in eqs. (3.2)–(3.4). As we saw previously, this large-order behaviour is intimately linked to the existence of branch cut singularities on the Borel plane, and resurgence analysis explicitly reveals the analytic properties of this link. It is well known15 that if we multiply our asymptotic series Φ0(u) by an overall factor u−β for a specific value of β such that the Borel transform removes the exact leading factorial growth of its coefficients, then this Borel transform will present a logarithmic cut at its leading singularity. 15We are within the realm of simple resurgent functions of Gevrey-1 type, see e.g. [21, 57]. 3.2 A Borel analysis of large-order relations For the particular case of the hydrodynamic sector and the leading singularity at ξ = A1 (corresponding to the first large-order contribution of (3.2)) we can write B h uβe1 Φ0 i (ξ) ξ=A1 = S0→e1 B h uβe1 Φe1 i (ξ −A1) log(ξ −A1) 2π i + regular terms . (3.12) (3.12) This means that if we analyse each branch cut of the Borel plane separately, we can recover the coefficients of the sector associated to that branch cut (in this case Φe1). It is vital that we know the factorial growth corresponding to each of the branch points (as given in – 17 – the large-order relations) with as high an accuracy as possible, as this determines the exact type of branch cut one finds in the Borel plane. This is necessary to be able to transform it into the logarithmic cut shown in eq. (3.12) above. the large-order relations) with as high an accuracy as possible, as this determines the exact type of branch cut one finds in the Borel plane. This is necessary to be able to transform it into the logarithmic cut shown in eq. (3.12) above. From the knowledge of eq. (3.12), we can transform the logarithmic behaviour into a leading square root branch cut by further multiplying the original sector by u−1/2 B h uβe1−1/2 Φ0 i (ξ) ξ=A1 = S0→e1 2 B h uβe1−1/2 Φe1 i (ξ −A1) + regular terms (3.13) = S0→e1 2√ξ −A1 ε(e1) 0 Γ(1/2) + ε(e1) 1 (ξ −A1) Γ(3/2) + ε(e1) 2 (ξ −A1)2 Γ(5/2) + · · · ! . (3.13) JHEP02(2019)073 This result can be demonstrated through a simple extension of an analogous proof contained in section 4 of [21] (for the case of the quartic integral), see also appendix B. Various changes of variables have been used in the literature to more effectively analyse the Borel plane singularity structure, in particular through the use of conformal maps, see e.g. [58–60], as well as recent applications [61].16 To further simplify our results, we can now perform a change of variables effectively transforming the branch cut into a simple pole. To this end we define ξ = A1 −(ζ −A1)2. 16We would like to thank O. Costin for useful discussions on this subject, as well as on on-going work on applications of conformal maps as an efficient method of retrieving asymptotic information on the Borel plane [62]. 17In fact we have access to the combination S0→e1 ε(e1) 0 , but we normalise the fundamental sectors such that their first non-zero coefficient is 1. 3.2 A Borel analysis of large-order relations In terms of this new variable we can write the above equation as B h uβe1−1/2 Φ0 i (ζ) ζ=A1 = S0→e1 2i (ζ −A1) ε(e1) 0 Γ(1/2) −ε(e1) 1 (ζ −A1)2 Γ(3/2) + · · · ! . (3.14) (3.14) From this expression we can see that by calculating the Borel transform B  uβe1−1/2Φ0(u)  (ζ) and analysing its residue at ζ = A1 we have direct access to the Borel residue S0→e1.17 Moreover, by subtracting the simple pole from the Borel transform: B h uβe1−1/2 Φ0 i (ζ) ζ=A1 − S0→e1 2i (ζ −A1) ε(e1) 0 Γ(1/2) = −ε(e1) 1 S0→e1 2i Γ(3/2) (ζ −A1) + · · · , (3.15) (3.15) and multiplying everything by (ζ −A1)−2, we can once again use the residue theorem to predict the next coefficient ε(e1) 1 of the nonhydro sector Φe1. This procedure can be systematised to predict an arbitrary number of coefficients of the nonhydro sectors, as is shown in appendix B. In the following we will consider in some detail the singular behaviour of sectors Φ0, Φe1, and how this behaviour is governed by the other fundamental sectors, as well as mixed sectors. We will observe the remarkable accuracy of the predictions obtained via resurgence from the analysis of the Borel planes. In section 4 we look at leading contributions com- ing from fundamental sectors (associated directly with the AdS black-brane QNMs): we calculate the coefficients of fundamental sectors Φe1 and Φe2 from the Borel analysis of – 18 – the hydrodynamic sector Φ0 and compare them with the values obtained directly from the exponentially-suppressed contributions to the bulk gravity solution of section 2. In section 5 we will show that the mixed sectors, coming from non-linear couplings between QNMs, contribute non-trivially to the singular behaviour of the transseries sectors and cannot be ignored in a full description of the energy density. Our analysis will focus on the Borel analysis of sector Φe1 and the corresponding prediction of coefficients from sectors Φ2e1 and Φe1+e1. The results presented in sections 4 and 5 provide significant further evidence that the energy density of N = 4 SYM can at late times be described by the resurgent transseries solution given in eq. (1.3) and discussed in this section. JHEP02(2019)073 4 Resurgence of QNMs in the hydrodynamic expansion We will now use the techniques explained in the previous section to analyse the resurgence relations between different sectors in the transseries shown in eq. (1.3). We will focus on the hydrodynamic gradient expansion and the appearance of the QNMs in the large-order behaviour of its coefficients. The appearance of the these modes was already evident from the visual Borel analysis shown in the top plot of figure 1. By applying the methodology introduced in the last section (and thoroughly explained in appendix B) to the perturbative sector Φ0, we can predict the values of the coefficients of the sectors associated the leading (least damped) QNMs, associated to the transseries fundamental sectors Φe1 and Φe2. In figure 4 we can find the normalised error of the resurgence prediction and numerical gravity calculation of the first coefficients associated to these two sectors. This error is given by ∆nε(m) k ≡ ε(m) k |n−predicted −ε(m) k |numerical ε(m) k |numerical , k ≥1, (4.1) (4.1) where we defined ε(m) k |numerical as the coefficients associated to the sector Φm numerically determined from the gravity calculation of section 2, and ε(m) k |n−predicted being the same coefficients as predicted by the resurgence analysis of sector Φn, which is outlined by eqs. (3.14) and (3.15). The comparison of predicted and numerical coefficients for k ≥1 follows the calculation of the respective Borel residues S0→e1 and S0→e2, as detailed below. 18Branch points associated to more exponentially suppressed sectors are not shown due to the loss of accuracy of the Pad´e approximant. 19More explicitly, the sub-leading coefficients can be predicted from eq. (B.19). 4.1 Behaviour of the hydrodynamic series at the leading Borel singularity As mentioned earlier, the singularity structure of the Borel transform of the hydrodynamic (perturbative) series Φ0(u) can be found in the top plot of figure 1, where we have plotted the zeros of the denominator of the (diagonal) Borel-Pad´e approximant BPN [Φ0], of order N = 189. We clearly observe pole condensation indicative of branch point singularities at ξ = Ai, Ai with i = 1, 2, 3 shown in blue, red and green (filled) circles, respectively. These are the branch points associated to the first fundamental sectors Φei, Φei with i = 1, 2, 3. The purple diamonds correspond to mixed sectors, which will be the subject of section 5.18 – 19 –      - - - -   Figure 4. Comparison of the predicted results from resurgence techniques and the numerical ones obtained from solving Einstein equations. In the above plots we show this comparison for the coefficients of the fundamental sectors Φe1 and Φe2, as predicted by the large-order behaviour of the coefficients of the perturbative sector Φ0 (see eq. (4.1)).      - - - -   JHEP02(2019)073 Figure 4. Comparison of the predicted results from resurgence techniques and the numerical ones obtained from solving Einstein equations. In the above plots we show this comparison for the coefficients of the fundamental sectors Φe1 and Φe2, as predicted by the large-order behaviour of the coefficients of the perturbative sector Φ0 (see eq. (4.1)). The two leading nonhydrodynamic, fundamental sectors appearing in this plot are Φe1, Φe1, responsible for the singular points ξ = A1, A1. We can then perform the Borel plane analysis introduced in the previous section and thoroughly described in appendix B for any of these singular points. Consider for instance the sector associated to ξ = A1, given in eq. (1.2) with the characteristic exponent βe1 given on page 10. The leading growth of the coefficients of the hydrodynamic series ε(0) k associated to this sector is given by β = β0 −βe1, as shown in the first line of the large-order relations eq. (3.2). Then from a direct application of eq. (3.14) we can determine the Borel residue associated with this singular point: S0→e1 = −0.011131682120023118507124753501675427870232109210448+ + 0.030501348613000820066073498678990910567099376605738 i . (4.2) (4.2) The convergence plots of figure 3 show exactly this result. But we can now go further and analyse the sub-leading behaviour of the perturbative series at the singular point ξ = A1, as predicted by the procedure shown in eq. (3.15).19 The comparison between values predicted using resurgence and what was numerically calculated directly from the bulk gravity solution in sector Φe1 can be inspected on the left plot of figure 4 for the first 11 coefficients ε(e1) m . We can see that the error remains extremely small, thus confirming the resurgent properties of our transseries solution (1.3). The convergence plots of figure 3 show exactly this result. But we can now go further and analyse the sub-leading behaviour of the perturbative series at the singular point ξ = A1, as predicted by the procedure shown in eq. (3.15).19 The comparison between values predicted using resurgence and what was numerically calculated directly from the bulk gravity solution in sector Φe1 can be inspected on the left plot of figure 4 for the first 11 coefficients ε(e1) m . We can see that the error remains extremely small, thus confirming the resurgent properties of our transseries solution (1.3). As we have seen in the previous section, the Borel residue associated to S0→e1 corre- sponding with the singularity ξ = A1 is related to S0→e1 by eq. (3.9): S0→e1 = −S0→e1 . (4.3) (4.3) This was directly checked by repeating the above calculation for the singularity ξ = A1. – 20 – 20These coefficients are explicitly predicted by eq. (B.19). 4.2 Sub-leading exponentially suppressed behaviour of the hydrodynamic series We have now verified the resurgent behaviour of the perturbative series in the nonhydro- dynamic sector corresponding to the least damped QNM. We can ask if we can go further and analyse the next pair of complex-conjugate singularities of the hydrodynamic series at ξ = A2, A2, which correspond to the second QNM. The sectors associated with these singularities are Φe2 and Φe2, respectively, given in eq. (1.2) with the characteristic expo- nents given on page 10. As explained before, in order to analyse the resurgent behaviour at these singularities we need to first subtract the leading contributions coming from the first QNM. This can done by subtracting the resummed leading large-order behaviour (through a Borel-Pad´e-´Ecalle lateral summation), shown in (3.2), from the coefficients of the hydrodynamic series. We obtain the subtracted coefficients given in eq. (3.8), which now obey the large-order relation given in eqs. (3.3), (3.5): JHEP02(2019)073 δ1ε(0) k ≃−S0→e2 2πi Γ (k + β0 −βe2) A k+β0−βe2 2 χ0→e2(k) + c.c. . (4.4) (4.4) These large-order relations allow us to predict the coefficients ε(e2) k , through the same Borel analysis applied before to the leading singularities, but now applied to the series δ1Φ0(u) ≃u−β0 +∞ X k=1 δ1ε(0) k u−k = u−β0−1 +∞ X k=0 δ1ε(0) k+1 u−k. (4.5) (4.5) The singularity structure on the Borel plane for this asymptotic series was already shown figure 2, where we can clearly notice the absence of the leading singularities at ξ = A1, A Note that we have an extra shift of the characteristic exponent β0: this was because we cannot define a resummation procedure for k = 0 to determine δ1ε(0) 0 . Nevertheless, the asymptotic properties are encoded in the large-orders, and taking this shift into account, we can easily determine the Borel residue associated to the second exponential weight (i.e. the second QNM): S0→e2 = 0.17002438360768242627609799156749507590247289563179+ S0→e2 = 0.17002438360768242627609799156749507590247289563179+ 5 Nonhydrodynamic sectors and effects of QNM coupling 5 In the previous section we have analysed the resurgence relations revealed by the asymptotic hydrodynamic expansion of the energy density and the exponentially suppressed fundamen- tal sectors, directly associated with QNMs of the AdS black brane. This analysis already showed very strong evidence that the energy density has a representation as the resurgent transseries (1.3). However, a much more striking part of this transseries is the existence of mixed sectors, corresponding to non-trivial non-linear coupling between (different) QNMs, such as Φ2e1 or Φe1+e1. The occurrence of these mixed sectors has already been seen in the gravity calculation of section 2; here we show that they are indeed essential to complete the resurgent picture of our transseries for the energy density. JHEP02(2019)073 The easiest way to show the non-linear coupling between quasinormal modes is by studying the large-order behaviour of the coefficients of the fundamental sector associated to the first QNM Φe1. The singularity structure of its Borel transform can be found on the middle left plot of figure 1. Here we already have direct evidence of the contribution of fundamental sectors such as Φe2, Φe1 (via the branch cut starting at ξ + A1 = A2 and ξ + A1 = A1, respectively), as well as the mixed sectors Φ2e1 and Φe1+e1 (via the branch cuts starting at ξ + A1 = 2A1 and ξ + A1 = A1 + A1). The resurgent properties of these contributions can be explicitly written in the form of the large-order behaviour of coefficients ε(e1) k (analogous to eq. (3.5), see [21]): ε(e1) k ≃ k≫1 −Se1→e2 2πi Γ(k+βe1−βe2) (A2−A1)k+βe1−βe2 χe1→e2+ (5.1) −Se1→2e1 2πi Γ(k+βe1−β2e1) A k+βe1−β2e1 1 χe1→2e1−Se1→e1+e1 2πi Γ(k+βe1−βe1+e1) A1 k+βe1−βe1+e2 χe1→e1+e2+ (5.1) The values of the characteristic exponents appearing above can be found on page 10. The first line of eq. (5.1) gives the leading growth of these coefficients, governed solely by the sector Φe2 (closest singularity on the Borel plane), while the second line shows the sub- leading exponentially suppressed growth governed by the mixed sectors Φ2e1 and Φe1+e1.21 Applying the methodology introduced in section 3 (and explained in detail in appendix B) to the coefficients of sector Φe1 we have predicted the values of the coeffi- cients of the fundamental sector Φe2, as well as of the mixed sectors Φ2e1, Φe1+e1. 5 Nonhydrodynamic sectors and effects of QNM coupling The comparison between the resurgence predictions and the numerical gravity calculations can be found on the plots of figure 5, where we have used the notation for the comparison of coefficients introduced in eq. (4.1). The following subsections discuss these developments in more detail. + 0.09746084799999641974938699072923874879800635398686 i . (4.6) (4.6) Also, from the direct analysis of the branch cut at ξ = A2 we have checked that the relation between Borel residues S0→e2 = −S0→e2 is satisfied as expected. Also, from the direct analysis of the branch cut at ξ = A2 we have checked that the relation between Borel residues S0→e2 = −S0→e2 is satisfied as expected. We can now apply the method proposed in the previous section to predict the sub- leading resurgent behaviour of the hydrodynamic series’ coefficients at the singular point ( ) We can now apply the method proposed in the previous section to predict the sub- leading resurgent behaviour of the hydrodynamic series’ coefficients at the singular point ξ = A2. This corresponds to the resurgent prediction of the coefficients ε(e2) m .20 ξ = A2. This corresponds to the resurgent prediction of the coefficients ε(e2) m .20 The right plot of figure 4 shows the comparison between prediction based on resurgence and the direct numerical calculation starting from the bulk gravity solution for the first 8 of these coefficients. We can see that the error remains small (although we have lost accuracy due to the resummation process), confirming once more the resurgent properties of the transseries solution (1.3). – 21 – 21At the same exponentially suppressed level, the branch cut related to the hydrodynamic series will al contribute, but our focus will be on the contribution of mixed sectors. 5.1 Resurgence of different fundamental sectors The first evidence of non-trivial relations between different QNMs comes from the fact that the leading large-order behaviour of the fundamental sector Φe1 associated to the least 21At the same exponentially suppressed level, the branch cut related to the hydrodynamic series will also contribute, but our focus will be on the contribution of mixed sectors. – 22 –       - - - - -      - - - - -       - - - - - - Figure 5. Comparison of the predicted results from resurgence techniques and the numerical ones obtained from solving Einstein equations. In the above plots we show this comparison for the coefficients of the fundamental sector Φe2 and mixed sectors Φ2e1 and Φe1+e1, as predicted by their relation to sector Φe1 (following eq. (4.1)).       - - - - -       - - - - - -      - - - - - JHEP02(2019)073 Figure 5. Comparison of the predicted results from resurgence techniques and the numerical ones obtained from solving Einstein equations. In the above plots we show this comparison for the coefficients of the fundamental sector Φe2 and mixed sectors Φ2e1 and Φe1+e1, as predicted by their relation to sector Φe1 (following eq. (4.1)). damped QNM is governed by the coefficients of the fundamental sector Φe2, associated to the second QNM. The precise relation can be analysed by focusing on the leading singularity ξ = A2 −A1 of the Borel transform of Φe1. Applying an approach almost identical to eq. (3.14) we can determine the Borel residue associated with this singular point: Se1→e2 = 2.6127578014515638725739302086318633648979300592843− −10.6770578911184045100721836940875685761278306458495 i. (5.2) 5.2 Mixed sectors Until this moment we have dealt solely with the resurgent relations between fundamen- tal sectors appearing in our transseries for the energy density, eq. (1.3). However, the full transseries (1.3) also includes mixed sectors which do not correspond to single QNMs: they are in fact expansions in sectors whose exponential weight is given by linear combi- nations of the QNM frequencies as a result of the coupling between different fundamental sectors. The presence of such mixed sectors was already shown by the direct gravity cal- culation of section 2, and it can be further justified via their resurgent relations to the fundamental sectors. JHEP02(2019)073 To determine the sub-leading, exponentially suppressed, behaviour of the Φe1 sector shown on the second line of eq. (5.1), we want to analyse the singularities on the Borel plane at ξ + A1 = 2A1 and ξ + A1 = A1 + A1. As explained in section 3 and applied in section 4, we can do so by performing a resummation of the contributions from leading singularity ξ + A1 = A2, shown on the first line of eq. (5.1), and determine a subtracted series: δ1Φe1 ≃u−βe1 +∞ X k=1 δ1ε(e1) k u−k , (5.3) (5.3) where the subtracted coefficients are given by the laterally resummed series δ1ε(e1) k ≡ε(e1) k + Se1→e2 2πi Γ(k + βe1 −βe2) A k+βe1−βe2 1 S0+χe1→e2 , (5.4) (5.4) whose large-order behaviour is now given by the second line of eq. (5.1). Through the anal- ysis of the leading singularities of B [δ1Φe1] (ξ) at ξ + A1 = 2A1, A1 + A1, we can now de- termine the Borel residues associated with contributions of the mixed sectors Φ2e1, Φe1+e1 to the large-order behaviour of Φe1: whose large-order behaviour is now given by the second line of eq. (5.1). Through the anal- ysis of the leading singularities of B [δ1Φe1] (ξ) at ξ + A1 = 2A1, A1 + A1, we can now de- termine the Borel residues associated with contributions of the mixed sectors Φ2e1, Φe1+e1 to the large-order behaviour of Φe1: Se1→2e1 = 2S0→e1 ; Se1→e1+e1 = S0→e1 = −S0→e1 . (5.5) (5.5) These relations between Borel residues were expected as they are some of the many such relations which can be found from the direct resurgent analysis of the transseries in eq. (1.3) (see sections 2 and 5 of the review [21] for more details). Se1→e2 = 2.6127578014515638725739302086318633648979300592843− −10.6770578911184045100721836940875685761278306458495 i. (5.2) (5.2) This Borel residue appears as the proportionality constant for the leading large-order growth shown in eq. (5.1). Using the procedure shown in eq. (3.15) we can further analyse the sub-leading large- order behaviour of the series Φe1 at the singular Borel plane point ξ = A2 −A1 and obtain a prediction of the coefficients of sector Φe2. The comparison between the values of the first 11 coefficients ε(e2) m predicted using resurgence and their values calculated from the bulk solution is found in the top plot of figure 5. The error observed there is very small, confirm- ing the presence of nontrivial resurgence relations between different fundamental sectors, and consequently different QNMs. This provides further evidence for the correctness of the picture based on resurgence theory. – 23 – 6 Outlook Through the AdS/CFT correspondence the problem of plasma equilibration can be viewed equivalently as the process by which a dynamical black object evolves toward its static state. The dissipative character of the black-brane horizon translates into the dissipative properties of the late-time asymptotic solution whose leading term was found in ref. [15]. Some of the subleading terms were determined analytically in refs. [46–48] and many more numerically in refs. [8, 16] through a power series in the variable u = τ 2/3. In this paper we presented a novel transseries solution for the bulk geometry which supplements the asymptotic power-series by including contributions which are non-perturbative (exponen- tially damped) in the late time expansion. These additional transseries sectors can be interpreted either as linearised perturbations of the late-time bulk solution or as non-linear couplings of these perturbations. They become most relevant at early times and naturally encode information about the initial data, which is “lost” as the system evolves toward equilibrium. These perturbations can be identified with quasinormal modes of the static AdS black-brane solution, modified by gradient corrections. We were able to explicitly com- pute the bulk solution expanded around several transseries sectors to high orders in the late-time expansion. Since the AdS/CFT correspondence maps QNMs of the black brane to nonhydrodynamic modes of the dual gauge theory plasma, from the bulk solution we derived a transseries for the energy density of N = 4 SYM plasma undergoing Bjorken flow. JHEP02(2019)073 On the field theory side, our bulk solution provides an asymptotic form of the energy density as a function of proper time together with corrections which depend on the initial state. The physical meaning of this object is the same as at the level of hydrodynamics [9, 19]. Even though the energy density depends on a scale parameter (which we have fixed as in ref. [8]), it is independent of other features of the initial state, which enter only through the exponentially suppressed contributions. Thus, the transseries for the energy density has a similar interpretation as the transseries for quantities which are universal in the sense of ref. [63] — it describes the dissipation of initial state information in the process of hydrodynamization [9, 19, 63, 64]. From a mathematical perspective our results provide a novel example where resurgence is at work: the late-time expansion of a strongly coupled QFT computed in a microscopic theory. 5.2 Mixed sectors We can continue our procedure further and predict the coefficients ε(2e1) k and ε(e1+e1) k of mixed sectors Φ2e1, Φe1+e1, respectively. The two bottom plots of figure 5 show the comparison between resurgence prediction and numerical calculation for the first 8/9 of these coefficients. While the errors are somewhat larger here (with some loss of accuracy due to the resummation process), they are still small enough to be confident that the presented picture, including the presence of mixed sectors in the full solution for the energy density (1.3), is fully consistent with expectations based on the theory of resurgence. – 24 – 6 Outlook The intricate large-order relations which express the resurgent properties of our transseries solution provide strong links between the original hydrodynamic series and all the non-hydrodynamic sectors. The predictive power of these relations is such that one can in principle recover all nonhydro sectors directly from the large-order behaviour of the coefficients of the hydro sector (the limiting factor being the actual number of coefficients determined). One can ask how is this possible, given that the Borel plane of the hydro sector (top plot of figure 1) does not directly show all the mixed sectors? The answer is that this is an iterative process: the hydro series returns full information about all fundamental sectors, which in turn provide all the information about the mixed sectors which was previously not visible. The asymptotic series which arise in this way show similarities with those arising in the context of the hydrodynamic model of ref. [18], whose asymptotic behaviour was – 25 – investigated in ref. [19]. In particular, the large-order behaviour of these series involve mul- tiple, competing factorial growths dependent of different complex parameters (exponential weights and characteristic exponents), which make it impossible to apply standard tools used in many recent studies of asymptotic series. We have however succeeded in developing an equivalent method for extracting information from asymptotic series directly at the level of the Borel plane singularities, which has made it possible to effectively calculate Borel residues (Stokes constants) and verify large-order relations. In sections 4 and 5 presented extremely accurate numerical evidence that both nonhydro fundamental and mixed sectors need to be included in the full solution in order to have a complete picture of the fluid’s energy density. Although natural from the point of view of resurgent transseries, the exis- tence of these mixed sectors, interpreted as the result of coupling between different QNMs, is completely novel. JHEP02(2019)073 Finally, we comment on the applicability of our approach to early time dynamics. Our transseries solution, once supplemented by initial data, provides a full non-perturbative description of the energy density of the fluid. Thus it naturally extends the regime of validity of the original hydrodynamic expansion. Although the transseries was formally defined for large-proper times, one can use a summation prescription on every (asymptotic) sector to obtain the energy density for smaller values of proper time. 6 Outlook Naturally the smaller the proper time, the more nonhydro sectors one will need to consider to obtain an accurate result. Of possible summation prescriptions the most widely used in the so-called Borel- Pad´e-´Ecalle summation procedure (see [21]) as discussed, for example, in [16, 18, 29, 65–68]. Such results may help to shed light on the question which initial conditions correspond to the early-time attractor in N = 4 SYM [10, 11]. Alternative summation procedures can also be adopted to analyse the early-time regime, such as trans-asymptotics and analytic transseries summation [69–71]. Finally, let us note that the explicit identification of nonhydrodynamic modes with the quasinormal mode spectrum suggests that a generalisation of the gradient expansion through the fluid-gravity duality [72] should exist. Such a solution would systematically map exponentially suppressed corrections in the bulk to nonhydrodynamic mode contri- butions in the dual Yang-Mills theory even without the imposition of the symmetries of Bjorken flow. A Leading singularities in the Borel plane • mixed sector Φ2e1: ξ + 2A1 = Ak, Ak, k = 1, · · · (singularities associated to funda- mental sectors); ξ = (n + 1)A1, 2A1, 2An, 2An, A1 + A1, A1 + An, A1 + An, 2A1 + k A1, 2A1+k An, 2A1+k An, k = 1, · · · , n = 2, · · · (singularities associated to mixed sectors). A Leading singularities in the Borel plane The theory of resurgence can help us predict which singularities will appear in the complex Borel plane associated to each asymptotic sector. This can be done by following the pro- cedure explained in section 5 of [21], applicable to a multi-parameter resurgent transseries with many fundamental sectors (many Stokes directions) as well as the corresponding mixed sectors. The techniques of resurgence theory thus allow us to predict which singular branch points will be visible in the Borel planes of different asymptotic sectors of the transseries for the energy density given by eq. (1.3). Acknowledgments We would like to thank Jorge Casalderrey-Solana, Ovidiu Costin, Micha l Heller, Romuld Janik, Marcel Vonk and Przemys law Witaszczyk for useful discussions. IA has been sup- ported in part by the Polish National Science Centre (NCN) grants 2012/06/A/ST2/00396 and 2015/19/B/ST2/02824, the FCT-Portugal grant PTDC/MAT-OUT/28784/2017, and the U.K. Engineering and Physical Sciences Research Council (EPSRC) Early Career Fel- lowship EP/S004076/1. JJ was supported by the NCN grant 2016/23/D/ST2/03125. BM is a Commonwealth Scholar and is also supported by the Oppenheimer Fund Scholarship. MS is supported by the NCN grant 2015/19/B/ST2/02824. – 26 – A Leading singularities in the Borel plane JHEP02(2019)073 In this appendix we list the expected branch points appearing in the Borel plane ξ of the asymptotic sectors discussed in the main text: • hydrodynamic sector Φ0: ξ = Ak, Ak, k = 1, · · · (singularities associated to fun- damental sectors); ξ = n Ak, Ak, k = 1, · · · , n = 2, · · · (singularities associated to mixed sectors); • hydrodynamic sector Φ0: ξ = Ak, Ak, k = 1, · · · (singularities associated to fun- damental sectors); ξ = n Ak, Ak, k = 1, · · · , n = 2, · · · (singularities associated to mixed sectors); • hydrodynamic sector Φ0: ξ = Ak, Ak, k = 1, · · · (singularities associated to fun- damental sectors); ξ = n Ak, Ak, k = 1, · · · , n = 2, · · · (singularities associated to mixed sectors); • fundamental sector Φe1: ξ + A1 = A1, Ak, Ak, k = 2, · · · (singularities associated to fundamental sectors); ξ = n A1, A1+k A1, A1+k An, A1+k An, k = 1, · · · , n = 2, · · · (singularities associated to mixed sectors); • fundamental sector Φe2: ξ + A2 = A1, A1, A2, Ak, Ak, k = 3, · · · (singularities asso- ciated to fundamental sectors); ξ = (n−1) A2, A2 +k A1, A2 +k A1, A2 +k A2, A2 + k An, A2 + k An, k = 1, · · · , n = 3, · · · (singularities associated to mixed sectors); • fundamental sector Φe2: ξ + A2 = A1, A1, A2, Ak, Ak, k = 3, · · · (singularities asso- ciated to fundamental sectors); ξ = (n−1) A2, A2 +k A1, A2 +k A1, A2 +k A2, A2 + k An, A2 + k An, k = 1, · · · , n = 3, · · · (singularities associated to mixed sectors); • mixed sector Φ2e1: ξ + 2A1 = Ak, Ak, k = 1, · · · (singularities associated to funda- mental sectors); ξ = (n + 1)A1, 2A1, 2An, 2An, A1 + A1, A1 + An, A1 + An, 2A1 + k A1, 2A1+k An, 2A1+k An, k = 1, · · · , n = 2, · · · (singularities associated to mixed sectors). B.1 Behaviour of an asymptotic series at the first Borel singularity We assume an asymptotic series expansion (for u ≫1) of the form Φ(0)(u) ≃u−β0 +∞ X n=0 F (0) n 1 un , (B.1) (B.1) whose coefficients F (0) n show large-order factorial growth. This growth will contain possibly multiple contributions, each of which growing as Γ(n+β) A−n−β for n ≫1 for some possibly complex parameters β0, β, A. The parameters β are the so-called characteristic exponents, defining the type of branch point singularities that we will find in the Borel plane, while A determines location of these singularities. In the context of the present paper we could just write εn instead of Fn here, but the actual method is clearly more general than the present context. JHEP02(2019)073 Given the factorial growth of the coefficients in the hydrodynamic (in general: pertur- bative) series in eq. (B.1), it is convenient to define a one-parameter family of series22 Φ(k) α (u) = u−α−β+β0Φ(k)(u) , (B.2) (B.2) whose Borel transform (defined via the rule u−δ 7→ξδ−1/Γ(δ)) will have a singularity structure dependent on the choice of α (see section 4 of [21] for more on this subject). In the vicinity of the singular point ξ = A the Borel transform for the particular case of α = 0 has the standard behaviour of simple resurgent functions:23 B h Φ(0) 0 i (ξ) = +∞ X n=0 F (0) n Γ (n + β)ξn+β−1 ∼S0→1 B h Φ(1) 0 i (ξ −A) log(ξ −A) 2πi + regular. (B.3) (B.3) In this equation S0→1 is a Borel residue, given by the first Stokes constant of the problem, and Φ(1) 0 is defined via eq. (B.2) with Φ(1)(u) ≃u−β1 +∞ X n=0 F (1) n 1 un . (B.4) (B.4) This asymptotic series will be associated with the first nonhydrodynamic (in general: non- perturbative) sector appearing in our transseries (with |A2| > |A|) F (u, σi) = Φ(0)(u) + σ1 e−A u Φ(1)(u) + σ2 e−A2 u Φ(2)(x) + · · · . (B.5) (B.5) The key point of this procedure is that given the behaviour in eq. (B.3), we can transform the logarithmic behaviour into a square root behaviour. To see this, it will be very convenient to define Ψ(0) (ξ) ≡ξ 1 2 −βB  Φ(0) 1 2  (ξ), (B.6) (B.6) which, as we now proceed to show, has a square root branch point singularity at ξ = A. B Large-order predictions from Borel plane residues In this appendix we will present some of the details of the large-order analysis of transseries sectors directly from the Borel plane point of view. This approach leads to a systematic pro- cedure where by taking residues of singularities associated with a particular sector one can predict the coefficients of the other non-perturbative sectors. This method is especially use- ful in situations where we have complex-conjugate singularities in the Borel plane A, A and multiple contributions characterised by different factorial growths Γ (k + βi) for k ≫1 with βi ∈C. This leads to intricate oscillatory large-order behaviour of the coefficients of our asymptotic series, from which the usual acceleration methods (such as Richardson trans- forms) cannot be effectively applied. This is a major complication if one wishes to calculate the Stokes constants or expansion coefficients appearing in nontrivial transseries sectors. – 27 – B.1 Behaviour of an asymptotic series at the first Borel singularity B.1 Behaviour of an asymptotic series at the first Borel singularity which, as we now proceed to show, has a square root branch point singularity at ξ = A. 22This definition is appropriate for analysing the hydrodynamic sector, but it can be trivially modified so it can be used for the nonhydrodynamic sectors, as done in section 5. 23 23Depending on the value of β one might need the exclude any constant term in the series, as the Borel transform is only defined for positive powers of 1/u. Such a constant term should then be added after the summation procedure is performed. – 28 – We begin by observing that in the vicinity of that point one has B  Φ(0) 1 2  (ξ)|ξ=A = S0→1 2 B  Φ(1) 1 2  (ξ −A) + regular . (B.7) (B.7) The validity of this relation can be verified using a simple generalisation of the methods described in section 4 of the review [21] for the case of the quartic integral.24 On the other hand, by definition, B  Φ(1) 1 2  (ξ) = ξβ1−β0+β−1 2 +∞ X n=0 F (1) n Γ n + β1 −β0 + β + 1 2  ξn , (B.8) (B.8) JHEP02(2019)073 for general values of βi this will have a complicated leading behaviour at ξ = 0. However, when Φ(0) and Φ(1) are two different sectors of a transseries solution of the form of eq. (B.5), the factorial growth of the coefficients of the original series (B.1) will obey β = β0 −β1. In this case the previous expression becomes B  Φ(1) 1 2  (ξ) = ξ−1 2 +∞ X n=0 F (1) n Γ n + 1 2  ξn . (B.9) (B.9) Using eq. (B.9) and eq. (B.7) we find that in the vicinity of ξ = A Ψ(0) (ξ) |ξ=A = S0→1 A 1 2 −β 2√ξ −A +∞ X n=0 (ξ −A)n × n X k=0 (−1)k k! (2A)k F (1) n−k Γ n −k + 1 2  k−1 Y j=0 (2β + 2j −1) (B 10) (B.10) where we also used that where we also used that ξ 1 2 −β ξ=A = A 1 2 −β +∞ X k=0 (−1)k k! (2A)k (ξ −A)k k−1 Y j=0 (2β + 2j −1) . (B.11) (B.11) As anticipated, eq. (B.10) has a square root branch point singularity. As anticipated, eq. B.1 Behaviour of an asymptotic series at the first Borel singularity (B.10) has a square root branch point singularity. The next step involves changing the Borel plane variable in order to more easily analyse the behaviour around each singular branch point. Such changes of variables have been used in the literature, in particular through the use of conformal maps, see e.g. [58–60], as well as recent applications [61]. In our case, it will be useful to change variables to transform the square root behaviour into a pole. This in turn will make it possible to extract information from eq. (B.1) by calculating residues. The required change of variable is ξ = A + eiπ (ζ −A)2 . (B.12) (B.12) Note that the point ξ = A corresponds to ζ = A. With this change of variables it follows from (B.10) that Note that the point ξ = A corresponds to ζ = A. With this change of variables it follows from (B.10) that Ψ(0) (ζ) |ζ=A = S0→1 2i A 1 2 −β (ζ −A) +∞ X n=0 (ζ −A)2n C(0→1) n , (B.13) (B.13) 24In general, one can obtain eq. (B.3) from eq. (B.7) by evaluating the semi derivative associated with B h Φ(0) 1 2 i (ξ) term by term, expanding the result about (ξ −A) and isolating the contributions that are linear in log (ξ −A). The reverse will also be true, so eq. (B.7) will always hold. – 29 – – 29 – where C(0→1) n ≡ n X k=0 (−1)n−k k! (2A)k F (1) n−k Γ n −k + 1 2  k−1 Y j=0 (2β + 2j −1) . (B.14) (B.14) The C(0→1) n depend solely on the coefficients of the non-perturbative series (B.4), with each C(0→1) n depending on all the coefficients F (1) k for k ≤n, for example: C(0→1) 0 = F (1) 0 Γ(1/2) ; C(0→1) 1 = −F (1) 1 Γ (3/2) + F (1) 0 Γ (1/2) (2β −1) 2A . (B.15) (B.15) JHEP02(2019)073 It is now straightforward to see that evaluating residues of eq. (B.6) will give us direct access to these coefficients, as well as the Borel residue (or, equivalently, the Stokes con- stant). To effectively perform this analysis, we need to use the hydrodynamic expansion coefficients to calculate the series Ψ(0) (ξ) and perform the change of variable in eq. (B.12). B.1 Behaviour of an asymptotic series at the first Borel singularity Expanding the result around ζ0 = A− √ A (which is the regular point of the Borel transform corresponding to ξ = 0) one finds Ψ(0) (ζ) = +∞ X n=0 F (0) n 2A1/2n Γ n + β + 1 2  +∞ X k=0 (−1)k 2A1/2k n! k!(n −k)! (ζ −ζ0)k+n = +∞ X n=0 (ζ −ζ0)n n X k=0 F (0) n−k 2A1/2n−2k (−1)k Γ n −k + β + 1 2  (n −k)! k!(n −2k)!. (B.16) (B.16) Assuming that we have N terms for this series, we define an analytic continuation of this truncation using the Pad´e approximant BP N 2  Ψ(0) (ζ). By virtue of the arguments pre- sented earlier, this quantity should have an isolated pole at ζ = A. This can be verified directly, and we can then calculate the residue at that point. The result can be com- pared with the value obtained from the asymptotic calculation (B.13), which leads to the conclusion that 1 Assuming that we have N terms for this series, we define an analytic continuation of this truncation using the Pad´e approximant BP N 2  Ψ(0) (ζ). By virtue of the arguments pre- sented earlier, this quantity should have an isolated pole at ζ = A. This can be verified directly, and we can then calculate the residue at that point. The result can be com- pared with the value obtained from the asymptotic calculation (B.13), which leads to the conclusion that 1 Resζ=ABPN h Ψ(0)i (ζ) = A 1 2 −β 2 i Γ(1/2) S0→1 F (1) 0 . (B.17) (B.17) This gives explicit access to the Borel residue S0→1, since all the remaining quantities appearing in eq. (B.17) are known (recall that we normalize the leading coefficients in the fundamental nonhydrodynamic sectors to 1). (1) This gives explicit access to the Borel residue S0→1, since all the remaining quantities appearing in eq. (B.17) are known (recall that we normalize the leading coefficients in the fundamental nonhydrodynamic sectors to 1). (1) Once the Borel residue is known, we can gain access to higher coefficients F (1) m of the non-perturbative series Φ(1) (which first appear in C(0→1) m of (B.13)), by calculating the residue of BmΦ(0) ≡ ( 2 i Aβ−1 2 S0→1 BPN h Ψ(0)i − m−1 X n=0 (ζ −A)2n−1 C(0→1) n ) (ζ −A)−2m . B.2 Behaviour of an asymptotic series at its second Borel singularit B.2 Behaviour of an asymptotic series at its second Borel singularity We have analysed the properties of the leading branch cut (closest to the origin) appearing in the Borel plane of the perturbative series. The position of the branch point was ξ = A. In order to differentiate between different branch point in the Borel plane, let us redefine A = A1. We would now like to analyse the behaviour around the singularities in the Borel plane further away from the origin, such as the one at ξ = A2 (where |A2| > |A1|). As it was explained in section 3, in order to analyse the branch cut at this singularity, we need to first subtract the contributions from the closer one at ξ = A1. We saw that this can done by removing the leading large-order behaviour from the coefficients of the perturbative series. In much the same way as in eqs. (3.2)–(3.4), this leading behaviour is given by (see [21] for further details): JHEP02(2019)073 F (0) n ≃−S0→1 2πi Γ (n + β0 −β) An+β0−β 1 χ0→1(n) + O |A2|−n , (B.21) (B.21) where where χ0→1(n) ≃ +∞ X h=0 Γ (n + β0 −β −h) Γ (n + β0 −β) Ah 1 F (1) h . (B.22) (B.22) The sum appearing in χ0→1(n) is to be taken as a series in n−1 for large-order n, and is asymptotic. We can then perform a Borel-Pad´e-´Ecalle resummation for each value of n.25 However, given that the singularities of the corresponding Borel transform also fall on the path of integration (the real axis), we will need to perform a lateral summation S0+χ0→1(n), as defined in eq. (3.7) (for a Pad´e approximant of order N). The subtracted coefficients δ1F (0) n ≡F (0) n + S0→1 2πi Γ (n + β0 −β) An+β0−β 1 S0+χ0→1(n) (B.23) (B.23) obey new large-order relations governed by the coefficients F (2) k of the next asymptotic sector Φ(2) (u) in the transseries solution to our problem (B.5). 25We determine its Borel transform, approximate it via the method of Pad´e approximants, and do a Borel summation of the result along the real axis, as n ∈N. See the reviews [21, 38]. B.1 Behaviour of an asymptotic series at the first Borel singularity (B.18) (B.18) Indeed this residue will give Resζ=ABmΦ(0) = C(0→1) m . (B.19) (B.19) – 30 – The case m = 0, corresponds to the calculation of the Borel residue found in eq. (B.17). With the value of this Borel residue one can predict the value of the coefficients F (1) m , with m > 0. For example, for m = 1 we can use eq. (B.15) to find F (1) 1 = F (1) 0 Γ (3/2) Γ (1/2) (2β −1) 2A −Γ (3/2) Resζ=AB1Φ(0) . (B.20) (B.20) B.2 Behaviour of an asymptotic series at its second Borel singularit To check the large-order behaviour of these subtracted coefficients we perform the analysis described above, but now applied to Borel transform of the series To check the large-order behaviour of these subtracted coefficients we perform th analysis described above, but now applied to Borel transform of the series δ1Φ(0)(u) ≃u−β0−1 +∞ X n=0 δ1F (0) n+1 u−n, (B.24) (B.24) and compare the residues Resζ=A2 Bmδ1Φ(0) to the corresponding coefficients C(0→2) m . Note that the resummation process can only be done for positive n, and we cannot determine the coefficient δ1F (0) 0 . Thus, the leading term of the subtracted series δ1Φ(0)(u) is now u−β0−1. – 31 – Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. References [1] W. 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Content of the tables should be written using Times New Roman 10 single space and the remarks of tables placed in the bottom with Times New Roman 9, single space. The title of tables should be written first with Times New Roman 10 pt. Content of the tables should be written using Times New Roman 10 single space and the remarks of tables placed in the bottom with Times New Roman 9, single space. Table 1. NRA, leaves greenish, stomatal density and width openings, photosynthesis rate, and productivities of nine PGL clones Clones NRA (µmol NO2g- 1hour-1) Leaves Greenish (SPAD Unit) Stomatal density (stomata mm-2) Stomatal width openings (µm) Photosynthesi s rate (µmol CO2 m-2s-1) Productivity (g m-2 plucking-1) PGL 1 0.678 a 59.39 a 100.22 g 0.015 e 98.66 a 9.250 ab PGL 3 0.857 a 60.99 a 120.67 e 0.022 c 97.66 a 10.379 ab PGL 4 1.042 a 59.54 a 110.00 f 0.028 b 105.58 a 10.301 ab PGL 7 0.654 a 57.79 a 79.33 h 0.011 f 97.03 a 6.969 b PGL 10 1.068 a 61.73 a 135.33 d 0.019 d 101.33 a 10.385 ab PGL 11 1.056 a 63.09 a 155.78 c 0.023 c 102.26 a 10.726 ab PGL 12 1.144 a 63.14 a 173.56 b 0.031 a 109.23 a 13.409 a PGL 15 1.279 a 63.26 a 224.67 a 0.029 ab 120.73 a 13.547 a PGL 17 0.907 a 59.59 a 117.11 e 0.022 c 102.90 a 10.305 ab Mean 0.965 60.95 135.19 0.022 103.93 10.585 CV (%) 14.26 5.30 2.80 6.270 13.78 24.789 Remark : The means in one column followed by the same letter were not significantly different according to DMRT (α 5%) Table 1. NRA, leaves greenish, stomatal density and width openings, photosynthesis rate, and productivities of nine PGL clones
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The cumulative dose-dependent effects of metformin on the development of tuberculosis in patients newly diagnosed with type 2 diabetes mellitus
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The Cumulative Dose-Dependent Effects of Metformin on the Development of Tuberculosis in Patients Newly Diagnosed with Type 2 Diabetes Mellitus Eunyoung Heo  SNU-SMG Boramae Medical Center Eunyoung Kim  Kyungpook National University Eun Jin Jang  Andong National University Chang-Hoon Lee  (  kauri670@empal.com ) Seoul National University College of Medicine, Seoul National University Hospital Research Article Keywords: diabetes mellitus, tuberculosis, metformin, prevention, effect Posted Date: June 22nd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-620529/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License.   Read Full License The Cumulative Dose-Dependent Effects of Metformin on the Development of Tuberculosis in Patients Newly Diagnosed with Type 2 Diabetes Mellitus Eunyoung Heo  SNU-SMG Boramae Medical Center Eunyoung Kim  Kyungpook National University Eun Jin Jang  Andong National University Chang-Hoon Lee  (  kauri670@empal.com ) Seoul National University College of Medicine, Seoul National University Hospital Research Article Keywords: diabetes mellitus, tuberculosis, metformin, prevention, effect Posted Date: June 22nd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-620529/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/13 Page 1/13 Abstract Background: Diabetes mellitus (DM) is a well-known risk factor for tuberculosis (TB). Metformin, which is an essential anti-diabetic drug, has been shown to exhibit anti-TB effects in patients with DM. Its effect on preventing the development of TB among patients who are newly diagnosed with DM remains unclear. We evaluated the protective effect of metformin on the development of TB among newly diagnosed patients with type 2 DM. Methods: This was a retrospective cohort study using the claims database. The study population included newly diagnosed type 2 DM patients between January 2003 and March 2011. A metformin user was defined if a patient had taken metformin for more than 28 days within the first 6 months after the initial cohort entry. Primary outcome was the development of TB within 2 years after the index date. Results: Metformin use was not associated with the prevention of TB development (Metformin user: 44/12,916 (0.34%) vs. Metformin non-user: 40/12,916(0.31%); HR, 1.17; 95% CI, 0.75-1.83; P = 0.482). There was, however, a reduction in the development of TB among patients taking a higher cumulative dose of metformin. Patients in the highest quartile of cumulative metformin dose had only a 10% risk of developing TB compared to metformin non-users. In contrast, patients in the second quartile had a higher risk of developing TB than patients in the first quartile. Conclusions: The highest cumulative doses of metformin were protective against the development of TB among newly diagnosed type 2 DM patients. Data source This was a retrospective cohort study of the claims database of the He Assessment Service (HIRA, Seoul, South Korea), a government-affiliate accuracy of claims for the National Health Insurance (NHI, which cove population) and National Medical Aid (which covers ~3.5% of the Sout claims data that had been submitted by health care providers between 2013. Anonymized identifiers were provided by the HIRA to protect priv Protection of Personal Information Maintained by Public Agencies. Th demographics and all medical services performed along with the diagn Classification of Disease, Tenth Revision [ICD-10]), procedures, prescrip name, prescription date, days of supply, dose, and route of administrat (outpatient visit, hospital or emergency department admissions). This was a retrospective cohort study of the claims database of the Health Insurance Review and Assessment Service (HIRA, Seoul, South Korea), a government-affiliated agency that examines the accuracy of claims for the National Health Insurance (NHI, which covers ~97% of the South Korean population) and National Medical Aid (which covers ~3.5% of the South Korean population). We used claims data that had been submitted by health care providers between 1 January 2002 and 31 December 2013. Anonymized identifiers were provided by the HIRA to protect privacy according to the Act on the Protection of Personal Information Maintained by Public Agencies. This database contains demographics and all medical services performed along with the diagnostic code (International Classification of Disease, Tenth Revision [ICD-10]), procedures, prescription drugs (brand name, generic name, prescription date, days of supply, dose, and route of administration) and type of medical utilization (outpatient visit, hospital or emergency department admissions). Background Tuberculosis (TB) is a major global health problem. Approximately one in three individuals worldwide have a latent TB infection, most of whom never develop active TB during their lifetime. Active TB affects approximately 10 million individuals per year with a mortality rate of more than 1 million individuals per year [1]. Certain risk factors increase the probability that latent TB will progress to active TB; diabetes mellitus (DM) is one such risk factor [2, 3]. The association between DM and TB has been well documented. Patients with diabetes have a two- to three-fold higher risk of developing TB compared to individuals who have not been diagnosed with diabetes [4, 5]. Treatment failure and TB recurrence also are more frequent among patients with DM [4, 6–11]. Patients with DM have an impaired immune response, which facilitates both primary infection with Mycobacterium tuberculosis and reactivation of latent TB [12]. Diabetic hosts are slow to mount an innate response to the alveolar macrophages initially infected with Mycobacterium tuberculosis. This delay in innate immune response subsequently leads to downstream delays in adaptive immunity in the lung during the logarithmic growth phase of M. tuberculosis replication, which results in a higher plateau of lung bacterial load once effective control has been exerted. This higher plateau is associated with an increased severity of immune pathology and worse outcomes in patients with DM who develop TB [12, 13]. Metformin is generally prescribed as a first-line anti-diabetic agent due to its association with weight loss and its lack of association with hypoglycemic complications. Beyond its hypoglycemic action, many experimental and clinical studies have reported on the pleiotropic effects of metformin, including in the prevention of atherosclerosis and the treatment of certain cancers and infections [14–17]. Metformin as a treatment for TB also has been actively studied. In particular, metformin is associated with the prevention of TB development, improvements in the successful treatment of TB, and decreases in the recurrence of TB among patients with DM [18–23]. These prior studies have some limitations, however, including small sample sizes, uncontrolled potential confounders, and cohorts that included both newly diagnosed and long-term patients with DM. In this study, therefore, we evaluated the protective effect of metformin on the development of TB among newly diagnosed patients with type 2 DM. We used data from a large national database and controlled for several confounders. Outcome variables The main outcome variable was the incidence of TB among newly diagnosed patients with type 2 DM within 2 years after the index date. The diagnosis of TB was defined as both the use of ICD-10 codes for TB (A15–A19, U88.0–U88.1) and prescription of at least one of the following anti-TB drugs: 1) INH and RMP, 2) EMB, 3) PZA, 4) PTH, 5) CS, 6) PAS, 7) Tubis®, 8) delamanid, and 9) bedaquiline more than once within 90 days of the TB diagnosis. The event date was defined as the first day on which the anti-TB drug was prescribed. Study population The study population included newly diagnosed patients with type 2 DM (ICD-10 codes E11-14) who were treated with anti-diabetic drugs between 1 January 2003 and 31 March 2011 and who were ≥20 years old at cohort entry (Figure 1). Individuals with incident type 2 DM were included according to the following eligibility criteria: (1) had at least two claims with ICD-10 code E11-14 within one year or (2) at least one claim for a prescription for an anti-diabetic medication (ICD-10 code E11-14) during the study period. Anti-diabetic drugs included biguanides (metformin), sulfonylureas, meglitinides, a-glucosidase inhibitors, insulin, thiazolidinediones, dipeptidyl peptidase-4 inhibitors, and glucagon-like peptide-1 (GLP- 1) analogues (incretin). We excluded patients with type 1 DM, which was defined as those who had at Page 3/13 least one claim with an ICD-10 E10 code and who were prescribed only insulin without any oral anti- diabetic drugs. The principal exposure variable was metformin use. Metformin user is defined as a patient who had taken metformin for more than 28 days within the first 6 months after the initial cohort entry. A metformin non-user is defined as a patient who had never been treated with metformin during the study was conducted. Patients who had taken a metformin but did not meet the criteria of being a metformin user were excluded from the analysis. The index date for a metformin user was the 28th day after they started to take metformin within 6 months of entry. The index date for a metformin non-user was the 28th day after diagnosis with DM. We subsequently excluded patients who had received a TB diagnosis and had taken anti-TB drugs based on ICD-10 codes for TB (A15–A19) within 1 year before the index date. Anti-TB drugs included isoniazid (INH), rifampicin (RMP), ethambutol (EMB), pyrazinamide (PZA), prothionamide (PTH), cycloserine (CS), para-aminosalicylic acid (PAS), Tubis® (INH, RMP, EMB and PZA combination drug), delamanid, and bedaquiline. Data Analysis We compared the baseline characteristics of metformin users and non-users. The distributions of these characteristics were compared using Student’s t tests, Chi-square tests, or Fisher’s exact tests as appropriate. To account for significant differences in patient characteristics, we performed 1:1 propensity score (PS) - matched analysis. The PS was calculated by binary logistic models that included metformin use as the dependent variable and the following covariates as independent variables: age, sex, Charlson comorbidity index (CCI), healthcare utilization, anti-diabetic treatment, immunosuppressive treatment, and other comorbidities. Cox proportional hazard regression models were used to evaluate whether TB incidence differed between metformin users and PS-matched metformin non-users. We also investigated the relationship between metformin cumulative dose and TB incidence. Cumulative dose was categorized according to quartile (i.e., Q1, Q2, Q3, Q4). Characteristics that were significantly different between metformin users and non-users prior to the PS-matching were adjusted in the Cox proportional hazard regression models. We report hazard ratios (HRs) and 95% confidence intervals (CI) for metformin use both unadjusted and adjusted for age, sex, and other variables. All statistical analyses were performed using SAS version 9.4 (Statistical Analysis Software Institute; Cary, NC, USA). Page 4/13 Ethical Approval This study involved the use of existing data coded in a manner that prevented the identification of patients either directly or through identifiers. The study protocol received a determination of exemption after review by the Institutional Review Board of Seoul National University Hospital (IRB No. 1906-048- 1038). Results In total, 76,973 patients were newly diagnosed with type 2 DM between January 1, 2003 and March 31, 2011. After excluding participants who did not meet the metformin-use definition or had a previous TB diagnosis, 66,132 patients were included in the analysis: 13,396 metformin users and 52,736 metformin non-users (Figure 1). Baseline demographic and clinical characteristic data are shown in Table 1. Metformin users were younger and more male. Only 25% of metformin non-users were being treated with other anti-diabetic drugs, whereas the 89% of metformin users were concomitantly being treated with other anti-diabetic drugs. Comorbid risk factors for TB development, such as malignancy, malabsorption, chronic kidney disease, dialysis, gastrectomy, and organ transplantation, were more frequent among metformin non-users. Metformin non-users also were more likely to have chronic respiratory diseases and to be taking corticosteroids or other immunosuppressants. Metformin users had less frequent health care utilization compared to metformin non-users at baseline; this finding did not change significantly over the follow-up study period. Due to these differences in baseline characteristics between metformin users and non-users, propensity score (PS) matching was performed (Table 2) to generate two groups in which the standardized difference (STD) for any covariate was less than 10%. PS-matched Cox proportional hazard regression models showed that metformin use was not associated with the prevention of TB development among patients who were newly diagnosed with type 2 DM (HR, 1.17; 95% CI, 0.75-1.83; P = 0.482) (Table 3). A trend towards the prevention of TB development was observed for higher cumulative doses of metformin at the two highest quartiles (Q3 and Q4) compared to metformin non-users (P-value for trend = 0.059) (Figure 2). The risk of TB development was only 10% among patients in the highest quartile (Q4) compared to metformin non-users. In contrast, however, the risk of TB was higher in patients in the 2nd quartile (Q2) compared to patients in the 1st quartile (Q1). Discussion In our study, metformin use was not associated with the prevention of TB development among patients who were newly diagnosed with type 2 DM. Our data suggest, however, that a higher cumulative dose of metformin may protect against the development of TB. Any metformin use was not significantly associated with the prevention of the development of TB in multivariate analysis (adjusted HR, 0.93; 95% CI, 0.65-1.34) (Supplementary Table 1) and in PS-matched participants (HR, 1.17; 95% CI, 0.75-1.83) across the total study period (Table 3). Intriguingly, however, we observed two phases of metformin cumulative dose that were associated with the development of TB. Among patients in the 2nd quartile Page 5/13 Page 5/13 (Q2) of metformin cumulative dose, the HR for TB development was 1.69 (95% CI, 1.05-2.71; P = 0.030) (Figure 2).In contrast, the HR for the development of TB trended towards a reduction among patients in the 3rd quartile (Q3) of metformin cumulative dose (HR, 0.49; 95% CI, 0.20-1.21, P=0.030) and a significant reduction among patients in the 4th quartile (Q4) of metformin cumulative dose (HR, 0.10; 95% CI, 0.01-0.70, P = 0.021). The effects at these two different time periods may have combined to produce null findings in the overall model. These findings may reveal different effects of metformin on the development of TB. During the early phase of metformin treatment, metformin may disturb anti-TB immunity. According to one report, metformin downregulated TNF-α production and excretion, which is an important cytokine for both macrophage activation and granuloma formation in obese mice [24] and macrophages [25]. Similarly, a study in a mouse model of TB reported that bacillary load increased within the first 2 weeks of metformin treatment, followed by an anti-TB effect thereafter [26]. In another experimental model, metformin did not initially improve the sterilizing activity of a first-line anti-TB treatment in mice; however, after 3.5 months of treatment, the addition of metformin to standard therapy reduced mean lung bacillary load by 0.18 log10 compared to a group receiving standard therapy only (P = 0.039) [27]. In human in vitro and in vivo studies, metformin inhibited a type I interferon (IFN) response induced by M. tuberculosis, and both IFN-¡ and TNF-α were reduced for up to 21 days after metformin intake. In this same study, however, metformin increased phagocytic activity and reactive oxygen species production [28]. Discussion Based on these results, there are two possible roles of metformin during the early phases of treatment: (1) metformin may disturb anti- TB immunity by down-regulating IFN-¡ and TNF-α; and (2) metformin may suppress the sterilizing effects of anti-TB agents during early metformin treatment. In contrast, during later phase of treatment, metformin may restrict mycobacterial growth by inducing mitochondrial reactive oxygen species production and phagocytic activity [26]. Recently, Lin et al. [19] reported that metformin use independently reduced the risk of the development of TB (RR, 0.24; 95% CI, 0.11-1.87), which is not consistent with our results. This study analyzed 5,026 PS- matched metformin users and non-users among newly diagnosed patients with type 2 DM from the Taiwan claims database between 1998 and 2010. In contrast to our study, however, the clinical characteristics between metformin users and non-users remained unbalanced even after PS-matching. For example, 56.9% of metformin users had been treated with statins, which may independently protect against the development of TB [29]. Moreover, the time since DM diagnosis was not well controlled in their study population; the risk of developing TB was more than two-fold higher among patients who had DM for over six years compared to patients who had DM for less than six years. Our study controlled for these potential confounders by balancing statin treatment between metformin users and non-users and only enrolling patients who were newly diagnosed with DM. Despite the results reported above, a different retrospective study using claims data from Taiwan showed that metformin use was an independent factor for preventing the development of TB compared to sulfonylurea use (HR, 0.337; 95% CI, 0.169-0.673) [21].This study did not compare metformin users to non-users, however. This study showed there were more rural residents with less statin users in the Page 6/13 sulfonylurea group, which is associated with the development of TB. However, even after PS matching was conducted to control for the differences, they remained statistically significant between the two groups. In addition, it has been reported that sulfonylurea may increase the risk of infection [30]. Sulfonylurea reduced primary human monocyte functions in response to TB in an in vitro study of patients with type 2 DM. Treatment with sulfonylurea therefore may result in an increased susceptibility to TB among patients with type 2 DM [31]. Conclusion Metformin use was not associated with the prevention of the development of TB in patients who were newly diagnosed with type 2 DM in our study. Among patients with lower cumulative doses, metformin trended towards an increased risk of TB, whereas higher cumulative doses decreased the risk of TB. These findings suggest that higher doses and longer durations of metformin may prevent the development of TB in patients with type 2 DM. Discussion Our study controlled for these confounders by including other drugs, including anti-diabetic drugs and immunosuppressives, in PS matching between metformin users and non-users. Ethics approval and consent to participate This study was conducted ethically in accordance with the World Medical Association Declaration of Helsinki. The study protocol received a determination of exemption after review by the Institutional Review Board of Seoul National University Hospital (IRB No. 1906-048-1038). Abbreviations TB, tuberculosis; DM, diabetes mellitus; PS, propensity score; HIRA, the Health Insurance Review and Assessment Service; NHI, the National Health Insurance; ICD, International Classification of Disease; GLP- 1, glucargon-like peptide-1; INH, isoniazid; RMP, rifampicin; EMB, ethambutol; PZA, pyrazinamide; PTH, prothionamide; CS, cycloserine; PAS, para-aminosalicylic acid; CCI, Charlson comorbidity index; HR, hazard ratios; CI, confidence intervals; STD, standardized difference. Consent for publication Not applicable Funding No funding for this study was received. Availability of data and materials Page 7/13 The datasets generated and analyzed during the current study are not publicly available due [REASON WHY DATA ARE NOT PUBLIC] but are available from the corresponding author on reasonable request. Competing Interests The authors have no conflicts of interest to declare. Author Contributions EYH: Interpretation of data and writing the manuscript, EYK: The acquisition and analysis of data, EJJ: Analysis and interpretation of data, CHL: Design of the work, writing the manuscript and final approval of the manuscript References Organization: Global Tuberculosis Report 2017. Geneva. 2017, WHO(2017). 1. World Health Organization: Global Tuberculosis Report 2017. Geneva. 2017, WHO(2017). 1. World Health Organization: Global Tuberculosis Report 2017. Geneva. 2017, WHO(2017). 2. Horsburgh CR, Jr., Rubin EJ: Clinical practice. Latent tuberculosis infection in the United States. N Engl J Med 2011, 364(15):1441–1448. 2. Horsburgh CR, Jr., Rubin EJ: Clinical practice. Latent tuberculosis infection in the United States. N Engl J Med 2011, 364(15):1441–1448. 3. Ferrara G, Murray M, Winthrop K, Centis R, Sotgiu G, Migliori GB, Maeurer M, Zumla A: Risk factors associated with pulmonary tuberculosis: smoking, diabetes and anti-TNFalpha drugs. Curr Opin Pulm Med 2012, 18(3):233–240. 3. Ferrara G, Murray M, Winthrop K, Centis R, Sotgiu G, Migliori GB, Maeurer M, Zumla A: Risk factors associated with pulmonary tuberculosis: smoking, diabetes and anti-TNFalpha drugs. Curr Opin Pulm Med 2012, 18(3):233–240. 4. Jeon CY, Murray MB: Diabetes mellitus increases the risk of active tuberculosis: a systematic review of 13 observational studies. PLoS medicine 2008, 5(7):e152. 4. Jeon CY, Murray MB: Diabetes mellitus increases the risk of active tuberculosis: a systematic review of 13 observational studies. PLoS medicine 2008, 5(7):e152. 5. Dooley KE, Chaisson RE: Tuberculosis and diabetes mellitus: convergence of two epidemics. The Lancet Infectious diseases 2009, 9(12):737–746. 5. Dooley KE, Chaisson RE: Tuberculosis and diabetes mellitus: convergence of two epidemics. The Lancet Infectious diseases 2009, 9(12):737–746. 6. Badawi A, Sayegh S, Sallam M, Sadoun E, Al-Thani M, Alam MW, Arora P: The global relationship between the prevalence of diabetes mellitus and incidence of tuberculosis: 2000–2012. Glob J Health Sci 2014, 7(2):183–191. 6. Badawi A, Sayegh S, Sallam M, Sadoun E, Al-Thani M, Alam MW, Arora P: The global relationship between the prevalence of diabetes mellitus and incidence of tuberculosis: 2000–2012. Glob J Health Sci 2014, 7(2):183–191. 7. Sanghani RN, Udwadia ZF: The association of diabetes and tuberculosis: impact on treatment and post-treatment outcomes. Thorax 2013, 68(3):202–203. 7. Sanghani RN, Udwadia ZF: The association of diabetes and tuberculosis: impact on treatment and post-treatment outcomes. Thorax 2013, 68(3):202–203. 8. Prakash BC, Ravish KS, Prabhakar B, Ranganath TS, Naik B, Satyanarayana S, Isaakidis P, Kumar AM: Tuberculosis-diabetes mellitus bidirectional screening at a tertiary care centre, South India. Public Health Action 2013, 3(Suppl 1):S18-22. 8. Prakash BC, Ravish KS, Prabhakar B, Ranganath TS, Naik B, Satyanarayana S, Isaakidis P, Kumar AM: Tuberculosis-diabetes mellitus bidirectional screening at a tertiary care centre, South India. References Public Health Action 2013, 3(Suppl 1):S18-22. Page 8/13 Page 8/13 9. Jali MV, Mahishale VK, Hiremath MB: Bidirectional screening of tuberculosis patients for diabetes mellitus and diabetes patients for tuberculosis. Diabetes Metab J 2013, 37(4):291–295. 10. Lin Y, Li L, Mi F, Du J, Dong Y, Li Z, Qi W, Zhao X, Cui Y, Hou F et al: Screening patients with diabetes mellitus for tuberculosis in China. Trop Med Int Health 2012, 17(10):1302–1308. 11. Li L, Lin Y, Mi F, Tan S, Liang B, Guo C, Shi L, Liu L, Gong F, Li Y et al: Screening of patients with tuberculosis for diabetes mellitus in China. Trop Med Int Health 2012, 17(10):1294–1301. 12. Kumar Nathella P, Babu S: Influence of diabetes mellitus on immunity to human tuberculosis. Immunology 2017, 152(1):13–24. 13. Vallerskog T, Martens GW, Kornfeld H: Diabetic mice display a delayed adaptive immune response to Mycobacterium tuberculosis. Journal of immunology (Baltimore, Md: 1950) 2010, 184(11):6275– 6282. 14. Vasamsetti SB, Karnewar S, Kanugula AK, Thatipalli AR, Kumar JM, Kotamraju S: Metformin inhibits monocyte-to-macrophage differentiation via AMPK-mediated inhibition of STAT3 activation: potential role in atherosclerosis. Diabetes 2015, 64(6):2028–2041. 15. Forouzandeh F, Salazar G, Patrushev N, Xiong S, Hilenski L, Fei B, Alexander RW: Metformin beyond diabetes: pleiotropic benefits of metformin in attenuation of atherosclerosis. J Am Heart Assoc 2014, 3(6):e001202. 16. Kajiwara C, Kusaka Y, Kimura S, Yamaguchi T, Nanjo Y, Ishii Y, Udono H, Standiford TJ, Tateda K: Metformin Mediates Protection against Legionella Pneumonia through Activation of AMPK and Mitochondrial Reactive Oxygen Species. J Immunol 2018, 200(2):623–631. 17. Mallik R, Chowdhury TA: Metformin in cancer. Diabetes research and clinical practice 2018, 143:409–419. 18. Ma Y, Pang Y, Shu W, Liu YH, Ge QP, Du J, Li L, Gao WW: Metformin reduces the relapse rate of tuberculosis patients with diabetes mellitus: experiences from 3-year follow-up. Eur J Clin Microbiol Infect Dis 2018, 37(7):1259–1263. 19. Lin Shang-Yi TH-P, Lu Po-Liang: Metformin is associated with a lower risk of active tuberculosis in patients with type 2 diabetes. Respirology 2018, 23(11):10. 19. Lin Shang-Yi TH-P, Lu Po-Liang: Metformin is associated with a lower risk of active tuberculosis in patients with type 2 diabetes. Respirology 2018, 23(11):10. 20. Sekhar MS, Unnikrishnan MK, Vyas N: Protective effect of metformin against tuberculosis in diabetic patients. J Infect Public Health 2017, 10(2):242–243. 20. Sekhar MS, Unnikrishnan MK, Vyas N: Protective effect of metformin against tuberculosis in diabetic patients. Tables Due to technical limitations, tables are only available as a download in the Supplemental Files section. References Kang YA CN, Seong JM, Heo EY, Koo BK, Hwang SS, Park BJ, Yim JJ, Lee CH.: The effects of statin use on the development of tuberculosis among patients with diabetes mellitus. Int J Tuberc Lung Dis 2014, 18(6):8. 29. Kang YA CN, Seong JM, Heo EY, Koo BK, Hwang SS, Park BJ, Yim JJ, Lee CH.: The effects of statin use on the development of tuberculosis among patients with diabetes mellitus. Int J Tuberc Lung Dis 2014, 18(6):8. 30. Mor A, Petersen I, Sorensen HT, Thomsen RW: Metformin and other glucose-lowering drug initiation and rates of community-based antibiotic use and hospital-treated infections in patients with type 2 diabetes: a Danish nationwide population-based cohort study. BMJ Open 2016, 6(8):e011523. 30. Mor A, Petersen I, Sorensen HT, Thomsen RW: Metformin and other glucose-lowering drug initiation and rates of community-based antibiotic use and hospital-treated infections in patients with type 2 diabetes: a Danish nationwide population-based cohort study. BMJ Open 2016, 6(8):e011523. 31. Kewcharoenwong C, Prabowo SA, Bancroft GJ, Fletcher HA, Lertmemongkolchai G: Glibenclamide Reduces Primary Human Monocyte Functions Against Tuberculosis Infection by Enhancing M2 Polarization. Frontiers in Immunology 2018, 9(2109). 31. Kewcharoenwong C, Prabowo SA, Bancroft GJ, Fletcher HA, Lertmemongkolchai G: Glibenclamide Reduces Primary Human Monocyte Functions Against Tuberculosis Infection by Enhancing M2 Polarization. Frontiers in Immunology 2018, 9(2109). References J Infect Public Health 2017, 10(2):242–243. 21. Pan SW, Yen YF, Kou YR, Chuang PH, Su VY, Feng JY, Chan YJ, Su WJ: The Risk of TB in Patients With Type 2 Diabetes Initiating Metformin vs Sulfonylurea Treatment. Chest 2017, 153(6):11. 21. Pan SW, Yen YF, Kou YR, Chuang PH, Su VY, Feng JY, Chan YJ, Su WJ: The Risk of TB in Patients With Type 2 Diabetes Initiating Metformin vs Sulfonylurea Treatment. Chest 2017, 153(6):11. 22. Marupuru S, Senapati P, Pathadka S, Miraj SS, Unnikrishnan MK, Manu MK: Protective effect of metformin against tuberculosis infections in diabetic patients: an observational study of south Indian tertiary healthcare facility. Braz J Infect Dis 2017. 22. Marupuru S, Senapati P, Pathadka S, Miraj SS, Unnikrishnan MK, Manu MK: Protective effect of metformin against tuberculosis infections in diabetic patients: an observational study of south Indian tertiary healthcare facility. Braz J Infect Dis 2017. 23. Lee YJ, Han SK, Park JH, Lee JK, Kim DK, Chung HS, Heo EY: The effect of metformin on culture conversion in tuberculosis patients with diabetes mellitus. Korean J Intern Med 2018, 33(5):933– 940. 23. Lee YJ, Han SK, Park JH, Lee JK, Kim DK, Chung HS, Heo EY: The effect of metformin on culture conversion in tuberculosis patients with diabetes mellitus. Korean J Intern Med 2018, 33(5):933– 940. Page 9/13 Page 9/13 24. Lin PL, Plessner HL, Voitenok NN, Flynn JL: Tumor necrosis factor and tuberculosis. J Investig Dermatol Symp Proc 2007, 12(1):22–25. 25. Hyun B, Shin S, Lee A, Lee S, Song Y, Ha NJ, Cho KH, Kim K: Metformin Down-regulates TNF-alpha Secretion via Suppression of Scavenger Receptors in Macrophages. Immune Netw 2013, 13(4):123– 132. 26. Singhal A, Jie L, Kumar P, Hong GS, Leow MK, Paleja B, Tsenova L, Kurepina N, Chen J, Zolezzi F et al: Metformin as adjunct antituberculosis therapy. Science translational medicine 2014, 6(263):263ra159. 27. Dutta NK, Pinn ML, Karakousis PC: Metformin Adjunctive Therapy Does Not Improve the Sterilizing Activity of the First-Line Antitubercular Regimen in Mice. Antimicrob Agents Ch 2017, 61(8). 28. Lachmandas E, Eckold C, Bohme J, Koeken V, Marzuki MB, Blok B, Arts RJW, Chen J, Teng KWW, Ratter J et al: Metformin Alters Human Host Responses to Mycobacterium tuberculosis in Healthy Subjects. J Infect Dis 2019, 220(1):139–150. 29. Figures Page 10/13 gure 1 Figure 1 Flow chart Figure 2 Adjusted Hazard Ratio (HR) of TB development according to cumulative dose of metformin *adjusted for age, sex, the use of insulin, sulfonylurea, other anti-diabetic treatment excluding metformin, systemic corticosteroid, other immunosuppressants, and comorbidities including malignancy, malabsorption, CKD, dialysis, gastrectomy, HIV/AIDS and organ transplantation, CCI, number of hospitalization, and outpatient visit days. HR=Hazard Ratio; CI=confidence interval; CKD = chronic kidney disease; CCI = Charlson Comorbidity Index Figure 1 Page 11/13 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to download. Page 12/13 Page 12/13 SupplementaryTable1190708.docx Tables.docx Page 13/13
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https://recyt.fecyt.es/index.php/retos/article/download/41928/31129, https://dialnet.unirioja.es/descarga/articulo/5841341.pdf, https://www.redalyc.org/pdf/3457/345750049008.pdf
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Adaptación y validación del cuestionario Epod 2.1 a usuarios libres de centros deportivos (Adaptation and validation of the Epod 2.1 questionnaire in free customers of sport centers)
Retos digital/Retos
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cc-by
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2017, Retos, 31, 40-45 © Copyright: Federación Española de Asociaciones de Docentes de Educación Física (FEADEF) ISSN: Edición impresa: 1579-1726. Edición Web: 1988-2041 (www.retos.org) Adaptación y validación del cuestionario Epod 2.1 a usuarios libres de centros deportivos Adaptation and validation of the Epod 2.1 questionnaire in free customers of sport centers *Alberto Nuviala, **Javier Antonio Tamayo-Fajardo, ***Cesar Ruiz-Alejos Gómez, ****Román Nuviala Nuviala, ***Josep María Dalmau Torres *Universidad Pablo de Olavide (España), **Universidad de Huelva (España), *** Universidad de La Rioja (España), ****Universidad de Cádiz (España) Resumen. Los servicios deportivos han evolucionado de forma considerable en los últimos años. Se ha visto incrementado el tipo, número de actividades y servicios ofrecidos a los clientes. Al mismo tiempo han aparecido en las organizaciones deportivas usuarios que realizan sus prácticas físico deportivas de forma libre. Los instrumentos creados para conocer las valoraciones del servicio recibido por parte de los usuarios han sido diversos. Sin embargo, ninguno de ellos ha sido diseñado para medir específicamente los juicios de valor de estos clientes que realizan sus actividades sin la presencia ni dirección de un técnico deportivo. El objetivo de esta investigación ha sido validar un instrumento que permita conocer la calidad percibida, valor percibido y satisfacción de usuarios que realizan actividades de forma libre. La población del estudio está conformada por 591 usuarios que realizan este tipo de actividad física en organizaciones deportivas públicas. Se realizó un análisis estadístico de los ítems, un análisis factorial exploratorio, un análisis factorial confirmatorio, pruebas de invarianza factorial, así como pruebas de fiabilidad y validez. Los resultados garantizan la validez y fiabilidad del instrumento para medir los juicios de valor de usuarios libres referentes a los tres constructos que conforman el cuestionario de análisis del servicio recibido, compuesto finalmente por 22 ítems agrupados en 8 dimensiones. Palabras clave. Usuarios libres, calidad, valor, satisfacción, validación. Abstract. Sport services have considerably evolved in recent years. There has been an increase in the type and number of activities and services offered to customers. At the same time, an increased number of clients of sports organizations do physical-sport activities on their own. Several instruments were created in order to assess customers’ evaluation of services received. However, none of them has been designed to specifically measure services appreciation of those customers who exercise without any support or presence of a sport specialist. The objective of this research was to validate an instrument that allows to analyze perceived quality, perceived values, and satisfaction from customers who workout on their own. The study population was composed by 591 customers of sport organizations who exercise on their own. Statistical analysis of items, Exploratory Factor Analysis, Confirmatory Factor Analysis, and factor invariance test were run, as well as reliability and validity were tested. Results demonstrated the validity and reliability of the instrument measuring free-use customers’ perception of the three constructs that constitute the questionnaire, whose final version was composed by 22 items grouped in 8 dimensions. Key words. Free-use users, quality, value, satisfaction, validation. Introducción La creciente preocupación por el conocimiento y valoración de la percepción que tienen los usuarios de los distintos servicios no es ajena al ámbito de las organizaciones deportivas, puesto que el mayor conocimiento de sus consumidores va a resultar de gran utilidad para mejorar la gestión de estos servicios (García-Fernández, Fernández-Gavira, & Velez-Colón, 2015; Martínez-Tur, Peiró, Ramos, & Tordera, 2000). Una mejor gestión del servicio pasa por una perfecta coordinación de las funciones, lo que se traduce en una mejor calidad y por tanto en una mayor valoración del servicio (Ramos-Carranza et al., 2015) El conocimiento de los clientes y de sus juicios es una de las vías principales para la mejora de la satisfacción de los usuarios (Afthinos, Theodorakis, & Nassis, 2005; Nuviala, Teva-Villén, Pérez-Ordás, Grao-Cruces, TamayoFajardo, & Nuviala, 2014; Westerbeek & Shilbury, 2003), lo que se traduce en un incremento de la lealtad y fidelización de los mismos (Calabuig, Burillo, Crespo, Mundina, & Gallardo, 2010; MartínezLemos & Romo-Perez, 2015). En los estudios sobre práctica deportiva realizados en los últimos años ha aparecido la tipología de usuarios que realizan actividades físico deportivas libres (no dirigidas por técnicos deportivos) en el ámbito de las organizaciones deportivas (Bernal, 2013; Nuviala, Grao-Cruces, Teva-Villén, Pérez-Turpin, Pérez-Ordás, & Tamayo-Fajardo, 2013a; Teva-Villén, Pérez-Ordás, Grao-Cruces, Tamayo-Fajardo, Nuviala, & Nuviala, 2014). A pesar de que existen numerosos autores que analizan los hábitos y preferencias de práctica físico deportiva (Alexandris, Zahariadis, Tsorbatzoudis, & Grouios, 2004; Dagger & Sweeney, 2006; García & Llopis, 2011; González, Martín, Jiménez, Campos, & Del Hierro, 2010; Infante, Axpe, Revuelta, & Ros, 2012; Moreno, Águila, & Borges, 2011; Martínez, Fernández, & Camacho 2010; Pavón & Moreno, 2006; Reverter & Barbany, 2007), muy pocos determinan o Fecha recepción: 04-12-15. Fecha de aceptación: 01-06-16 Alberto Nuviala anuvnuv@upo.es - 40 - referencian en sus análisis la actividad física libre o actividad física dirigida. Pavón y Moreno (2006) en un estudio, en el que utilizaron el cuestionario de motivaciones e intereses hacia las actividades físicodeportivas (M.I.A.F.D.), sobre las características de la práctica físicodeportiva en estudiantes universitarios, contemplaron en la variable «tipo de práctica», los ítems: «por mi cuenta solo; por mi cuenta con amigos; como actividad dirigida en un club; como actividad dirigida en un gimnasio»; lo cual demuestra el interés por diferenciar entre actividad física libre y actividad física dirigida. Castillo y Giménez (2011), en una investigación sobre los hábitos de práctica de actividad física del alumnado de la Universidad de Huelva, incluyen en su cuestionario cerrado de 7 dimensiones, dentro de la denominada «práctica físico deportiva», dos ítems referidos a qué tipo de actividad física practican y cómo la practican, con opciones de respuesta «actividad libre» y «por mi cuenta», aspectos que se pueden relacionar con actividad libre, a pesar de que no hay ninguna opción de respuesta como «actividad dirigida». González et al. (2010) utilizaron como técnica de investigación la entrevista estructurada, siendo la población objeto de estudio personas mayores, se les preguntó el modo de realización de la práctica física dentro de dos opciones de respuesta: «con presencia de profesor o sin presencia de profesor»; en otra de las cuestiones demandadas se les interrogó sobre el nivel de organización de la actividad física realizada, haciendo referencia a la práctica físico deportiva organizada o dirigida o por el contrario auto organizada, es decir de carácter libre. Castillo y Giménez (2011) ponen a la luz que la mayor parte de los alumnos universitarios por ellos estudiados (43.2%) practican actividad física libre frente a los que realizan actividad física reglada (19.7%). García y Llopis (2011) en la Encuesta de Hábitos Deportivos en España 2010 muestran, todavía con mayor claridad, esta tendencia, al poder comparar datos referentes a los últimos años de forma longitudinal. De hecho, son más del 75 % los que realizan actividad física no organizada y no competitiva en 2010, cuando en el año 2000 eran un 66 % y en 2005 un 68 %. Todo ello reafirma los resultados obtenidos en otros estudios realizados en esta misma línea de investigación, tanto a nivel Retos, número 31, 2017 (1º semestre) nacional como internacional (Pavón, Moreno, Gutiérrez, & Sicilia, 2004; De Figueiredo, 2005). Por lo que, teniendo en cuenta estos datos, puede darse como cierta la afirmación que realizan García y Llopis (2011) cuando dicen que en la actualidad tres de cada cuatro practicantes en España hacen deporte por su cuenta. Existen diversos instrumentos diseñados para evaluar la calidad y satisfacción por parte de los usuarios de los servicios deportivos como pueden ser el ideado por Afthinos et al. (2005) para usuarios de servicios de fitness griegos, el elaborado por Bodet (2006) para centros deportivos de fitness franceses, NEPTUNO diseñado por Calabuig, Quintanilla y Mundina (2008) para evaluar servicios náuticos, el ideado por Howat, Crilley y McGrath (2008) para analizar los centros públicos acuáticos australianos, QUESC creado por Kim y Kim (1995) para evaluar centros deportivos coreanos, el diseñado por Mañas, Jiménez, Muyor, Martínez y Moliner (2008) para un centro deportivo privado almeriense, ICPAF de Morales, Hernández-Mendo y Blanco (2005) dirigido a evaluar los servicios públicos de un municipio español, el creado por Sanz, Redondo, Gutiérrez y Cuadrado (2005) para evaluar la satisfacción en los practicantes de spinning, el Qsport-10, el diseñado por Rial, Varela, Rial y Real (2010) para usuarios de centros deportivos o EPOD2 de Nuviala, Grao-Cruces, Tamayo-Fajardo, Nuviala, Álvarez y Fernández-Martínez (2013b) que evalúa la calidad, valor y satisfacción de los usuarios de servicios públicos y privados, recreativos y/o competitivos. Ninguno de ellos está pensado y/o adaptado para usuarios de servicios deportivos que realizan actividades deportivas de forma libre, es decir, sin la presencia o dirección por parte de un técnico. Por ello, ante la falta de un instrumento adaptado para este tipo de clientes, que representan un número creciente e importante, es preciso crear o adaptar un nuevo instrumento que de forma específica valore el servicio deportivo recibido por parte de este nuevo grupo de usuarios. Se ha decido adaptar el cuestionario EPOD2 (Nuviala et al, 2013b) a este tipo de usuarios porque incluye los constructos calidad percibida, valor percibido y satisfacción, constructos relacionados con la lealtad de los usuarios (Murray & Howat, 2002; Theodorakis, Howat, Ko, & Avourdiadou, 2014) y porque está orientado a evaluar cualquier tipo de servicio deportivo. Por lo tanto, el objetivo de este trabajo se centra en validar el instrumento EPOD 2.1, así como constatar la fiabilidad del mismo como medio de evaluación de los servicios y organizaciones deportivas a través de los constructos calidad percibida, valor percibido y satisfacción, de los usuarios de actividades físico-deportivas libres. Método Participantes Los participantes de este estudio fueron 591 usuarios que realizaban actividad física no dirigida en servicios deportivos públicos de La Rioja. Hombres eran el 50.30% y el 49.70% mujeres, con una edad media de 38.02 ± 13.84 años. En cuanto al nivel socio cultural el 7.4% tiene estudio primarios, el 43% posee estudios secundarios y el 38.3% tenía estudios superiores. El tiempo medio de práctica diaria es de 73.58 ± 30.48 minutos y la frecuencia para el 58.50% es dos o tres veces por semana. La población participante en el estudio fue dividida en dos grupos de manera aleatoria con el fin de realizar estudios paralelos que permitieran corroborar y verificar los resultados obtenidos. El grupo 1 se conformó con 297 personas, de los cuales el 48.80% eran hombres y el 51.20% mujeres. La edad media era de 38.13 ± 14.08 años y el 40.40% poseían estudios superiores. El tiempo medio de práctica diaria es de 74.88 ± 29.93 minutos y la frecuencia para el 48% es dos o tres veces por semana. Por su parte el grupo 2 estuvo compuesto por 294 usuarios, 51.70% hombres y 48.30% mujeres, con una edad media de 37.90 ± 13.62 años, poseían estudios universitarios el 37.00%. El tiempo medio de práctica diaria es de 72.28 ± 31.01 minutos y la frecuencia para el 52% es dos o tres veces por semana. Retos, número 31, 2017 (1º semestre) Instrumento El instrumento utilizado fue el cuestionario EPOD2 (Nuviala et al., 2013b). Inicialmente está compuesto por 25 ítems de respuesta alternativa Likert, que oscilan de 1 (muy en desacuerdo) a 5 (muy de acuerdo). Se centra en tres áreas de evaluación: 1. Calidad percibida (20 ítems, 6 factores: técnicos, personal de servicios, material, espacios, comunicación y actividad) 2. Satisfacción (4 ítems, un solo factor) 3. Valor del servicio (1 ítem, un solo factor) Al cuestionario original se le quitaron un total de 3 ítems, uno de la dimensión técnicos (creo que el monitor/a o instructor/a adapta las clases a los intereses-necesidades de los clientes) y dos de la dimensión actividad (las actividades finalizan en el tiempo indicado; me ha resultado sencillo incorporarme en la actividad que participo). De la misma forma en el cuestionario se procedió a sustituir las expresiones técnicos deportivos por técnico de sala, obteniéndose de esta forma un instrumento conformado por un total de 22 ítems. Todo ello se realizó para conseguir que las escalas estuviesen adaptadas a usuarios que realizan actividades no dirigidas, libres. Procedimiento El trabajo de campo se realizó mediante un cuestionario autoadministrado con presencia del encuestador. Se solicitó a los participantes que lo cumplimentaran y que consultaran cualquier duda que tuvieran con los ítems. El tiempo invertido en la realización fue de unos 10 minutos. Antes de proceder a la recogida de datos, se pidió permiso a los responsables de las diferentes organizaciones que participaron en el estudio. De la misma forma todos los usuarios aceptaron participar voluntariamente en el estudio. Análisis de datos El análisis se realizó en dos fases. En la primera se efectuó un análisis estadístico de los ítems y posteriormente un análisis factorial exploratorio. En la segunda se ejecutó un análisis factorial confirmatorio y de invarianza factorial, con el fin de obtener una prueba que presente las mejores propiedades para la conformación de la valoración de la calidad percibida y satisfacción de los usuarios libres de centros deportivos. Para determinar el número mínimo de factores comunes capaces de reproducir las correlaciones observadas entre los ítems del instrumento, se realizaron sendos análisis factoriales exploratorios en los grupos 1 y 2, a partir del método de factores principales mediante el procedimiento de rotación varimax. Mediante el coeficiente α de Cronbach se estimó la consistencia interna para cada factor retenido como una medida de su fiabilidad. El Análisis Factorial Confirmatorio (AFC) se realizó con el programa AMOS 20.1. El método de estimación empleado fue el de Máxima Verosimilitud. Para evaluar la bondad del ajuste se revisaron los siguientes indicadores: estadístico chi-cuadrado (x2); la razón entre x2 y el número de grados de libertad (x2/gl); índices de ajuste de carácter absoluto: GFI, RMR y RMSEA; índices de ajuste incremental: TLI, CFI e IFI. Todo ello se realizó en las dos poblaciones. A continuación se calculó la invarianza factorial con el objeto de comprobar la estabilidad del modelo en diferentes poblaciones. Finalmente se calculó la fiabilidad del instrumento resultante mediante el coeficiente alfa de Cronbach, así coma la validez convergente. Resultados Análisis estadístico de los ítems En la Tabla 1 se muestran los estadísticos descriptivos de los ítems, tanto de la escala referente a calidad percibida como a satisfacción. Se puede observar que para los ítems de ambas escalas la escala, en los dos grupos, los índices de asimetría y curtosis son próximos al valor cero y por debajo del valor 1.96, lo que indica semejanza con la curva normal. Estos resultados permiten la utilización de técnicas factoriales que se - 41 - Tabla 1. Media (M), desviación típica (DT), asimetría, curtosis, correlación ítem-total (R IT-c) y alfa si algún ítem es eliminado (α sin ítem). Grupo 1 Grupo 2 α sin α sin M D.T. Asimetría Curtosis R IT-c M D.T. Asimetría Curtosis R IT-c ítem ítem Calidad percibida del servicio 1. Estoy contento/a con el trato recibido por 3.83 .96 -.293 -.578 .612 .878 3.92 .97 -.488 -.456 .485 .894 el técnico de sala. 2. Creo que presta una atención adecuada a los problemas de los usuarios-alumnos desde 3.63 .98 -.148 -.627 .595 .879 3.78 1.00 -.375 -.534 .549 .892 el primer día. 3. Considero que el técnico de sala anima 3.53 1.05 -.120 -.645 .548 .881 3.57 1.14 -.178 -.914 .497 .894 suficientemente. 4. Los vestuarios están suficientemente 3.56 .98 -.432 -.091 .504 .882 3.69 .96 -.424 -.170 .571 .891 limpios. 5. Los vestuarios son lo suficientemente 3.11 1.14 .084 -.894 .444 .886 3.13 1.12 -.063 -.734 .419 .897 amplios. 6. Las instalaciones están suficientemente 3.63 .97 -.458 .056 .552 .880 3.74 .93 -.593 .448 .618 .889 limpias. 7. Se dispone de suficiente material para las 3.16 .97 -.039 -.286 .492 .883 3.34 .96 -.069 -.036 .606 .890 actividades. 8. El material está en condiciones óptimas 3.27 .92 -.070 -.187 .506 .882 3.41 .93 .036 -.236 .606 .890 para su uso. 9. El material es moderno. 3.16 .93 -.052 .037 .448 .884 3.35 .91 -.032 -.188 .555 .892 10. La actividad que realizo es amena. 3.66 .86 -.033 -.149 .574 .880 3.80 .80 .207 -1.114 .575 .891 11. Las tareas que desarrollo en el entrenamiento son lo suficientemente 3.65 .90 -.161 -.374 .534 .881 3.61 .89 -.055 -.220 .564 .891 variadas. 12. Con esta actividad obtengo los resultados 3.77 .82 -.038 -.539 .529 .882 3.81 .86 -.113 -.544 .561 .891 que esperaba. 13. Disponen las instalaciones de algún .86 .368 .942 .386 .886 3.19 .92 -.068 .406 .471 .894 medio para transmitir sus sugerencias (buzón 3.00 de sugerencias. tablón de anuncios). 14. La información sobre las actividades que 3.30 .84 .092 .179 .528 .881 3.46 .82 .264 -.126 .587 .891 se desarrollan en el centro es adecuada. tante está conformada por un solo factor en ambos grupos, que explica un 89.71% de la varianza del grupo 1 y del 90.50% del grupo 2. La fiabilidad fue de .961 para el grupo 1 y de .964 para el grupo 2. Análisis factorial confirmatorio e invarianza factorial Los parámetros fueron estimados mediante el método de máxima verosimilitud. En la Tabla 3 se recoge la información proporcionada por los índices de ajuste utilizados para las dos escalas que conforman el cuestionario EPOD 2.1, en los dos grupos, obteniendo resultados satisfactorios. Posteriormente se analizó la invarianza de esta estructura factorial a través del análisis multigrupo. Se trataba de com15. La oferta de actividades/servicios se 3.26 .88 -.010 .180 .533 .881 3.37 .86 .395 -.305 .527 .892 actualiza. probar que no hubiera diferen16. El trato del personal de la instalación es 3.80 .91 -.237 -.489 .628 .878 3.81 .90 -.077 -.817 .629 .889 cias significativas entre un modeagradable. 17. Hay buena relación entre el personal de lo sin invarianza y diferentes 3.70 .90 -.014 -.523 .623 .878 3.74 .86 .109 -.683 .641 .889 la instalación. modelos con invarianza en alguSatisfacción 18. Haber elegido esta organización ha sido nos parámetros. 3.91 .86 -.217 -.605 .875 .952 3.93 .84 .016 -1.391 .913 .953 una buena decisión. En las Tablas 4 y 5 aparecen 19. Estoy conforme por haberme 3.89 .87 -.239 -.443 .931 .935 3.92 .83 .107 -1.496 .935 .947 matriculado/inscrito en esta organización. los índices de ajuste para los cua20. Tuve una buena idea al decidir tro modelos comparados en el incorporarme a realizar actividades 3.92 .89 -.334 -.441 .923 .938 3.93 .83 .017 -1.346 .929 .948 deportivas en esta organización. análisis de invarianza en los mo21. Estoy complacido por haberme 3.84 .92 -.306 -.356 .864 .956 3.84 .89 -.144 -.694 .873 .965 matriculado en esta organización delos de calidad percibida y saValor tisfacción. En ambos análisis se 22. Estoy satisfecho/a con la relación 3.74 1.04 3.66 .98 calidad/precio encontraron diferencias significativas en chi cuadrado entre el modelo Modelo 1 y el Modelo realizarán a continuación. La fiabilidad de la escala de calidad percibida 4. Es necesario añadir que el Δ CFI entre los modelos es muy pequeño evaluada con alfa de Cronbach es de .888 para el grupo 1 y de .897 para por lo que se puede sugerir que la estructura de los dos modelos para el 2. La consistencia interna de la escala de satisfacción es .961 para el ambos grupos es invariante. grupo 1 y de .964 para el grupo 2. Análisis de la fiabilidad Análisis de la estructura interna La fiabilidad el instrumento resultante que estudia la calidad percibida Para conocer la estructura factorial de la escala de calidad percibida, Tabla 3. Indicadores de ajuste y error del análisis factorial confirmatorio. se realizó un análisis factorial exploratorio sobre los 17 ítems resultantes Escala RMR RMSEA GFI IFI TLI CFI x2 Gl x2/gl del análisis estadístico de los ítems por el método de extracción de Calidad .037 .042 .943 .981 .974 .980 157.621 104 1.516 Percibida Grupo 1 componentes principales y posterior rotación Varimax. Antes de realiSatisfacción .047 .068 .978 .994 .990 .994 13.138 4 3.284 zar el análisis, se calculó la medida de adecuación muestral de KaiserCalidad .040 .047 .937 .977 .970 .977 172.111 104 1.655 Meyer-Olkin (KMO) y el test de esfericidad de Bartlett. Para el grupo Percibida Grupo 2 1, el índice KMO mostró un valor de .831 y el test de Bartlett resultó Satisfacción .020 .022 .989 .998 .998 .998 6.471 4 1.618 estadísticamente significativo (x2136 =2805.590; p<.001). El grupo 2 Tabla 4. obtuvo los siguientes resultados, KMO mostró un valor de .853 y el Estadísticos de ajuste para los modelos de calidad percibida. Comparación entre modelos usando 2 el modelo 1 como correcto. test de Bartlett resultó estadísticamente significativo (x 136 =3056.520; Dif. Dif x2 Gl x2/gl CFI RMSEA RMR P Modelo x2 GL p<.001), lo que llevó a concluir que la aplicación del análisis factorial Modelo 1 329.732 208 1.585 .979 .032 .039 resultaba pertinente en ambos casos. La estructura dimensional resulModelo 2 347.192 219 1.585 .978 .032 .041 11 17.460 .095 Modelo 3 369.290 240 1.539 .977 .030 .049 32 39.558 .168 tante está conformada, para los dos grupos, por seis factores (técnicos, Modelo 4 407.370 257 1.585 .974 .032 .049 49 77.637 .006 personal de servicios, comunicación, actividad, material y espacios) que Modelo 1 que no tiene restricciones de ningún tipo. Modelo 2 tiene restricciones en el peso de medida. Modelo 3 tiene restringidos los pesos de medida y covarianzas. Modelo 4 tiene conjuntamente explican un 78.38% de la varianza para el grupo 1 y restricciones en los pesos de medida, covarianzas y residuos de medida. 80.10% para el grupo 2 (Tabla 2). Tabla 5. Estadísticos de ajuste para los modelos de satisfacción. Comparación entre modelos usando el Se realizó el mismo procedimiento para los cuatro ítems de la escala modelo 1 como correcto. de satisfacción. El índice KMO mostró un valor de .843 y el test de CFI RMSEA RMR Dif. Dif x2 Modelo P Gl x2/gl x2 GL 2 Bartlett resultó estadísticamente significativo (x 6 =1407.948; p<.001) Modelo 1 4.456 2 2.228 .999 .046 .003 Modelo 2 8.052 5 1.610 .999 .032 .016 3 3.596 .309 para el grupo 1. Mientras que, para el grupo 2 el índice KMO mostró un Modelo 3 8.467 6 1.411 .999 .026 .023 4 4.011 .405 valor de .859 y el test de Bartlett resultó estadísticamente significativo Modelo 4 21.591 11 1.963 .996 .040 .029 9 17.135 .047 Modelo 1 que no tiene restricciones de ningún tipo. Modelo 2 tiene restricciones en el peso de 2 (x 6 =1498.079; p<.001) lo que llevó a concluir que la aplicación del medida. Modelo 3 tiene restringidos los pesos de medida y covarianzas. Modelo 4 tiene restricciones en los pesos de medida, covarianzas y residuos de medida análisis factorial resultaba pertinente. La estructura dimensional resul- 42 - Retos, número 31, 2017 (1º semestre) Tabla 2. Estructura factorial rotada, comunalidades, autovalores, alfa de Cronbach y porcentaje de varianza explicada por cada factor. Grupo 1 1 2 3 4 5 Grupo 2 6 Extracción 1 2 3 4 5 6 Extracción 1. Estoy contento/a con el trato .849 recibido por el técnico de sala. .842 .904 .867 2. Creo que presta una atención adecuada a los problemas de .900 los usuarios-alumnos desde el primer día. .893 .862 .849 3. Considero que el técnico de sala anima suficientemente. .847 .898 .856 4. Los vestuarios están suficientemente limpios. .875 demuestra la existencia de este tipo de validez. Una segunda evidencia de validez convergente se determina con el índice de fiabilidad y el promedio de varianza extractada AVE. Los valores obtenidos son, para la fiabilidad, > .6, y para el AVE, > .5 (Tabla 6). Discusión .868 .817 .807 .804 Los resultados de este trabajo apoyan la validez y fiabilidad 6. Las instalaciones están .802 .762 .749 .798 suficientemente limpias. del EPOD 2.1 como instrumen7. Se dispone de suficiente to adecuado para aplicarlo en la .824 .757 .742 .712 material para las actividades. valoración de la calidad, valor y 8. El material está en condiciones óptimas para su .888 .849 .879 .875 satisfacción de usuarios deportiuso. vos que realizan actividades por 9. El material es moderno. .836 .764 .867 .834 su cuenta. Se ha seguido el proce10. La actividad que realizo es .792 .748 .785 .727 amena. dimiento establecido por Carre11. Las tareas que desarrollo tero-Dios y Pérez (2005). En prien el entrenamiento son lo .810 .750 .817 .769 suficientemente variadas. mer lugar se llevó a cabo el análi12. Con esta actividad obtengo sis estadístico de los ítems con el .828 .761 .826 .776 los resultados que esperaba. objeto de excluir aquellos que no 13. Disponen las instalaciones de algún medio para transmitir cumpliesen con los criterios que sus sugerencias (buzón de .791 .653 .818 .736 permitirían realizar análisis sugerencias. tablón de anuncios). factoriales posteriores. La primera 14. La información sobre las condición de selección fue que los actividades que se desarrollan .830 .787 .806 .801 en el centro es adecuada. valores de asimetría y curtosis 15. La oferta de deben estar próximos al valor actividades/servicios se .803 .747 .800 .744 cero y por debajo del valor 1.96, actualiza. 16. El trato del personal de la lo que indica semejanza con la .834 .889 .838 .891 instalación es agradable. curva normal. A continuación, 17. Hay buena relación entre el .834 .888 .812 .874 para conservar un ítem se valoró personal de la instalación. % Varianza explicada 15.28 14.01 13.70 13.11 12.51 9.76 78.38 15.73 14.48 13.73 13.72 12.68 9.75 80.10 que el coeficiente de correlación Autovalor 6.19 1.93 1.66 1.42 1.21 1.09 6.61 2.22 1.48 1.27 1.18 1.00 corregido ítem-total que fuese un Alfa de Cronbanch .913 .769 .856 .836 .806 .884 .888 .912 .808 .871 .838 .828 .874 .897 valor mayor o igual a .35 (Cohen & Manion, 2002; Nurosis, 1993). es .870. Para los factores resultantes oscila entre .913 (técnicos) y .705 El resultado fue la no eliminación de ningún ítem de las escalas propues(material). La fiabilidad para la escala de satisfacción es .961 (Tabla 6). tas. La fiabilidad de las mismas, en ambos grupos, se midió con el alfa de Cronbach obteniéndose unos valores excelentes. Validez convergente Se procedió a valorar la estructura interna mediante un análisis La validez convergente fue calculada por los coeficientes de correfactorial exploratorio tomando como base el criterio de Kaiser (Costello lación de Pearson entre la puntuación de la calidad percibida del servicio, & Osborne, 2005), mediante el procedimiento de rotación varimax, a la satisfacción y el valor del servicio. El resultado muestra una correlapesar de que se aconseja para casos en que los factores no están relacioción significativa entre los factores que componen el instrumento, lo que nados. Se optó por éste debido al interés teórico por separar, en la medida de lo posible, los factores resultantes, a pesar de Tabla 6. constatar la relación de los factores (Carretero-Dios & Pérez, Factores, ítems por factor y media y desviación típica. Correlaciones entre los factores de la EPOD 2.1 y consistencia 2007). El resultado en la escala de calidad percibida fue la interna (en la diagonal). Calidad percibida 7. Satisfacción 8. Valor percibido extracción de seis factores (técnicos, personal de servicios, Escala Factor Ítems M 1 2 3 4 5 6 7 8 AVE FC comunicación, actividad, material y espacios) lo que ha per1 mitido comprobar y reforzar la configuración de la escala de 1. Técnicos 2 3.71±.94 (.913).266** .282** .398** .244** .448** .451** .218** .78 .91 3 acuerdo al modelo teórico propuesto inicialmente por Nuviala 4 2. Espacios 5 3.49±.96 (.709) .361** .378** .329** .401** .404** .291** .59 .81 et al. (2013b), modelo similar al que propone Gálvez-Ruiz y 6 Morales-Sánchez (2015) quienes proponen un modelo de 7 3. Material (.705) .308** .339** .309** .379** .236** .69 .81 8 3.31±.92 calidad con cinco dimensiones entre las que no se incluye el Calidad 9 percibida personal de servicios. Todo ello con una varianza explicada 10 4. Actividad (.836) .404** .477** .527** .269** .63 .83 11 3.72±.74 de alrededor del ochenta por ciento en ambos grupos. Luego 12 13 mediante el coeficiente α de Cronbach se estimó la consisten5. (.819) .466** .483** .328** .61 .78 14 3.26±.74 Comunicación cia interna para cada factor retenido como una medida de su 15 16 6. P. Servicios 3.76±.84 (.879) .627** .367** .52 .68 fiabilidad (Elosua & Zumbo, 2008; Nunnally & Bernstein, 17 18 1995), estando comprendida su fiabilidad entre .769 y .913. 19 7. Satisfacción 3.90±.82 (.961) .491** .89 .97 Para la escala de satisfacción se realizó el mismo proceso Satisfacción 20 21 obteniendo como resultado la extracción de un solo factor Valor 8. Valor 22 3.70±1.01 percibido percibido que explicaba el noventa por ciento de la varianza, con ** Correlación significativa al nivel p < .01 (bilateral). fiabilidades próximas a uno en ambos grupos. AVE: Análisis Varianza Extraída 5. Los vestuarios son lo suficientemente amplios. .681 .571 .809 .702 FC: Fiabilidad Compuesta Retos, número 31, 2017 (1º semestre) - 43 - Debe destacarse que en ambas escalas y en los dos grupos, la varianza explicada supera el 60%, límite teórico establecido como límite inferior en ciencias sociales (Hair, Anderson, Tatham, & Black, 2004; Henson & Roberts, 2006). Resultado que puede considerarse muy positivo al superar ese límite teórico de forma amplia. A continuación, para comprobar la estructura factorial de las escalas, calidad percibida y satisfacción, se llevó a cabo un análisis factorial confirmatorio. Los parámetros fueron estimados mediante el método de máxima verosimilitud, siguiendo la recomendación de Thompson (2004). Para evaluar la adecuación de los modelos sometidos a prueba (modelo extraído del análisis factorial exploratorio) se optó por la valoración conjunta de un grupo de índices. Fueron seleccionados algunos de los índices de ajuste más utilizados, considerándose aceptables valores en el caso del GFI, IFI, TLI y CFI, por encima de .90 y en el caso del RMR y RMSEA, entre .05 y .08. Por su parte, los valores y en el cociente entre x2 y los gl, un modelo considerado perfecto su valor sería de 1.00 y las ratios por debajo de 2.00 se considerarán como indicadores de un muy buen ajuste del modelo, mientras que valores por debajo de 5.00 son considerados como aceptables (Hu & Bentler, 1999). Los modelos estudiados, integrados por 17 ítems la escala de calidad percibida y 4 ítems la escala de satisfacción, presentaron unos índices de ajuste correctos, mostrando índices en conformidad con los valores críticos, respectivamente, más de .90 por AGFI, GFI, CFI y valores por debajo de .08 para el RMR y RMSEA (Bollen, 1990; MacCallum, Widaman, Preacher, & Hong, 2001; Yuan, 2005). Todos los índices, excepto RMSEA y x2/gl, en la escala de satisfacción para el grupo 1, han mostrado valores excelentes siguiendo el criterio establecido por SchermellehEngel, Moosbrugger y Müller (2003), lo que demuestra un más que aceptable ajuste de los modelos. Se analizó la invaianza de la estructura factorial a través del análisis multigrupo en ambos grupos siguiendo las recomendaciones de Abalo, Lévy, Rial y Varela (2006). Se trataba de comprobar que no hubiera diferencias significativas entre un modelo sin invarianza y diferentes modelos con invarianza en algunos parámetros. El primer grupo estaba compuesto por 297 usuarios con una edad media de 38.13 ± 14.08, de los cuales el 48.80% eran hombres, y el segundo por 294 con una edad media de 37.90 ± 13.62 años, siendo el 51.70% hombres. Se encontraron diferencias significativas en chi cuadrado entre el modelo sin restricciones (Modelo 1) y el modelo que tiene restricciones en los pesos de medida, covarianzas y residuos de medida (Modelo 4). No obstante, dado que el coeficiente chicuadrado es sensible al tamaño de la muestra, se empleó también el criterio establecido por Cheung y Rensvold (2002) respecto al ÄCFI. Según estos autores, valores de ÄCFI inferiores o iguales a -.01 indican que no se puede rechazar la hipótesis nula de la invarianza. Los valores de ÄCFI encontrados en este estudio en la comparación del modelo sin restricciones con el resto de modelos sugieren que la estructura factorial de la escala de calidad percibida y de satisfacción para usuarios de servicios deportivos por libre (EPOD 2.1) es invariante. La validez convergente se determinó por las correlaciones entre los factores de EPOD 2.1 a través del coeficiente de Pearson. Las correlaciones entre ellos son positivas, moderadas y están significativamente relacionadas, lo que demuestra este tipo de validez, ya que los resultados manifiestan que se trata de constructos similares pero conceptualmente diferentes. Siguiendo el criterio de Luque (2000), por el cual ninguna de las correlaciones es mayor que .9, viene a corroborarse la existencia de este tipo de validez. Una segunda prueba de validez convergente del instrumento viene determinada con el índice de fiabilidad y el promedio de varianza extractada AVE. Los valores aceptables son, para la fiabilidad, > .6 y, para el AVE, > .5 (Fornell & Larcker, 1981; Baggozi & Yi, 1988). En conclusión, los resultados permiten presentar una herramienta capaz de evaluar la calidad percibida, valor percibido y satisfacción del servicio que prestan las organizaciones deportivas a usuarios que realizan actividades no dirigidas, por su cuenta, de forma sencilla y breve, contemplando las diferentes dimensiones que conforman la prestación de los servicios deportivos. Tras los análisis factoriales confirmatorios - 44 - se ha obtenido un cuestionario reducido compuesto por ocho dimensiones, seis relativas a calidad percibida, con 17 ítems, una referente al valor percibido, 1 ítem, y una sobre satisfacción compuesta por cuatro ítems, con unas grandes proporciones de varianza explicado, con fiabilidades altas. 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Defence mechanisms: the role of physiology in current and future environmental protection paradigms
Conservation physiology
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Chris N. Glover1,2,* 1Faculty of Science and Technology and Athabasca River Basin Research Institute, Athabasca University, Canada 2Department of Biological Sciences, CW 405, Biological Sciences Bldg. University of Alberta Edmonton, Alberta, Canada T6G 2E9 *Corresponding author: 1 University Drive, Athabasca, Alberta, Canada T9S 3A3. Tel: +(587) 985 8007. Email: cglover@athabascau.ca Ecological risk assessments principally rely on simplified metrics of organismal sensitivity that do not consider mechanism or biological traits. As such, they are unable to adequately extrapolate from standard laboratory tests to real-world set- tings, and largely fail to account for the diversity of organisms and environmental variables that occur in natural environ- ments. However, an understanding of how stressors influence organism health can compensate for these limitations. Mechanistic knowledge can be used to account for species differences in basal biological function and variability in envir- onmental factors, including spatial and temporal changes in the chemical, physical and biological milieu. Consequently, physiological understanding of biological function, and how this is altered by stressor exposure, can facilitate proactive, predictive risk assessment. In this perspective article, existing frameworks that utilize physiological knowledge (e.g. biotic ligand models, adverse outcomes pathways and mechanistic effect models), are outlined, and specific examples of how mechanistic understanding has been used to predict risk are highlighted. Future research approaches and data needs for extending the incorporation of physiological information into ecological risk assessments are discussed. Although the review focuses on chemical toxicants in aquatic systems, physical and biological stressors and terrestrial environments are also briefly considered. Adverse outcomes pathway, biotic ligand model, metabolism, risk assessment, toxicokinetics, toxicodynam Key words: Adverse outcomes pathway, biotic ligand model, metabolism, risk assessment, toxicokinetics Volume 6 • 2018 10.1093/conphys/coy012 Perspective Volume 6 • 2018 10.1093/conphys/coy012 Perspective 10.1093/conphys/coy012 1 © The Author(s) 2018. Published by Oxford University Press and the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction to assessing threats to the environment. For example, eco- logical risk assessment seeks to determine whether a given chemical, physical (i.e. climatic, land-use or hydrological change) or biological (e.g. invasive species or disease) stressor will result in a negative ecological outcome. Historically, environmental risk has been largely determined by monitoring the toxicity of stressors to individual model species, in controlled laboratory settings, using crude metrics such as mortality (Maruya et al., 2016). These data have then been extrapolated Physiology provides the mechanism that underpins our under- standing of ecology, evolution, health and disease. It also facil- itates the translation of molecular and cellular responses of individual organisms, to changes observed at the population, community and ecosystem scale (Somero, 2000). As such there is significant crossover between physiology as a discip- line, and the goals of conservation, particularly as they pertain 1 © The Author(s) 2018. Published by Oxford University Press and the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Conservation Physiology • Volume 6 2018 Perspective to discern levels of stressors that will have no, or only a minor, environmental impact. As such, risk assessments take individual-level impacts, which result from suborganismal per- turbation, and seek to translate these effects to larger scales (Rohr et al., 2016). It is, therefore, surprising that environmen- tal risk assessment has traditionally eschewed physiological approaches that would appear to contribute directly to regula- tory goals. For example, mechanistic knowledge of how a stressor impacts an organism facilitates predictive, and thus proactive, approaches to assessing risk; it allows an under- standing of processes such as acclimation that could affect stressor impact; and it enables evaluation of risk under real- world scenarios where multiple stressors are simultaneously acting upon an organism. Hence, physiological knowledge can provide the basis for versatile and robust risk assessment tools that utilize an understanding of stressor impacts at an individ- ual level, but which are applicable to the prediction of effects across biomes, across species, and in the presence of multiple stressors (Segner et al., 2014). The current Perspective initially outlines traditional risk assessment paradigms, highlighting that these approaches generate values that have a limited mechanistic basis. Species sensitivity distributions A more integrative tool for assessing risk is the species sensi- tivity distribution (SSD). Here, end-point values for multiple species are combined, a distribution is fitted to the data, and a hazard concentration is determined (e.g. a stressor concen- tration that protects 95% of tested species; Wheeler et al., 2002). As such, SSDs contribute directly to risk assessment goals (e.g. protect 95% of the species, 95% of the time; van Straalen and van Rijn, 1998). While SSDs are clearly more robust than single end-point values alone (Hahn et al., 2014), they suffer from similar criticisms. For example, stud- ies are still performed in a relatively few species, and under standard test conditions. Furthermore, the outcomes of SSD analyses depend on the number of data points used to con- struct them, and the nature of the distribution fitted (Forbes and Calow, 2002). Some SSD approaches do normalize end- point values on the basis of bioavailability (van Sprang et al., 2016), and as bioavailability is driven by organismal physi- ology, then such analyses do incorporate mechanistic under- standing. However, while SSDs can show that differences in sensitivity between species exist, and can identify which of the test species are more sensitive, the mechanisms under- lying these differences are not directly investigated. Regulatory background Most current risk assessment practices are designed with a focus on the stressor. In the case of anthropogenic toxicants, approaches seek to determine the ‘safe’ concentration of a given chemical, and give little consideration to the biological receptor (Maruya et al., 2016; Burton, 2017). Traditional risk assessment is also characterized by its reductionist approach, condensing toxicological information down to a single value or set of values (Jager, 2016). This simplification is understandable given the ever-growing complexity of the chemical milieu, the variety of human-induced alterations in physicochemical properties of ecosystems, and the diverse nature of these ecosystems and the biota therein. Introduction It then provides a generic overview of more modern approaches that have the potential to incorporate mechanistic information, and predictive capacity, into risk assessment frameworks. Thereafter, selected case studies illus- trating how mechanistic information has been utilized within the context of the stressor-organism nexus are outlined. Finally, opportunities for future physiologically based research efforts that could contribute significantly to an improved understanding of the drivers of toxicological impact are identi- fied. Overall, the aim of this work is to offer insight into the regulatory environment from a physiological perspective, and to identify where physiological knowledge has, and could fur- ther, contribute to risk assessment approaches. stipulated by a regulatory body, and is almost invariably a standard toxicological model. These are organisms that are amenable to laboratory culture, but which may hold little relevance to an environmental scenario (Segner and Baumann, 2015). These tests, conducted over acute or chronic timeframes, generate values that summarize the sen- sitivity of the species towards a stressor. When examining chemical toxicants, these values include the median lethal concentration (LC50), and the lowest tested concentration at which no (no observable adverse effect concentration; NOEC) or some (lowest observable adverse effect concentra- tion; LOEC) effect is noted. Sometimes, rather than mortal- ity, an alternative end-point may be used (median effect concentration; EC50), such as growth in assessments of algal toxicity, or reproduction in chronic assays. Measures of sur- vival, growth and reproductive capacity are all derivations of the interaction of the stressor with organism, and thus reflect physiological impairment. However, these simple end-points do not consider at all the mechanism by which species sur- vival/function has been compromised (Chapman et al., 1998). As such there is no understanding of why end-point values may differ between species, life-stages or populations, and thus there is little predictive power in such metrics. Single end-point values The weaknesses in simple end-point analyses and SSD approaches have been recognized for some time (Forbes and Calow, 2002). For example, a contributing factor to the per- ceived failure of pesticide risk assessments in tropical regions of Australia is that SSD methods resulted in trigger values significantly greater than concentrations at which sub-lethal The standard approach to assessing the environmental risk of a stressor is laboratory toxicity testing (Chapman et al., 1998). Taking a single species, toxicity tests over fixed time intervals are performed under standard conditions (e.g. tem- perature, water chemistry). The choice of species is often ................................................................................................................................ ................................................................................................................................. 2 .............................................................................................................................................................. Conservation Physiology • Volume 6 2018 Perspective Conservation Physiology • Volume 6 2018 Perspective Water chemistry Physiology of absorption Physiology of effect Molecular Initiating Event Toxicity Community, population, ecosystem effect Number and properties of available transporters Mucus secretion and unstirred water layer effects Epithelial reductases Membrane fluidity Bioreactivity Metabolism, detoxification and sequestration Characteristics of impacted pathways BLM AOP MEM Organismal physiology Figure 1: Diagrammatic representation of modelling frameworks (BLM, biotic ligand models; AOP, adverse outcome pathways; MEM, mechanistic effect models) incorporating physiological considerations for the purposes of risk assessment. Figure 1: Diagrammatic representation of modelling frameworks (BLM, biotic ligand models; AOP, adverse outcome pathways; MEM, mechanistic effect models) incorporating physiological considerations for the purposes of risk assessment. longer-term (e.g. 96 h) toxic effect (e.g. mortality, inhibition of ion uptake), to determine whether the concentration of a metal in a given water body is likely to be harmful (Di Toro et al., 2001). Consequently, the BLM approach relies abso- lutely on physiological understanding of uptake pathways and mechanism of toxicity. Importantly, because of its mech- anistic underpinning, this is a model that has been shown to have relevance in different environmental matrices (e.g. saline waters, Arnold et al., 2005; sediments, Di Toro et al., 2005; soils, An et al., 2012), and has been applied to the complex issue of stressor mixtures (Iwasaki et al., 2015). Because of robust validation, and its capacity for predicting toxicity proactively, the BLM approach has been approved as an acceptable regulatory tool in many jurisdictions world- wide (Rüdel et al., 2015). effects may be induced (Holmes, 2014). This flaw would likely have been mitigated by a better understanding of bio- logical mechanisms of pesticide impact. Single end-point values Consequently, driven by the need for risk assessment methods that integrate bio- logical knowledge, and which facilitate predictive, proactive assessment, a number of mechanism-based tools that incorp- orate physiological measurements have been proposed/devel- oped. These include biotic ligand models (BLM), adverse outcome pathways (AOP) and a variety of mechanistic effects models (MEM). While current BLMs are specific for chemical toxicants, AOP and MEM approaches are applic- able to a broader range of stressors. The general framework of each of these tools, and their integration of physiological information, is outlined in Fig. 1, and discussed below. Biotic ligand model Recent work, seeking to expand the BLM approach from standard North American fish species to key Southern Hemisphere fish, has shown that while the mechanisms of effects are generally conserved, there are important differ- ences. For example, Galaxias maculatus, a widely distributed southern temperate fish species, has a scaleless skin surface which performs a number of physiological roles (Glover et al., 2013), including ion transport (Harley, 2015). It has been proposed that the skin of these species may serve both as an alternative pathway of toxicant uptake, but also as a rescue pathway, taking on physiologically important func- tions that may be compromised at the gill of metal-exposed fish (Glover et al., 2016; McRae et al., 2016). This observa- tion does, however, further serve to highlight the importance of physiological knowledge in risk assessment, particularly when applying guidelines across biomes. Figure 2: Growth in peer-reviewed journal publications incorporating physiological considerations for the purposes of risk assessment, from 2000 to 2016 (ISI Web of Science). It is also worth noting that although BLM approaches have been adopted by regulatory authorities worldwide, they are not always employed in the manner for which they were designed. The BLM approach was developed to assess acute toxicity, but is often used as a predictor of chronic toxicity (Niyogi and Wood, 2004). This is a practice that assumes mechanisms of toxicity, and the relationships between accu- mulation and effect, are independent of exposure duration. This assumption remains unverified for many toxicants (Niyogi and Wood, 2004). Furthermore, chronic BLMs are often developed by implying, rather than measuring, a toxi- cant burden at the sensitive site (Villavicencio et al., 2011). If coupled with methodologies where the chronic toxicity itself is a calculation based on acute-to-chronic extrapolation (USEPA, 2007), then this has the potential to greatly dimin- ish the mechanistic basis of the BLM approach. impacts of stressor mixtures (Kienzler et al., 2016). To date, AOPs have been postulated for a number of important envir- onmental contaminants (e.g. nanoparticles, Muller et al., 2015; metals, Brix et al., 2017; endocrine disrupting chemicals, Song et al., 2017). The AOP approach shares many weaknesses with the BLM, including the assumption that the AOP for a given stressor in one species is likely to be applicable to others, and a limited scope to account for acclimation effects (Rohr et al., 2016). Biotic ligand model There are, however, acknowledged weaknesses to the BLM approach. For example, it does not account easily for toxicants sourced from the diet (Niyogi and Wood, 2003). The diet is the major route of metal exposure, but metals absorbed by this pathway can have distinct bioavailability and bioreactivity owing to the presence of absorbable toxicant-organic ligands, thus distorting the relationship between body burden and toxicity. Current BLMs are also limited in that they do not account for acclimation, wherein the uptake route and/or the toxicologically sensitive pathway change in response to prolonged and/or prior exposure (Niyogi and Wood, 2003). One further important assump- tion of the BLM is that mechanisms of uptake and toxicity are conserved between life-stages and species. However, test- ing of these assumptions has focussed on a relatively narrow The BLM approach is based on an understanding of the rela- tionship between water chemistry, toxicant speciation, crit- ical tissue burden, and mechanisms of toxicant uptake and effect (Di Toro et al., 2001; Niyogi and Wood, 2004). The best developed BLMs are those applied to assessing metal toxicity in freshwaters. Based on physiological studies that show metal toxicants are mimics of essential ions, the BLM approach uses water chemistry to establish the amount of bioavailable metal (i.e. that which is ionic and thus capable of being absorbed through epithelial transporters; Bury et al., 2003). This information is then coupled with experi- mental analysis of the relationship between bioavailability, the short-term (e.g. 3 h) accumulation of metal at a sensitive site (gills of fish, or whole bodies of small invertebrates), and ................................................................................................................................ 3 Conservation Physiology • Volume 6 2018 Perspective Year 2000 2002 2004 2006 2008 2010 2012 2014 2016 Number of publications per year 0 20 40 60 80 100 120 140 Biotic ligand model Adverse outcome pathway Mechanistic effect model Figure 2: Growth in peer-reviewed journal publications incorporating physiological considerations for the purposes of risk assessment, from 2000 to 2016 (ISI Web of Science). Year 2000 2002 2004 2006 2008 2010 2012 2014 2016 Number of publications per year 0 20 40 60 80 100 120 140 Biotic ligand model Adverse outcome pathway Mechanistic effect model range of test species, a legacy of standard toxicity tests. Adverse outcome pathway Another mechanistic approach to risk assessment is the AOP framework (Ankley et al., 2010), which has received signifi- cant attention from the research community since its concep- tion (Fig. 2). An AOP seeks to build mechanistic knowledge of how a stressor impacts the biology of an exposed organ- ism. The theoretical framework of the AOP is that an initial perturbation of molecular function (termed a ‘molecular ini- tiating event’), causes changes at a biochemical and cellular level, resulting in a physiological consequence (Ankley et al., 2010). Theoretically, stressors that induce similar changes in molecular and/or biochemical responses, will have similar physiological and toxicological consequences. Thus, the identification of shared pathways of effect acts as a mechan- ism by which grouping of stressors by their mechanism of action can occur, simplifying risk assessment, and facilitating prediction of toxic impact. Furthermore, knowledge of the properties of impacted physiological pathways in receptor species will allow prediction of their sensitivity. Importantly, the AOP framework is of significant potential utility in solving many of the issues associated with predicting the toxicological Biotic ligand model Furthermore, altered function at a molecular level may not directly translate to meaningful change at an organismal level. For example, compensation of functions by alternate pathways may mean that changes in the expression of specific genes or proteins do not translate to physiological changes, and consequently may have limited value for envir- onmental protection (Feder and Walser, 2005). Similarly, not all physiological changes that may be induced by expos- ure to a stressor will be relevant to measures of fitness, and even for those changes that are pertinent, there may be lim- ited knowledge of the thresholds at which these changes result in ecologically relevant effects. It has also been sug- gested that the large number of molecular changes that may be induced by stressor exposure, may confound identification of biologically meaningful events, and lead to AOPs that do not truly reflect cause and effect pathways that facilitate pre- diction (Escher et al., 2017). For this reason, top-down approaches, where an observed adverse outcome initiates the elucidation of causative mechanisms, may have more predict- ive value than bottom-up approaches, where known mechan- isms of stressor effect are linked to adverse outcomes. Applications of physiological knowledge to risk assessment There is a strong theoretical justification for incorporating mechanistic knowledge into risk assessment. Indeed, in prac- tice there have been some significant advances towards this goal. Selected diverse case studies where physiological under- standing has already made a contribution to predictive risk assessment are described below. The MEMs of perhaps the greatest physiological rele- vance, are toxicokinetic-toxicodynamic (TKTD) models (Ashauer and Escher, 2010). Simply stated, toxicokinetic processes are those through which the organism can affect the toxicant. These therefore include absorption, distribu- tion, metabolism and excretion. As these are factors strongly controlled by organism physiology, then greater predictive power can be gained by the incorporation of physiologically based toxicokinetic (PB-TK) models, which account for the critical molecular, biochemical and physiological processes that impact kinetics (Grech et al., 2017). Toxicodynamic processes are those wherein the toxicant impacts the organ- ism, and therefore incorporate mechanisms of toxicity. When combined into TKTD or PB-TKTD models, factors such as exposure concentration, exposure duration, organ- ism size, metabolic rate, molecular, cellular and whole organ- ism physiological parameters can be used to predict toxicity. Importantly, these models are able to account for temporal variability in exposures, and factors such as organism accli- mation to exposure over time (Ashauer and Escher, 2010). They are also easily adjusted to account for physiological dif- ferences in model parameters between species (Nyman et al., 2014). Case study A—Silver toxicity to freshwater biota Knowledge of how silver causes toxicity in aquatic animals is an excellent example of how mechanistic understanding can contribute directly to regulatory risk assessment. Waterborne silver is toxic to aquatic biota, but only in its ionic form (Ag+). The physiological basis for this observation is that ionic silver mimics sodium, and therefore gains access to the animal through sodium transport pathways (Wood, 2012). Other chemical species of silver are not bioavailable, and thus do not contribute to toxicity. This understanding revo- lutionized risk assessment for silver in the environment, help- ing to modify regulations based on total metal which were overly restrictive, to those that accounted for metal speci- ation, eventually leading to approaches such as the BLM (Adams et al., 2002). Once absorbed, the main mechanism of silver toxicity is the inhibition of ion transport, through impairment of the basolateral sodium pump, which maintains the electrochem- ical gradient that achieves ion regulation. Mechanistic effect model This is an umbrella term that covers a range of different approaches that incorporate modelling to extrapolate effects Conservation Physiology • Volume 6 2018 Perspective effect was likely mediated by a decline in offspring quality, a factor unaccounted for in traditional testing protocols. However, an MEM approach incorporating multiple end- points was able to accurately predict the effects of Dispersogen A on population size (Gabsi et al., 2014). at an organism level, to broader community, population and ecosystem settings (Forbes and Calow, 2012). These are usu- ally frameworks that have been developed for ecological modelling, adapted to account for the presence of a stressor. As such they provide a means of relating traditional laboratory-based studies, to impacts at levels of organization of greatest relevance to risk assessors (Grimm and Martin, 2013). A key feature of MEMs is their ability to assess inter- specific interactions. For example, MEM approaches show that when interspecies competition is accounted for in a sys- tem receiving a pulsed pesticide exposure, population recov- ery rates (based on physiological knowledge of reproductive parameters) could be as much as three-fold longer, compared to scenarios where competition effects are not considered (Kattwinkel and Liess, 2014). Pesticide toxicity and water permeability in aquatic insects Physiology can aid significantly in the prediction of how increasing global temperature trends are likely to impact upon species distributions (Evans et al., 2015). For most environmental stressors there is only a limited understanding of their temporal variability. For example, factors such as improved industrial processes, remediation, and complex natural degradation pathways, means that chemical stressor concentrations fluctuate and are not easily predicted (Iqbal and Oberg, 2013). In the case of climate change, owing to the global nature of the problem, intervention will have only a limited impact on temperatures in the immediate future. Thus, while the predictions of the extent of warming differ, there is little doubt as to the general trend in this stressor over time, and its climatic impacts (Heikkinen et al., 2006). Consequently, predictions of species responses to climate are likely to be of significant real-world value (Helmuth et al., 2005). Owing to the relationship between uptake and toxicity for most chemical toxicants, measures of accumulation can offer insight into toxicity. In a study examining ten aquatic insect taxa exposed to the organophosphate pesticide chlorpyrifos, it was shown that aerially respiring insects accumulated less toxicant than aquatic breathers (Buchwalter et al., 2002). Aerial respiration is problematic owing to the risk of desicca- tion, but this is compensated for by the higher oxygen con- tent relative to water. These factors are both drivers facilitating a reduced relative size of the respiratory surface in air breathing taxa (Maina, 2002). Thus, when aerially respiring animals are exposed to membrane-permeable toxi- cants such as chlorpyrifos, there is relatively less surface area across which absorption can take place, resulting in a rela- tively lower body burden, and thus reduced toxic impact. As such, knowledge of respiration mode is a strong predictor of pesticide toxicity in aquatic insects. However, it is important to note that, in the case of chlorpyrifos, there are other fac- tors which will also determine toxic impact (e.g. rate of metabolism, elimination rates, lipid content and body size; Rubach et al., 2012). Nevertheless, this example shows that knowledge of fundamental physiological characteristics, and how these vary between organisms, can be informative in understanding and predicting toxicity. Some of the most important studies utilizing physiological measures to enhance our understanding of biological responses to climate change have been performed in flying insects. Applications of physiological knowledge to risk assessment As silver toxicity correlates with silver burden at sensitive sites (gill of fish, whole body of invertebrates; Wood, 2012), then it holds that toxicity will be strongly shaped by the characteristics of sodium uptake pathways. This partly explains the high sensi- tivity of freshwater animals to silver, as these species must have high rates of sodium uptake to compensate for sodium lost via diffusion from their more concentrated bodies to the surrounding environment. Furthermore, it also explains the finding that small freshwater animals are especially sensitive to silver toxicity (Grosell et al., 2002). These species and life- stages have a high surface area to body volume ratio, and thus rely absolutely on effective sodium uptake to maintain homoeostasis. Mechanistic effects models can be very complex, a charac- teristic that can limit their utility in risk assessments (Hommen et al., 2016). However, when complexity is eschewed for more simple approaches, the integrity of the models is often compro- mised (Rowland et al., 2017). Furthermore, the strength and utility of a given model is dependent on both the quantity and quality of the data upon which it is based. Currently, the MEM approach is limited by insufficient knowledge of physio- logical parameters across receptor species (Ippolito et al., 2012). Despite this, there are examples where MEMs have provided improved risk prediction where traditional single end-point values and laboratory lethal toxicity tests have failed. For example, the pesticide additive Dispersogen A has low toxicity in a standard chronic Daphnia magna toxicity test with reproduction as an end-point, but exposure to a predicted NOEC in a population growth test resulted in a near 20% decline in population (Hammers-Wirtz and Ratte, 2000). This Importantly, because of the underlying physiological basis of silver toxicity, models that predict sensitivity of silver can be applied across settings that differ in their water chemistry (i.e. salinity), by geochemical modelling or direct measurement of ................................................................................................................................. 5 Conservation Physiology • Volume 6 2018 Perspective free silver ion (Nichols et al., 2006). Furthermore, assuming that mechanisms of silver toxicity are conserved, then BLMs developed in model species can be parameterized to non-model animals, with knowledge of sodium uptake mechanisms, or even body size (Veltman et al., 2014). Because of this utility, and the confidence associated with a robust understanding of underlying physiological mechanism, silver BLMs are import- ant regulatory tools in a number of jurisdictions worldwide (Wood, 2012). Applications of physiological knowledge to risk assessment anaerobic metabolites increase at thermal limits in the fruit fly (Drosophila melanogaster; Malmendal et al., 2006) and an Antarctic midge (Michaud et al., 2008), while hypoxia exposure reduces thermal tolerance in Drosophila (Lighton, 2007). The OCLTT is an appealing hypothesis as it strongly links physiological mechanisms (e.g. respiration, blood flow) to ecologically relevant effects (e.g. changes in animal distri- bution). The utility of physiological information for predict- ing species responses to a changing climate is not restricted just to terrestrial insects, but has value to other terrestrial organisms (Marin et al., 2014; Mathewson et al., 2017), and to aquatic biota (Martinez et al., 2015). Pesticide toxicity and water permeability in aquatic insects For example, the increasingly early emergence of the butterfly Heteronympha merope in recent years has been linked to knowledge of the role of temperature in develop- ment processes in early life-stages of this species (Kearney et al., 2010). Similarly, Buckley and colleagues (2011) incor- porated species-specific knowledge of the relationship between temperature and developmental rate to fit a model to historical data of butterfly distribution. When applied to a later dataset, this model was a better predictor of butterfly distributions than a model which assumed butterfly develop- ment as a function of temperature was fixed between species (Buckley et al., 2011), thus demonstrating the importance of specific physiological knowledge within climate change impact scenarios. In another example, assessment of critical thermal maxima (a physiological metric of thermal toler- ance), predicted the population responses of different bee species to urban heat-island effects (Hamblin et al., 2017). Finally, there is some evidence in flying insects that supports the Oxygen- and Capacity-Limited Thermal Tolerance (OCLTT) hypothesis, which suggests that oxygen acquisition is a limiting factor for animals as oxygen demand increases (e.g. as a function of elevated environmental temperature; Pörtner et al., 2017). Specifically, it has been shown that This example is one of several studies that have shown a strong relationship between aquatic insect physiology and their sensitivity to chemical stressors. Correlations between aquatic insect biological traits and chemical exposure, have also been investigated for inorganic toxicants such as zinc (Buchwalter and Luoma, 2005), cadmium (Buchwalter et al., 2008) and sulphate (Scheibener et al., 2017). Case study B—Climate change and flying insect distribution Pesticide toxicity and water permeability in aquatic insects Maximizing mechanism There is a growing understanding of the value of incorporat- ing biological traits into risk assessment approaches. This is reflected in the growth of publications that feature mechanis- tic modelling considerations (Fig. 2). However, there is sig- nificant further scope for the incorporation of mechanistic knowledge into assessments of environmental risk. Below, ................................................................................................................................ 6 Conservation Physiology • Volume 6 2018 Perspective selected examples of how physiological tools, frameworks, and data may contribute to a more robust understanding of the stressor-organism interaction are highlighted. allowing a specific and refined risk assessment analysis approach in environments at risk of copper contamination. There are several other examples of settings where long-term exposure to a stressor has resulted in enhanced tolerance (Weis et al., 1981; Uren Webster et al., 2013; Lindberg et al., 2017), but for very few of these instances is there a mechan- istic understanding of how tolerance is achieved. These examples do, however, demonstrate that extremophile spe- cies and environments with extreme characteristics, offer promise for examining mechanisms of stressor action. Applying Krogh models Among the founding doctrines of comparative physiology is Krogh’s principle, which states: ‘For a large number of pro- blems there will be some animal of choice or a few such ani- mals on which it can be most conveniently studied’ (Krogh, 1929). From a risk assessment perspective, Krogh’s principle could apply to those species that are most sensitive to a given stressor, as an NOEC established in the most sensitive spe- cies, will be protective of all. Water fleas (e.g. Daphnia mag- na) are often championed as excellent toxicological model species, owing in part to their high sensitivity to stressors (Baudo, 1987), and could therefore be considered a Krogh model. However, research utilizing a database of previous studies showed that, relative to Daphnia magna, 22% of taxa are more sensitive to organic toxicants, and 30% of taxa are more sensitive to trace metals (von der Ohe and Liess, 2004). This suggests that the most appropriate Krogh models may differ according to the stressors of relevance, and that identifying the most relevant toxicological Krogh model may not be a simple task (Cairns and Niederlehner, 1987; Rohr et al., 2016). Gathering mechanistic information from standard laboratory toxicity tests Gathering mechanistic information from standard laboratory toxicity tests While the weaknesses of standard toxicity tests are well recognized, they remain at the core of most risk assessment guidelines, and will likely remain so until new methodologies are embraced. There are, however, missed opportunities to gather mechanistic knowledge from these assessments. For example, simple measures of toxicant burden, can help link exposure to toxic effect (McCarty, 2015), and identify aspects of toxicant handling (e.g. distribution, metabolism, excretion) which can be informative of mechanisms of effect. Furthermore, adding measures of sub-lethal toxicity to stan- dardized assays can help to identify mechanisms of impair- ment. These can be used to detect pathways of effect that are conserved between species and stressors, to identify unknown mechanisms of stressor action, and to allow pre- diction of sensitivity under non-standard exposure condi- tions. This is particularly true for tests conducted in novel species and/or with novel stressors, for which very little prior information may be available. A simple tool-box incorporat- ing measurements of key end-points (e.g. bioaccumulation, metabolism, ion regulation, oxidative stress; see McRae et al., 2018), could be an important addition to standard sin- gle end-point measurements in laboratory studies. Furthermore, such analyses are of significant value from an animal ethics perspective, in that they add data without increasing animal use. However, physiological knowledge of the mechanisms by which stressors impact organismal health opens up new ave- nues for the application of Krogh’s principle. Such approaches might include the identification of environments that are par- ticularly susceptible to stressor impact. For example, the find- ing that sodium ion turnover strongly correlates with silver ion toxicity (Wood, 2012), means that organisms inhabiting set- tings that already challenge sodium homoeostasis might be particularly at risk to silver toxicity. Such habitats would include waters of low pH, wherein high proton concentration inhibits sodium uptake, and forces these species to display extreme transport characteristics (Glover et al., 2012). This could either make these environments home to particularly susceptible biota, or havens for species that have adaptations that would make them less sensitive to silver ion toxicity. Under either circumstance, these settings would be useful for identifying organisms most at risk, or for the identification of model species that would allow further elucidation of mechanisms of toxicity by identifying pathways that facili- tate tolerance. Delineating mechanisms of stressor action Knowledge of how stressors cause adverse outcomes is essen- tial for predicting sensitivity, and thus essential for proactive risk assessment. Recognizing this, some systems have been developed specifically for gathering mechanistic information related to stressor exposure. For example, an in vivo trans- ected animal method has been previously employed to moni- tor up to 26 biochemical and physiological end-points in fish acutely exposed to stressors (McKim et al., 1987). Although this technique has been largely superseded by the develop- ment of less invasive methods (Pettem et al., 2017; Viant et al., 2017), the underlying principle of collecting multiple end-points that will elucidate pathways of toxicant effect is one that has been widely adopted. Through these types of approaches, mechanisms of action are well understood for some stressors (e.g. ion mimicry for dissolved metals; A more direct application of Krogh models to risk assess- ment is the study of animals capable of withstanding envir- onments elevated in the stressor of interest. For example, populations of rainbowfish (Melanotaenia nigrans) have sur- vived for several decades in copper-enriched waters asso- ciated with mining leachate in Australia. The tolerance of these fish appears to be related to their capacity to limit branchial copper uptake (Gale et al., 2013). Characterization of the specific mechanisms involved could provide insight into factors that shape the sensitivity of species to copper, ................................................................................................................................ .................................................................................................................................. 7 Conservation Physiology • Volume 6 2018 Perspective areas of specific interest. Such areas could include sites of particular ecological value, and/or sites considered particu- larly at risk or already impacted by stressors. Field-collected data could therefore provide fundamental information regarding the physiology of species of greatest relevance to field settings, under natural exposure conditions. Combined with mechanistic knowledge of stressor action, this informa- tion may be used to proactively highlight sensitive species (i.e. species with extreme rates of basal functions targeted by stressors). In areas that are already impacted by stressors, comparisons with control sites could identify species traits that have led to extirpation (White et al., 2017), elucidating unknown mechanisms of stressor effect. Although there are a number of important practical considerations, including con- founding effects associated with exposure history and hand- ling (Harley and Glover, 2014), this general approach of applying field-based physiological methods to directly inter- rogate regions of interest has significant value. Delineating mechanisms of stressor action It facilitates the integration of individual-level measurements with eco- logical relevance, a key pitfall of current risk assessment approaches (Rohr et al., 2016). impaired energy metabolism in hypoxia; conserved mechan- isms of action of pharmaceuticals between target and non- target organisms, Brown et al., 2014). However, for many groups of chemicals, there is surprisingly little understanding of toxic mechanism. For organic chemical stressors, quantitative structure activity relationships (QSARs) have been used to deduce bio- logical activity on the basis of shared structural similarities. However, for some of these chemical groupings, little is known regarding their mechanisms of action. For example, a large number of chemical stressors induce a narcotic effect, an ill-defined ‘baseline toxicity’ (Veith et al., 1983), which is correlated with the hydrophobicity of the chemical toxicant (Escher and Hermens, 2002). This lack of mechanistic knowledge for narcotic chemicals hinders prediction of organism responses to these chemicals, both alone and as components of complex mixtures (Escher and Hermens, 2002). However, recent work has suggested that these toxi- cants may have a common mechanism of impairment. Exposure of Daphnia magna to chemicals that induce narco- sis, resulted in transcriptional profiles over-enriched in genes associated with calcium homoeostasis (Antczak et al., 2015). The magnitude of transcriptional change correlated to the hydrophobicity of the chemical, which in turn was related to the capacity of the chemical to specifically inhibit compo- nents of intracellular calcium homoeostasis (i.e. the sarco/ endoplasmic reticulum calcium ATPase). Importantly, altera- tions in calcium handling were then able to be related to changes in Daphnia heart rate (Antczak et al., 2015), an eas- ily monitored physiological end-point. This AOP provides valuable mechanistic information of relevance to risk assess- ment, and is thus an exemplar of experimental approaches that are required to enhance the mechanistic basis of risk assessments. Conclusions Current regulatory practices focus on deriving simple toxico- logical metrics, and as such do not consider the biological mechanisms driving toxicity (Jager, 2016). Furthermore, extrapolation of laboratory tests in model organisms to effects on biota in ecosystems does not account for the often distinct physiology of non-model species, which will alter the toxicological impact of the stressor. Knowledge of how stressors cause their effects facilitates predictive modelling by accounting for species differences and the influence of environmental vari- ables that impact biological function. Mechanistic information is also key for approaches assessing the impact of multiple stres- sors (Segner et al., 2014). Critically, predicting stressor sensitiv- ity on the basis of molecular, biochemical and physiological traits minimizes the need for extensive lethal toxicity testing. The identification of conserved physiological characteristics that underlie sensitivity to a given stressor will facilitate cross-species extrapolation without having to utilize large number of animals (as in the case of LC50 assessments), and which can be per- formed in the absence of the stressor (e.g. sodium uptake char- acteristics as a determinant of silver toxicity). This will have significant benefits in terms of cost and ethical considerations (Burden et al., 2015). Field-based physiological techniques There are a number of practical reasons why risk assess- ments are restricted to a small subset of organisms that are amenable to laboratory culture. Laboratory-reared animals have a known and constant exposure history and, as they are often inbred, have a reduced genetic diversity that mini- mizes variability in responses to toxicants (Ballatori and Villalobos, 2002; Segner and Baumann, 2015). However, laboratory models are not always ecologically relevant, and their responses may be poorly representative of other mem- bers of their taxonomic group (Cairns and Niederlehner, 1987). There are, however, significant challenges to integrating physiological measures into risk assessment approaches. As for toxicological studies, research on organismal physiology is often focussed on a few model species, and thus there is often only limited knowledge of physiological function in the most relevant species to an environment of concern. Model parametrization will rely absolutely on expanding fundamen- tal knowledge, and this is therefore a major obstacle to adopting effective mechanism-based approaches. Similarly, attaining the weight of evidence needed to link physiological There are a number of physiological end-points (e.g. metabolic rate, and in aquatic settings, ion regulation), that seem particularly valuable for assessing the risks posed by environmental stressors. These are also parameters that can be measured in the field, using non-lethal techniques (Wood et al., 2002; Butler et al., 2004; Mochnacz et al., 2017). Consequently, there is capacity to conduct simple physio- logical measurements on organisms directly sampled from 8 Conservation Physiology • Volume 6 2018 Perspective Brown AR, Gunnarsson L, Kristiansson E, Tyler CR (2014) Assessing variation in the potential susceptibility of fish to pharmaceuticals, considering evolutionary differences in their physiology and ecol- ogy. Phil Trans R Soc B 369: 20130576. changes to toxicity is challenging (Hahn, 2011). Consequently, for many stressors there is still significant research effort required to establish reliable pathways of effect. Finally, the complexity of environmental exposure scenarios is a major hurdle for the development of effective traits-based risk predic- tion. The spatial and temporal variation in stressor magnitude, the myriad of complicating environmental factors, and the cap- acity of the organism to acclimate to stressor exposure will all compromise effective prediction. Nevertheless, the successful implementation of physiology-based models such as the BLM (Rüdel et al., 2015), show that these challenges can be largely overcome, and that there is significant scope for, and value in, considering organism physiology in risk assessment approaches. References Adams W, Dwyer R, Gorsuch J, Janssen C, Lee C, Paquin P, Reiley M, Walsh P, Wood C (2002) Preface. Comp Biochem Physiol C 133: 1–2. 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Effects of postoperative delirium on parents’ illness uncertainty and quality of life in children after ventricular septal defect repair
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Jiang-shan Huang Department of Cardiac Surgery, Fujian Branch of Shanghai Children’s Medical Center, Fuzhou, China Yu-kun Chen Department of Cardiac Surgery, Fujian Branch of Shanghai Children’s Medical Center, Fuzhou, China Shi-hao lin Effects of postoperative delirium on parents’ illness uncertainty and quality of life in children after ventricular septal defect repair Effects of postoperative delirium on parents illness uncertainty and quality of life in children after ventricular septal defect repair Jiang-shan Huang  Department of Cardiac Surgery, Fujian Branch of Shanghai Children’s Medical Center, Fuzhou, China Yu-kun Chen  Department of Cardiac Surgery, Fujian Branch of Shanghai Children’s Medical Center, Fuzhou, China Shi-hao lin  Department of Cardiac Surgery, Fujian Branch of Shanghai Children’s Medical Center, Fuzhou, China Wen-hao Lin  Department of Cardiac Surgery, Fujian Branch of Shanghai Children’s Medical Center, Fuzhou, China Qiang Chen  Department of Cardiac Surgery, Fujian Branch of Shanghai Children’s Medical Center, Fuzhou, China Yi-Rong Zheng  (  zhengyirong2020@163.com ) Department of Cardiac Surgery, Fujian Branch of Shanghai Children’s Medical Center, Fuzhou, China Article Keywords: postoperative delirium, ventricular septal defect, parents’ illness uncertainty, quality of life, children, SF-36 Posted Date: April 25th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1576430/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Results In terms of clinical data, there were no significant differences in gender and size of ventricular septal defect between the two groups, but significant differences in age, operation time, CPB time, mechanical ventilation time and ICU time between the two groups. In terms of the illness uncertainty scores, there are statistically significant differences between the two groups on ambiguity, lack of clarity, lack of information and unpredictability. In terms of the parents' quality of life, at discharge, there were no significant differences in PH, SF, RP, BP, and GH between the POD group and the no-POD group. There was no statistical significance in PH, SF, RP, BP, GH and RE at third postoperation months. In terms of the relationship between Parents' illness uncertainty and quality of life, there was a negative correlation between illness uncertainty and quality of life in POD and no-POD group. Methods 178 patients were enrolled and assigned to POD (n = 77) and no-POD (n = 101) based on evaluation of delirium. Delirium was assessed by a CPAD. Chinese version of the Parents' Perception of Uncertainty Scale (PPUS) was used to evaluate the Parents' illness uncertainty. Quality of life was assessed with the SF-36 scale at discharge and third postoperation months. Conclusion Parents of children with delirium after ventricular septal defect repair had significantly higher levels of illness uncertainty than parents of children without delirium. Moreover, the higher the illness uncertainty, the worse the quality of life. Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Page 1/13 Page 1/13 Abstract The purpose of this study was to investigate the illness uncertainty and quality of life of parents of children with postoperative delirium. Patients This study is a retrospective analysis. Patients with ventricular septal defect (VSD) were enrolled between January 2020 and December 2022. This study is a retrospective analysis. Patients with ventricular septal defect (VSD) were enrolled between January 2020 and December 2022. A total of 178 cases were included according to inclusion and exclusion criteria. The inclusion criteria were simple ventricular septal defect. Exclusion criteria: 1) complicated with other heart malformations; 2) Preoperative complicated with severe pulmonary infection and organ failure; 3) Severe postoperative complications (conduction block; Secondary thoracotomy, etc.); 4) Postoperative residual shunt; 5) Chromosomal and other physical abnormalities; 6) Have other surgical experience before surgery; 7) Incomplete data and lost follow-up. Background Delirium is a common central nervous system dysfunction after surgery, which is mainly manifested as disturbance of consciousness, attention, cognitive function and perception.[1] A recent study put the prevalence in pediatric cardiac intensive care units at 49%. Adult delirium is associated with increased mortality, longer hospital stay, post-discharge morbidity, and neurocognitive decline.[2,3] Critically ill children often undergo multiple treatments, such as sedative drugs, and are at risk for hallucinations and altered thinking.[4] Thus, experiences of delirium during critical illness may prevent children and their Page 2/13 Page 2/13 families from returning to normal activities and expose them to constant worry and changed expectations about the future. As parents of children are uncertain about the complicated surgical process, treatment and prognosis, they will have a series of uncomfortable psychological experiences such as fear, helplessness, anxiety and guilt.[5] Fear of the risk of surgery, concern about the efficacy of treatment and lack of confidence in the prognosis may lead to their lack of ability to make decisions related to the disease, resulting in a sense of illness uncertainty. The adaptive ability of parents can affect the positive self-adjustment ability of children after trauma. [6] The mental health status of parents directly affects the outcome, prognosis and mental health status of children. However, few studies have been conducted on parents’ illness uncertainty and postoperative quality of life in children with postoperative delirium (POD). Therefore, the purpose of this study was to investigate the illness uncertainty and quality of life of parents of children with delirium after VSD repair. Measurement of delirium RASS is a reliable scale for assessing the depth and quality of sedation and is suitable for critically ill patients. Combined with Delirium is assessed using the Cornell Assessment of Pediatric Delirium scale (CAPD), it can improve the ability to assess delirium in severely ill children.[7] RASS scores range from -5 to +4, which can be divided into 10 grades. When the scores are -4 and -5, the patients are in severe sedation and coma. Children with a score ≥-3 can be evaluated for CAPD.[8] Each item of the CAPD score scale is scored 0-4 points according to the frequency of the children’s behaviors using Likert 5-level scoring method. The fewer symptoms and behaviors described in items 1-4, the higher the score (respectively: dose the child make eye contact with the caregiver; are the child’s actions purposeful; Is the child aware of his/her surroundings; dose the child communicate needs and wants); The more frequent the symptoms and behaviors described in items 5-8, the higher the score (respectively: is the child restless; is the child inconsolable; is the child underactive-very little movement while awake; does it take the child a long time to respond to interactions). CAPD score greater than 9 is considered delirium. Since the design of CAPD considers an extended observation period (rather than as a point-of-time screening), Page 3/13 Page 3/13 the assessment time is at the end of the nurse's shift. Therefore, before the start of the study, the bedside nurses received delirium education and CAPD training. Measurement of parents’ illness uncertainty We adopted the Chinese version of the Parents' Perception of Uncertainty Scale (PPUS), which included four dimensions: ambiguity, lack of clarity, lack of information and unpredictability, with a total of 28 items. Likert 5-level scoring method was used to assign 1-5 points from "strongly disagree" to "strongly agree". The higher the score is, the higher the level of illness uncertainty is. Individuals were considered to have a high level of illness uncertainty when the disease uncertainty score was greater than 50% of the total score. Measurement of parents' quality of life The SF-36 scale was used to objectively evaluate the parents' quality of life at discharge and third postoperation months (POM3). The SF-36 scale contains eight aspects: physical functioning, social functioning, role-physical, role-emotional, mental health, vitality, bodily pain and general health. Statistical method SPSS Statistics 23 was used for statistical analysis. The count data conformed to the normal distribution with the student’s t test, and did not conform to the normal distribution with the rank sum test. It was found that there was a certain negative correlation between uncertainty and quality of life by drawing scatter plot, and the data was found to conform to normal distribution by normal test, so Pearson linear correlation analysis was adopted. Table 3 summarizes the parents' quality of life. (1) At discharge, there were no significant differences in PH(P = 0.733), SF (P = 0.262), RP (P = 0.384), BP (P = 0.871), and GH (P = 0.811) between the POD group Result Based on inclusion and exclusion criteria, 178 patients were enrolled and divided into postoperative delirium (POD) (n = 77) and no-POD (n = 101) groups based on evaluation of delirium. In terms of clinical data, there were no significant differences in gender and size of ventricular septal defect between the two groups, but significant differences in age, operation time, CPB time, mechanical ventilation time and ICU time between the two groups. (See Table 1) Page 4/13 Table 1 General clinical data of patients Item POD No-POD p Number 77 101 / M/F 36/41 52/49 0.549 Age (m) 32.1 ± 11.4 36.4 ± 13.5 0.027 Size of VSD (mm) 5.6 ± 1.8 5.2 ± 1.8 0.226 CPB (min) 65.5 ± 10.3 61.5 ± 12.9 0.033 Operation time (min) 130.6 ± 36.9 116.0 ± 31.4 0.005 Mechanical ventilation 14.4 ± 4.1 12.0 ± 4.3 0.000 ICU(d) 3.3 ± 1.5 2.6 ± 1.7 0.017 Table 1 General clinical data of patients Table 2 summarizes the illness uncertainty scores of the POD and no-POD. (1) In terms of ambiguity, the score of POD group (35.7 ± 11.9) was significantly higher than that of no-POD group (31.9 ± 12.0), and the difference was statistically significant (P = 0.037). (2) In terms of lack of clarity, the score of POD group (26.0 ± 10.4) was higher than that of no-POD group (20.7 ± 11.9), and the difference between the two groups was statistically significant (P < 0.000). (3) In terms of lack of information, the score of POD group (17.1 ± 5.9) was higher than that of no-POD group (14.7 ± 6.2), and the difference between the two groups was statistically significant (P = 0.011). (4) In terms of unpredictability, the score of POD group (16.6 ± 7.3) was higher than that of no-POD group (11.6 ± 4.9), and the difference between the two groups was statistically significant (P < 0.000). (5) In the total score of illness uncertainty, the scores of both groups were 50% higher than the total score of 140, indicating that both groups had obvious illness uncertainty, and POD group (95.3 ± 19.5) was significantly higher than no-POD group (78.9 ± 17.3), there was a significant statistical difference between the two groups (P < 0.000). Table 2 summarizes the illness uncertainty scores of the POD and no-POD. Result (1) In terms of ambiguity, the score of POD group (35.7 ± 11.9) was significantly higher than that of no-POD group (31.9 ± 12.0), and the difference was statistically significant (P = 0.037). (2) In terms of lack of clarity, the score of POD group (26.0 ± 10.4) was higher than that of no-POD group (20.7 ± 11.9), and the difference between the two groups was statistically significant (P < 0.000). (3) In terms of lack of information, the score of POD group (17.1 ± 5.9) was higher than that of no-POD group (14.7 ± 6.2), and the difference between the two groups was statistically significant (P = 0.011). (4) In terms of unpredictability, the score of POD group (16.6 ± 7.3) was higher than that of no-POD group (11.6 ± 4.9), and the difference between the two groups was statistically significant (P < 0.000). (5) In the total score of illness uncertainty, the scores of both groups were 50% higher than the total score of 140, indicating that both groups had obvious illness uncertainty, and POD group (95.3 ± 19.5) was significantly higher than no-POD group (78.9 ± 17.3), there was a significant statistical difference between the two groups (P < 0.000). Table 2 Score of Parents' Perception of Uncertainty Scale in POD and no-POD groups Item clauses range POD No-POD p Total 28 28–140 95.3 ± 19.5 78.9 ± 17.3 0.000 Ambiguity 11 11–55 35.7 ± 11.9 31.9 ± 12.0 0.037 Lack of clarity 8 8–40 26.0 ± 10.4 20.7 ± 11.9 0.000 Lack of information 5 5–25 17.1 ± 5.9 14.7 ± 6.2 0.011 Unpredicatability 4 4–20 16.6 ± 7.3 11.6 ± 4.9 0.000 and the no-POD group, indicating that there was no difference in the quality of life between the POD group and the no-POD group in these aspects. There were significant differences in RE (P = 0.029), MH (P  < 0.000) and VT (P = 0.007) between the two groups. Overall quality of life was significantly lower in the POD group (81.3 ± 13.4) than in the no-POD group (83.7 ± 13.7), with statistically significant differences (P = 0.001). Figures 1 and 2 summarize the comparison of the two groups' quality of life at discharge and POM3. In Fig. 1, we found that the POD group had significantly improved quality of life at POM3 at RP (P = 0.020), RE (P = 0.001), MH (P = 0.033), and VT (P = 0.009). And overall quality of life in POD group was significantly higher at POM3 (84.1 ± 13.5) than at discharge (81.3 ± 13.4) (P = 0.001). In Fig. 2, we found a statistically significant improvement in quality of life at 3 months postoperatie in RE (P = 0.01) and VI (P  = 0.000) in the non-delirium group. The overall quality of life at 3 months after surgery (85.5 ± 13.2) was higher than that at discharge (83.7 ± 13.7) (P = 0.008). Result (2) At third postoperation months, there were no significant differences in PH (P = 0.596), SF (P = 0.327), RP (P = 0.172), BP (P = 0.370), GH (P = 0.770), RE (P = 0.086) between the POD group and the non-delirium group, indicating that there was no difference in quality of life between the delirium group and the non-delirium group in these aspects. There were significant differences in MH (P < 0.000) and VITA (P = 0.006) between the two groups, indicating that the quality of life in delirium group was significantly inferior to that in non-delirium group. In terms of overall quality of life, there was no statistical difference between the POD group (84.1 ± 13.) and the no-POD group (85.5 ± 13.2). Table 3 Quality of life of parents of children item At discharge Third postoperation months   POD No-POD P POD No-POD P Total 81.3 ± 13.4 83.7 ± 13.7 0.001 84.1 ± 13.5 85.5 ± 13.2 0.051 physical functioning 88.3 ± 13.9 87.5 ± 17.8 0.733 86.8 ± 11.1 87.6 ± 10.8 0.596 social functioning 86.0 ± 10.5 87.7 ± 9.8 0.262 85.9 ± 11.7 88.0 ± 15.4 0.327 role-physical 85.4 ± 11.3 87.1 ± 12.1 0.348 88.6 ± 11.8 86.2 ± 11.1 0.172 role-emotional 70.2 ± 12.3 74.3 ± 12.2 0.029 79.1 ± 12.3 82.2 ± 11.0 0.086 mental health 74.8 ± 11.0 82.6 ± 10.3 0.000 77.7 ± 9.7 84.6 ± 12.5 0.000 vitality 71.3 ± 9.8 76.0 ± 11.9 0.007 78.4 ± 8.4 82.1 ± 8.9 0.006 bodily pain 86.3 ± 10.4 86.1 ± 11.8 0.871 87.5 ± 19.8 85.2 ± 13.4 0.370 general health 87.9 ± 10.0 88.4 ± 13.4 0.811 88.7 ± 14.1 88.0 ± 18.4 0.770 Figures 1 and 2 summarize the comparison of the two groups' quality of life at discharge and POM3. In Fig. 1, we found that the POD group had significantly improved quality of life at POM3 at RP (P = 0.020), RE (P = 0.001), MH (P = 0.033), and VT (P = 0.009). And overall quality of life in POD group was significantly higher at POM3 (84.1 ± 13.5) than at discharge (81.3 ± 13.4) (P = 0.001). In Fig. 2, we found a statistically significant improvement in quality of life at 3 months postoperatie in RE (P = 0.01) and VI (P  = 0.000) in the non-delirium group. Discussion Pediatric delirium is an acute brain disorder that occurs in pediatric intensive care units.[9] The occurrence of delirium is not conducive to the stability of children's autonomic nervous system and endocrine system, and may lead to unplanned extubation, falling out of bed, prolonged hospital stay, and increased hospital mortality.[10,11,12] Children with delirium often have a reduced ability to maintain attention and clinical symptoms such as memory and language impairment.[13] Critically ill children often undergo multiple treatments, are treated with sedative drugs and are at risk for hallucinations. [4] As parents of children are uncertain about the complicated surgical process, treatment and prognosis, they will have a series of uncomfortable psychological experiences such as fear, helplessness, anxiety and guilt.[5] Fear of surgical risk, concern about the outcome of treatment and postoperative delirium(POD), and lack of confidence in the prognosis may lead to a sense of illness uncertainty. The psychological anxiety brought about by the illness uncertainty often affects the quality of life of the parents. Few articles have focused on that. Therefore, the purpose of this study was to investigate the illness uncertainty and quality of life of parents of children with delirium after VSD repair. Delirium is assessed using the Cornell Assessment of Pediatric Delirium scale (CAPD), a reliable and simple bedside tool for children of all ages.[14] The 2016 European Society of Pediatric and Neonatal Intensive Care (ESPNIC) recommended the use of the CAPD as an assessment tool for childhood delirium, with evidence level A.[15] The CAPD was developed by pediatric psychiatrist Gabrielle Sliver and Pediatric intensive Care unit physician Chani Traube and their team in 2012 based on the Pediatric Anesthesia Emergence Delirium scale (PAED) scale.[16] Appropriate sedation is often performed in patients with ventricular defect repair, so RASS in combination with RASS may improve the assessment of critically ill delirium. Children after ventricular defect repair often receive appropriate sedation, so the combination of RASS can improve the ability to evaluate delirium in critically ill children. The CAPD is designed to take into account a period of observation (rather than a point in time), therefore the evaluation time is at the end of the nurse's shift. Bedside nurses received extensive delirium education and delirium screening practice prior to the study. Result The overall quality of life at 3 months after surgery (85.5 ± 13.2) was higher than that at discharge (83.7 ± 13.7) (P = 0.008). Figures 1 and 2 summarize the comparison of the two groups' quality of life at discharge and POM3. In Fig. 1, we found that the POD group had significantly improved quality of life at POM3 at RP (P = 0.020), RE (P = 0.001), MH (P = 0.033), and VT (P = 0.009). And overall quality of life in POD group was significantly higher at POM3 (84.1 ± 13.5) than at discharge (81.3 ± 13.4) (P = 0.001). In Fig. 2, we found a statistically significant improvement in quality of life at 3 months postoperatie in RE (P = 0.01) and VI (P  = 0.000) in the non-delirium group. The overall quality of life at 3 months after surgery (85.5 ± 13.2) was higher than that at discharge (83.7 ± 13.7) (P = 0.008). In terms of the relationship between Parents' illness uncertainty and quality of life, there was a negative correlation between illness uncertainty and quality of life in POD group (Pearson correlation coefficient r=-0.593, P = 0.000), indicating that the higher the illness uncertainty, the worse the quality of life. Page 6/13 Page 6/13 Similarly, there was also a negative correlation between the two in the no-POD group (Pearson correlation coefficient r=-0.647, P = 0.000), which also indicated that the higher the uncertainty, the worse the quality of life. (See the Fig. 3) Similarly, there was also a negative correlation between the two in the no-POD group (Pearson correlation coefficient r=-0.647, P = 0.000), which also indicated that the higher the uncertainty, the worse the quality of life. (See the Fig. 3) Discussion Mishel, then a professor at the University of North Carolina, published a Measure of Uncertainty in 1981, after which he formally proposed the theory of uncertainty and elaborated on the exhaustive content of the theory during illness, treatment and hospitalization in 1988 by exploring a series of questions.[17] Mishel considers illness uncertainty to be defined when an individual has no capacity to determine the meaning of the disease-related event (symptom, diagnosis, treatment, prognosis) during the course of the illness and when there are cognitive feelings such as confusion, uncertainty about the development of the disease, lack of medical information and unpredictability of the future. Lan et al interviewed mothers of 12 children with congenital heart disease and found that they felt stressed because of uncertainty about Page 7/13 Page 7/13 the outcome of the procedure.[18] Whereas Mitchell found that anxiety made families of ICU patients’ hyper vigilance and interfered with patients' families developing a reasonable cognitive framework about the disease event, higher levels of uncertainty were positively associated with anxiety.[19] In our study, it was found that the scores of the two groups of parents' illness uncertainty were higher than 50% of the total score, indicating a higher sense of illness uncertainty. Demaso et al reported that when parents face surgery on their children, active coping can relieve the pressure of hospitalization and reduce the sense of illness uncertainty.[20] It also seems to be a reminder that we can guide parents of children with positive attitudes to reduce the uncertainty of the disease and thus reduce their anxiety levels. the outcome of the procedure.[18] Whereas Mitchell found that anxiety m hyper vigilance and interfered with patients' families developing a reason the disease event, higher levels of uncertainty were positively associated was found that the scores of the two groups of parents' illness uncertain total score, indicating a higher sense of illness uncertainty. Demaso et al surgery on their children, active coping can relieve the pressure of hospit illness uncertainty.[20] It also seems to be a reminder that we can guide attitudes to reduce the uncertainty of the disease and thus reduce their a The quality of life of patients was assessed using the SF-36 questionnaire, at the time of discharge and at the time of return to the hospital for follow-up three months after surgery. Discussion The SF-36 scale was developed by the Boston Health Institute, USA based on the MOS scale from the Stewartse’s study and contains 8 aspects: physical functioning, social functioning, role-physical, role-emotional, mental health, vitality, bodily pain, general health, which comprehensively reflects the patient's physical and psychological condition.[21] Moreover, SF-36 is an important scale for objectively assessing QOL, which has been widely used. Lawoko S et al ‍ found that parents of children with congenital heart disease had lower quality of life than parents of children with other diseases, and that their psychological disturbances, feelings of helplessness, and economic status had more severe effects on their quality of life than other aspects.[22] In this study, we found that the quality of life of the children's parents was lower, especially in the aspects of role-emotional, mental health and vitality. These aspects belong to the psychological quality of life and the reason for the analysis may be the presence of greater psychological stress on the parents. This result is similar to that of Arafa's study.[23] And in the comparison between the two groups, we found that the quality of life of the parents of children with delirium was significantly worse than that of the parents of the no-POD group, especially in terms of psychological quality of life. The reason for the analysis may be that the child has delirium, which increases the psychological burden on the parents of the child. Due to the lack of understanding of postoperative delirium, the worry about its prognosis may be the difference in the quality of psychological life. However, the quality of life at 3 months after surgery had improved significantly, although still not as good as in the non-delirium group, but significantly better than at the time of discharge. This may also indicate that with the extension of time, the children gradually recover, the parents' psychological pressure is gradually reduced, and the quality of life is gradually improved. This can also remind us that in the future in medical work, we should be more patient to explain the doubts about delirium, and actively guide the parents of the children, so that the parents can understand the disease, so as to reduce the illness uncertainty and relieve the negative emotions In the correlation analysis between parental illness uncertainty and quality of life, the stronger the parents' sense of illness uncertainty, the worse the quality of life. Conclusion Parents of children with delirium after ventricular septal defect repair had significantly higher levels of illness uncertainty than parents of children without delirium, and had a worse postoperative quality of life (especially mentally) than parents of children without delirium. Moreover, the higher the illness uncertainty, the worse the quality of life. Discussion The study by Ju HO et al. found that the higher the level of illness uncertainty of the parents of children, the more obvious the perceived anxiety and depression.[24] Paul IM and others believe that the psychological depression of the mother of the child will affect the emotional interaction between the mother and the child, and it is also very detrimental to the child's behavioral cognitive function and physical and mental development.[25] Therefore, as Page 8/13 Page 8/13 medical staff, this also reminds us to explain the disease situation of children more carefully in our daily work, reduce the uncertainty of parents of children as much as possible, and improve their quality of life, which is also something we cannot ignore. medical staff, this also reminds us to explain the disease situation of children more carefully in our daily work, reduce the uncertainty of parents of children as much as possible, and improve their quality of life, which is also something we cannot ignore. Limitations This article is a retrospective analysis of a small sample size from a single center, and specific medical habits (such as conversation habits) may be biased. In addition, regarding the illness uncertainty of the parents of the children, the survey data collected in this study at one time point cannot reflect the degree of uncertainty about the dynamic changes of the disease. Declarations Funding: The authors did not receive support from any organization for the submitted work. The authors have no fnancial or proprietary interests in any material discussed in this article. Conflict of interest: The authors have no financial or other conflicts of interest to disclose Conflict of interest: The authors have no financial or other conflicts of interest to disclose Ethical approval: This study was approved by the ethics committee of Fujian Maternity and Child Health Hospital and conformed to the Declaration of Helsinki. References 1. Bryant KJ. Pediatric Delirium in the Cardiac Intensive Care Unit: Identification and Intervention. Crit Care Nurse. 2018 Aug;38(4):e1-e7. 1. Bryant KJ. Pediatric Delirium in the Cardiac Intensive Care Unit: Identification and Intervention. Crit Care Nurse. 2018 Aug;38(4):e1-e7. 2. Watson RS, Choong K, Colville G, Crow S, Dervan LA, Hopkins RO, Knoester H, Pollack MM, Rennick J, Curley MAQ. Life after Critical Illness in Children-Toward an Understanding of Pediatric Post-intensive Care Syndrome. J Pediatr. 2018 Jul;198:16–24. 3. Traube C, Mauer EA, Gerber LM, Kaur S, Joyce C, Kerson A, Carlo C, Notterman D, Worgall S, Silver G, Greenwald BM. Cost Associated With Pediatric Delirium in the ICU. Crit Care Med. 2016 Dec;44(12):e1175-e1179. 4. Kaur S, Silver G, Samuels S, Rosen AH, Weiss M, Mauer EA, Gerber LM, Greenwald BM, Traube C. Delirium and Developmental Disability: Improving Specificity of a Pediatric Delirium Screen. Pediatr Crit Care Med. 2020 May;21(5):409–414. Page 9/13 Page 9/13 5. Płaszewska-Żywko L, Gazda D. Emotional reactions and needs of family members of ICU patients. Anaesthesiol Intensive Ther. 2012 Jul-Sep;44(3):145-9. 6. Hafstad GS, Gil-Rivas V, Kilmer RP, Raeder S. Parental adjustment, family functioning, and posttraumatic growth among Norwegian children and adolescents following a natural disaster. Am J Orthopsychiatry. 2010 Apr;80(2):248 − 57. 7. Wild D, Grove A, Martin M, Eremenco S, McElroy S, Verjee-Lorenz A, Erikson P; ISPOR Task Force for Translation and Cultural Adaptation. Principles of Good Practice for the Translation and Cultural Adaptation Process for Patient-Reported Outcomes (PRO) Measures: report of the ISPOR Task Force for Translation and Cultural Adaptation. Value Health. 2005 Mar-Apr;8(2):94–104. 8. Traube C, Silver G, Kearney J, Patel A, Atkinson TM, Yoon MJ, Halpert S, Augenstein J, Sickles LE, Li C, Greenwald B. Cornell Assessment of Pediatric Delirium: a valid, rapid, observational tool for screening delirium in the PICU*. Crit Care Med. 2014 Mar;42(3):656 − 63. 9. Van Tuijl SG, Van Cauteren YJ, Pikhard T, Engel M, Schieveld JN. Management of pediatric delirium in critical illness: a practical update. Minerva Anestesiol. 2015 Mar;81(3):333 − 41. 10. Smeets IA, Tan EY, Vossen HG, Leroy PL, Lousberg RH, van Os J, Schieveld JN. Prolonged stay at the paediatric intensive care unit associated with paediatric delirium. Eur Child Adolesc Psychiatry. 2010 Apr;19(4):389 − 93. 11. Pandharipande P, Shintani A, Peterson J, Pun BT, Wilkinson GR, Dittus RS, Bernard GR, Ely EW. References Quality of life among parents of children with heart disease. Health Qual Life Outcomes. 2008 Nov 3;6:91. 24. Ju HO, McElmurry BJ, Park CG, McCreary L, Kim M, Kim EJ. Anxiety and uncertainty in Korean mothers of children with febrile convulsion: cross-sectional survey. J Clin Nurs. 2011 May;20(9– 10):1490-7. 24. Ju HO, McElmurry BJ, Park CG, McCreary L, Kim M, Kim EJ. Anxiety and uncertainty in Korean mothers of children with febrile convulsion: cross-sectional survey. J Clin Nurs. 2011 May;20(9– 10):1490-7. 25. Paul IM, Downs DS, Schaefer EW, Beiler JS, Weisman CS. Postpartum anxiety and maternal-infant health outcomes. Pediatrics. 2013 Apr;131(4):e1218-24 25. Paul IM, Downs DS, Schaefer EW, Beiler JS, Weisman CS. Postpartum anxiety and maternal-infant health outcomes. Pediatrics. 2013 Apr;131(4):e1218-24 References Lorazepam is an independent risk factor for transitioning to delirium in intensive care unit patients. Anesthesiology. 2006 Jan;104(1):21 − 6. 12. Ely EW, Shintani A, Truman B, Speroff T, Gordon SM, Harrell FE Jr, Inouye SK, Bernard GR, Dittus RS. Delirium as a predictor of mortality in mechanically ventilated patients in the intensive care unit. JAMA. 2004 Apr 14;291(14):1753-62. 13. Holly C, Porter S, Echevarria M, Dreker M, Ruzehaji S. CE: Original Research: Recognizing Delirium in Hospitalized Children: A Systematic Review of the Evidence on Risk Factors and Characteristics. Am J Nurs. 2018 Apr;118(4):24–36. 14. Silver G, Traube C, Gerber LM, Sun X, Kearney J, Patel A, Greenwald B. Pediatric delirium and associated risk factors: a single-center prospective observational study. Pediatr Crit Care Med. 2015 May;16(4):303–309. 15. Harris J, Ramelet AS, van Dijk M, Pokorna P, Wielenga J, Tume L, Tibboel D, Ista E. Clinical recommendations for pain, sedation, withdrawal and delirium assessment in critically ill infants and children: an ESPNIC position statement for healthcare professionals. Intensive Care Med. 2016 Jun;42(6):972 − 86. 16. Silver G, Kearney J, Traube C, Hertzig M. Delirium screening anchored in child development: The Cornell Assessment for Pediatric Delirium. Palliat Support Care. 2015 Aug;13(4):1005-11. MH. Uncertainty in illness. Image J Nurs Sch. 1988 Winter;20(4):225 − 32. 18. Lan SF, Mu PF, Hsieh KS. Maternal experiences making a decision about heart surgery for their young children with congenital heart disease. J Clin Nurs. 2007 Dec;16(12):2323-30. Page 10/13 Page 10/13 19. Mitchell ML, Courtney M. Reducing family members' anxiety and uncertainty in illness around transfer from intensive care: an intervention study. Intensive Crit Care Nurs. 2004 Aug;20(4):223 − 31. 20. DeMaso DR, Snell C. Promoting coping in children facing pediatric surgery. Semin Pediatr Surg. 2013 Aug;22(3):134-8. 21. McHorney CA, Ware JE Jr, Lu JF, Sherbourne CD. The MOS 36-item Short-Form Health Survey (SF- 36): III. Tests of data quality, scaling assumptions, and reliability across diverse patient groups. Med Care. 1994;32:40–66. 22. Lawoko S, Soares JJ. Quality of life among parents of children with congenital heart disease, parents of children with other diseases and parents of healthy children. Qual Life Res. 2003 Sep;12(6):655 − 66. 23. Arafa MA, Zaher SR, El-Dowaty AA, Moneeb DE. Quality of life among parents of children with heart disease. Health Qual Life Outcomes. 2008 Nov 3;6:91. 23. Arafa MA, Zaher SR, El-Dowaty AA, Moneeb DE. Figures Page 11/13 Figure 1 The quality of life in group POD at discharge and POM3 The quality of life in group POD at discharge and POM3 The quality of life in group POD at discharge and POM3 Figure 2 The quality of life in group no-POD at discharge and POM3 Figure 2 The quality of life in group no-POD at discharge and POM3 The quality of life in group no-POD at discharge and POM3 Page 12/13 Page 12/13 Figure 3 The relationship between Parents' illness uncertainty and quality of life The relationship between Parents' illness uncertainty and quality of life The relationship between Parents' illness uncertainty and quality of life Page 13/13 Page 13/13
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Revista Brasileira de Engenharia Agrícola e Ambiental v.16, n.2, p.229–234, 2012 Campina Grande, PB, UAEA/UFCG – http://www.agriambi.com.br Protocolo 049.11 – 05/04/2011 • Aprovado em 09/12/2011 Revista Brasileira de Engenharia Agrícola e Ambiental v.16, n.2, p.229–234, 2012 Campina Grande, PB, UAEA/UFCG – http://www.agriambi.com.br Protocolo 049.11 – 05/04/2011 • Aprovado em 09/12/2011 Revista Brasileira de Engenharia Agrícola e Ambiental v.16, n.2, p.229–234, 2012 Campina Grande, PB, UAEA/UFCG – http://www.agriambi.com.br Protocolo 049.11 – 05/04/2011 • Aprovado em 09/12/2011 RESUMO O óleo mineral, originário do petróleo, é o fluido isolante tradicionalmente utilizado em transformadores elétricos. Diante do apelo por fontes de energia limpa e renovável o setor elétrico também é pressionado a apresentar alternativas ao fluido de origem fóssil. Neste estudo, óleos de algodão, babaçu, girassol, milho e soja, foram avaliados quanto ao seu potencial para utilização como fluido dielétrico. As propriedades avaliadas foram densidade, viscosidade, acidez, tensão de ruptura, fator de perda, teor de água e corrosividade. Os resultados obtidos foram comparados aos limites estabelecidos na NBR 15422. Os óleos vegetais apresentaram densidade e viscosidade dentro dos limites recomendados; além disso, não se mostraram corrosivos mas devem ser submetidos a tratamentos específicos que os conduzam a atender outras especificações; o tratamento dos óleos com solução de hidróxido de sódio diminuiu a acidez, melhorou a tensão de ruptura e diminuiu o fator de perda. Palavras-chave: óleo mineral, óleo isolante vegetal, transformador Caracterização físico-química e dielétrica de óleos biodegradáveis para transformadores elétricos Claudia R. Silva1, Maria W. N. C. Carvalho2, Líbia de S. Conrado2, Marcus V. L. Fook3 & Krsthianna P. dos S. Leite2 1 DEQ/UFSCar, Rod. Washington Luiz, km 235, Monjolinho, CEP 13565-905, São Carlos, SP. Fone: (16) 3351-8697. E-mail: claudiacr81@hotmail.com 2 UAEQ/UFCG, Av. Aprígio Veloso 882, Bodocongó, CEP 58109-970, Campina Grande, PB. Fone: (83) 2101-1115. E-mail (s): wilma@deq.ufcg.edu.br; libiac@deq.ufcg.edu.br; krsthianna_cg@hotmail.com 3 UAEMa/UFCG, Av.AprígioVeloso 882, Bodocongó,CEP58109-970, Campina Grande,PB. Fone:(83)2101-1115.E-mail:marcusvinicius@dema.ufcg.edu.br Key words: mineral oil, insulating vegetable oil, transformer INTRODUÇÃO Transformadores são equipamentos de extrema importância nos sistemas de conversão e distribuição de energia elétrica e estão presentes desde a planta geradora, elevando a tensão para níveis adequados à transmissão a longas distâncias, até a distribuição, reduzindo a tensão para níveis de consumo residencial. Tal aplicabilidade faz dos transformadores os equipamentos mais importantes do sistema elétrico de potência. Wilhelm et al. (2009) apresentaram uma análise comparativa de propriedades térmicas, físico-químicas e elétricas de algumas variedades de óleos vegetais com relação ao óleo mineral e constataram que os óleos vegetais têm viscosidades semelhantes às do óleo de silicone e muito inferior à do fluido comercial HMWH (High Molecular Weight Hydrocarbons), usados pelo setor elétrico. Baseados em tal comprovação, os autores defenderam que óleos vegetais podem ser facilmente utilizados sem necessidade de redução da viscosidade; todavia, destacaram a possibilidade de alterar essa propriedade para níveis desejados, mediante a adição de fluidos adequados. Para diagnosticar a qualidade de novos óleos isolantes ou em serviço, são realizados ensaios estabelecidos em normas. A NBR 15422 da ABNT (2006c) serve como guia para utilização de óleo vegetal isolante para equipamentos elétricos. Um componente básico do transformador é o óleo, que promove a refrigeração e o isolamento dos circuitos elétricos e magnéticos. A popularidade do óleo mineral, oriundo do petróleo se deve, nesta aplicação, à disponibilidade, ao baixo custo e às suas excelentes propriedades dielétricas e refrigerantes (Georgilakis, 2011). A partir do século 20 surgiram vários problemas de segurança ambiental (Ribeiro, 2010; Carioca et al., 2010; Mariano et al., 2008), com o desenvolvimento dos fluidos dielétricos de origem mineral (Life et al., 2010; Kwofie et al., 2011), tornando extremamente atrativo e importante o uso de produtos com alta biodegrabilidade (Meshram et al., 2011). Em decorrência da escassez do petróleo as grandes companhias mundiais do setor elétrico passaram a investigar alternativas para substituição do óleo mineral a partir de 1990 (Shogren et al., 2004; Essam & Al-Ammar, 2010). Desde então, algumas patentes foram publicadas e os resultados de pesquisas levaram ao desenvolvimento de fluidos, isolantes vegetais comerciais. O fluido BIOTEMP foi patenteado nos Estados Unidos, em setembro de 1999, pela ABB Power T&D Company, Inc. (Oommen & Claiborne, 1999). Outra patente foi emitida em 1999, como resultado de estudos com óleo de soja, sob o domínio da Waverly Light & Power (Cannon & Honary, 1999). ABSTRACT The mineral oil, originated from petroleum, is the insulating fluid traditionally used in electrical transformers. Responding to appeals for clean and renewable energy sources, the electrical sector is also under pressure to present alternatives to the fluid of fossil origin. In this study, cotton, `babassu’, sunflower, corn and soybean oils were evaluated for their potential of utilization as a dielectric fluid. The properties investigated were density, viscosity, acidity, breakdown voltage, loss factor, water content and corrosivity. The results were compared with the values of the limits established in NBR 15422. Vegetable oils showed density and viscosity within the limits set by standard, however, higher than those presented by the studied mineral oil. It was found that vegetable oils have to pass through improvements to meet other specifications required. The treatment of oils with sodium hydroxide solution reduced the acidity, improved the breakdown voltage thereby lowering the loss factor. Key words: mineral oil, insulating vegetable oil, transformer 230 Claudia R. Silva et al. isolamento de transformadores de alta potência. A característica de biodegradabilidade, alto ponto de fulgor (> 300 °C) e a possibilidade de aumentar a vida útil do papel isolante, são os pontos mais relevantes que podem ser abordados (Fofana et al., 2010). Diversos autores vêm reportando exemplos do uso de óleos vegetais como óleo isolante aplicado em transformadores de distribuição de energia, tais como óleo de palma, canola e oliva (Essam & Al-Ammar, 2010), óleo de milho e algodão (Shah & Tahir, 2011). INTRODUÇÃO Em março de 2000 a patente do fluido Envirotemp FR3 foi concedida à Cooper Industries (McShane et al., 2000). Essas razões justificam o desenvolvimento de fluidos dielétricos naturais. Com base no exposto considera-se imprescindível caracterizar óleos vegetais disponíveis no Brasil, no sentido de se chegar a alternativas viáveis uma vez que, em regra, materiais comerciais apresentam segredos de preparação. Assim, o objetivo do trabalho é apresentar os resultados da investigação de características físico-químicas e dielétricas dos óleos de algodão, babaçu, girassol, milho e soja da classe comestível. As propriedades avaliadas foram: densidade, viscosidade cinemática, índice de acidez, corrosividade, teor de água, rigidez dielétrica e fator de perdas. O conhecimento dessas propriedades é fundamental pois são elas, dentre outras, que garantem a eficiência do sistema de isolamento e a refrigeração em transformadores (Sidibé et al., 2010; Zhang et al., 2006). Os resultados obtidos para os óleos vegetais foram comparados aos limites indicados na NBR 15422; usou-se também, como comparativo, um óleo mineral regenerado. Ensaios realizados com fluidos dielétricos disponíveis comercialmente, demonstraram que o isolante de origem vegetal atinge ponto de combustão aos 360 °C e é 97% biodegradável em 21 dias. Destaca-se ainda que durante a combustão esses óleos só emitem dióxido de carbono e água sem derivados poliaromárticos ou silicatos prejudiciais, quando queimados. Em contraste, a queima do óleo mineral se dá aos 160 °C e sua biodegrabilidade é de apenas 25,2% para o mesmo intervalo de tempo, levando 15 anos para ser totalmente degradado (Ferreira Junior, 2006). Além disso, o óleo vegetal é atóxico, oferece menor risco de acidentes no manuseio e armazenamento, possui melhor tolerância à umidade, tem boa rigidez dielétrica, melhor eficiência na troca térmica pelas excelentes características térmicas e ainda amplia a vida útil do equipamento. Outras vantagens relacionadas ao uso do óleo vegetal é que ele permite ampliar a potência do transformador, que passa a comportar mais kVA por quilo e ajuda a diminuir as falhas técnicas. Por outro lado, uma desvantagem dos óleos vegetais é que apresentam menor estabilidade à oxidação em comparação com óleos minerais, sendo esta característica o principal obstáculo para usá-los como base para o desenvolvimento de fluidos dielétricos (Oommen & Claiborne, 1999). Ésteres naturais estão aumentando sua participação no mercado como fluido de R. Bras. Eng. Agríc. Ambiental, v.16, n.2, p.229–234, 2012. RESULTADOS E DISCUSSÃO A viscosidade cinemática dos óleos foi determinada com o uso do viscosímetro capilar Cannon-Fenske imerso em banho, com temperatura controlada. O ensaio de viscosidade foi realizado nas três temperaturas recomendadas pela NBR 15422 (ABNT 2006c): 20, 40 e 100 ºC e os experimentos foram realizados em triplicata. Os índices de acidez dos óleos estão dispostos na Tabela 1. Os óleos vegetais comestíveis apresentaram acidez superior à do limite estabelecido na NBR 15422 (ABNT 2006c), de 0,06 mgKOH g-1; em função disto, foram realizadas neutralizações (tratamento com solução de NaOH) dos óleos para deixá-los com os níveis recomendados de acidez. A acidez dos óleos foi determinada por meio da metodologia que utiliza solução de hidróxido de sódio como titulante. O cálculo do índice de acidez foi realizado de acordo com a Eq. 1: Tabela 1. Índice de acidez dos óleos comerciais após tratamento de neutralização Tabela 1. Índice de acidez dos óleos comerciais após tratamento de neutralização Índice de acidez (mg KOH g-1) Tipo de óleo Óleo comercial Óleo comercial tratado com NaOH Algodão 0,14 ± 0,00 0,05 ± 0,00 Babaçu 0,27 ± 0,01 0,04 ± 0,00 Girassol 0,14 ± 0,00 0,04 ± 0,00 Milho 0,22 ± 0,00 0,03 ± 0,00 Soja 0,20 ± 0,01 0,03 ± 0,00 p a.f.M.e Acidez de Índice  (1) p a.f.M.e Acidez de Índice  (1) (1) a - volume em mL da solução de hidróxido de sódio usado na titulação f - fator de correção da solução de hidróxido de sódio (determinado por padronização da solução) Os resultados apresentados na Tabela 1 indicam que, após o procedimento de neutralização, todos os óleos alcançaram índice de acidez inferior ao máximo recomendado na NBR15422. p - massa em gramas de óleo usada e - equivalente grama do hidróxido de potássio M - concentração molar da solução titulante São apresentados na Tabela 2 os valores de viscosidade e densidade dos óleos vegetais após processo de neutralização. Os resultados indicam que os óleos de algodão, girassol, milho e soja, são cerca de três vezes mais viscosos que o óleo mineral; contudo, nas três temperaturas a viscosidade está dentro dos limites estipulados na NBR 15422 (ABNT, 2006c). A densidade desses óleos também é superior à do óleo mineral; entretanto, os valores não excedem o valor máximo estabelecido. Não há diferença significativa entre as densidades e viscosidades desses óleos e as dos fluidos vegetais comerciais BIOTEMP, Envirotemp FR3. MATERIAL E MÉTODOS Foram avaliadas Cinco tipos de óleo vegetal refinados da classe comestível, disponíveis no mercado brasileiro, foram avaliados, dentre os quais os óleos de algodão, babaçu, girassol, milho e soja. Os óleos adquiridos apresentavam aspecto claro, límpido e isento de materiais em suspensão, como recomenda a NBR 15422 (ABNT 2006c). A observação do aspecto visual do óleo é item importante para fins de isolamento elétrico. A presença de materiais em suspensão, por exemplo, tem consequência direta nas características térmicas e dielétricas do fluido isolante. Cada amostra foi preservada em sua embalagem original, até o momento de uso. O óleo mineral regenerado foi doado pela Companhia Hidro- Elétrica do São Francisco (CHESF). Não se obtiveram R. Bras. Eng. Agríc. Ambiental, v.16, n.2, p.229–234, 2012. 231 Caracterização físico-química e dielétrica de óleos biodegradáveis para transformadores elétricos Os óleos vegetais objetos deste estudo, apresentaram acidez superior ao limite estabelecido na NBR 15422 (ABNT 2006c). Após esta constatação procedeu-se à neutralização de 1L de cada amostra de óleo, através do tratamento com solução de hidróxido de sódio; após a neutralização as caracterizações físico-químicas e dielétricas foram realizadas novamente a fim de verificar se o processo de neutralização modificou as características iniciais. informações a respeito das etapas pelas quais o óleo mineral foi submetido durante a regeneração; sabe-se, no entanto, que este processo é uma ação corretiva que se aplica para a retirada de água, compostos ácidos, gases dissolvidos e sedimentos do óleo envelhecido, recompondo suas características, deixando-os pronto para reutilização como fluido dielétrico. As propriedades avaliadas para cada espécie de óleo são descritas a seguir. A determinação da densidade dos óleos a 20 ºC foi realizada através do método da picnometria. Os experimentos foram realizados em triplicata. R. Bras. Eng. Agríc. Ambiental, v.16, n.2, p.229–234, 2012. RESULTADOS E DISCUSSÃO Densidade relativa e viscosidade cinemática dos óleos vegetais neutralizados Viscosidade (cSt) T = 20 ºC T = 40 ºC T = 100 ºC Tipo de óleo Limite NBR 15442 Densidade relativa* 0,96 máximo 150 máximo 50 máximo 15 máximo Algodão 0,9207 ± 000 66,81 ± 0,09 33,98 ± 0,09 8,04 ± 0,00 Babaçu - - 27,51 ± 0,04 6,50 ± 0,02 Girassol 0,9249 ± 0,00 70,16 ± 0,13 32,15 ± 0,09 8,14 ± 0,00 Milho 0,9221 ± 0,00 74,95 ± 0,13 34,80 ± 0,06 8,35 ± 0,05 Soja 0,9239 ± 0,00 67,99 ± 0,27 31,60 ± 0,00 8,27 ± 0,02 Mineral 0,8797 ± 0,01 20,90 ± 0,00 09,35 ± 0,01 2,74 ± 0,00 * densidade do óleo a 20 ºC em relação à água pura a 4 ºC ade relativa e viscosidade cinemática dos óleos vegetais neutralizados Tabela 2. Densidade relativa e viscosidade cinemática dos óleos vegetais neutralizados * densidade do óleo a 20 ºC em relação à água pura a 4 ºC corrosivo por se tratar de um fluido que passou por desgaste durante seu tempo de uso. a capacidade de dissipação de calor e levar o equipamento a sobrecargas de temperatura. Trata-se, portanto, de dois aspectos críticos para o funcionamento seguro de transformadores, especialmente em regiões de clima frio. Em função de densidade e viscosidade superior àquela apresentada pelo óleo mineral, o uso do óleo vegetal como isolante para equipamentos elétricos pode requerer mudanças no design e no sistema de bombeamento do fluido. girassol soja algodão babaçu milho mineral Figura 2. Fitas de cobre após ensaio de corrosividade dos óleos neutralizados girassol soja algodão girassol soja Na Figura 1 valores médios da viscosidade são comparados aos limites estabelecidos na NBR 15422 (ABNT, 2006c). Verifica- se que os óleos vegetais neutralizados (N) ou não, apresentam viscosidade dentro do limite estabelecido. Brock et al. (2008) encontraram resultados equivalentes. babaçu milho mineral Figura 2. Fitas de cobre após ensaio de corrosividade dos óleos neutralizados Figura 2. Fitas de cobre após ensaio de corrosividade dos óleos neutralizados Algodão Algodão (N) Babaçu Babaçu (N) Girassol Girassol (N) Milho Milho (N) Soja Soja (N) Mineral 0 30 60 90 120 150 Limite para Temp. 100°C NBR 15422 Limite para Temp. 40°C NBR 15422 Viscosidade Cinemática (cSt) Temp. 20°C Temp. 40°C Temp. 100°C Limite para Temp. 20°C NBR 15422 Figura 1. RESULTADOS E DISCUSSÃO Por outro lado, o óleo de babaçu é pastoso e opaco, mesmo a 25 °C, devido à elevada percentagem de triaciglicerídeos saturados naturalmente presentes na sua composição. A verificação da corrosividade do óleo por presença de compostos de enxofre foi realizada pela imersão de fitas de cobre em amostras de óleo. Os frascos contendo óleo de fita foram fechados e levados à estufa a 150 °C, por 48 h, conforme procedimento descrito na NBR 10505 (ABNT, 2006a). Passado o tempo do teste, realizou-se a avaliação da cor das fitas de cobre e se classificou o óleo como corrosivo ou não-corrosivo, de acordo com a escala de cores apresentada na norma mencionada. O experimento foi realizado em triplicata. O teor de água das amostras foi verificado pelo método Karl Fischer, usando-se o Titulador Water Content Measuring Test Equipment Aquameter KFM 3000 fabricado pela Baur e de acordo com os critérios recomendados pela NBR 10710 (ABNT, 2006b). Assim, na temperatura de ensaio de 20 °C o óleo não apresenta características fluidodinâmicas adequadas para a utilização como fluido isolante de transformadores; apesar disto, para as temperaturas mais elevadas (40 e 100 °C) o óleo de babaçu apresenta menor viscosidade que os demais óleos vegetais testados. Amostras de óleo foram submetidas a uma tensão elétrica sob condições prescritas no método de ensaio NBR 6869 (ABNT, 1989), exceto pelo fato de que o aumento de tensão foi de 30kV min-1, devido à limitação do equipamento usado: o Fully Automatic Insulating Oil Tester DTA 100 E fabricado pela Baur. As normas recomendam 3kV min-1. Como o resfriamento dos transformadores é realizado pelo fluxo convectivo do líquido isolante, tanto a viscosidade quanto a densidade do óleo são fatores importantes do ponto de vista da transferência de calor. Assim, baixas viscosidade e densidade do fluido isolante são desejáveis para facilitar sua circulação; ao passo em que forem excessivamente elevadas, podem inibir O fator de perdas dielétricas foi determinado através do Sistema Automático para a Medida do Fator de Dissipação e Resistividade DTL, fabricado pela Baur. A norma usada como referência para o ensaio foi a NBR 12133 (ABNT, 1991). R. Bras. Eng. Agríc. Ambiental, v.16, n.2, p.229–234, 2012. Claudia R. Silva et al. 232 Tabela 2. RESULTADOS E DISCUSSÃO Viscosidade dos óleos a 20, 40 e 100°C comparada aos limites estabelecidos na NBR 15422 Fonte: ABNT, 2006c O caráter não corrosivo apresentado pelo óleo vegetal é um aspecto bastante positivo (Castelo-Branco & Torres, 2011), haja vista que as reações de corrosão produzem sulfeto de cobre que tem elevada condutividade e, ao se dispersar sobre o papel isolante do transformador, reduz sua rigidez dielétrica. O ensaio NBR 10505 é um teste bastante usado no setor elétrico para verificar corrosividade dos óleos. Entretanto, trata- se de um teste puramente qualitativo que não deve ser considerado conclusivo para a presença ou não de compostos de enxofre. É interessante aliar este resultado em relação a outras técnicas de caracterização, para maior confiabilidade. O caráter não corrosivo apresentado pelo óleo vegetal é um aspecto bastante positivo (Castelo-Branco & Torres, 2011), haja vista que as reações de corrosão produzem sulfeto de cobre que tem elevada condutividade e, ao se dispersar sobre o papel isolante do transformador, reduz sua rigidez dielétrica. Viscosidade Cinemática (cSt) O ensaio NBR 10505 é um teste bastante usado no setor elétrico para verificar corrosividade dos óleos. Entretanto, trata- se de um teste puramente qualitativo que não deve ser considerado conclusivo para a presença ou não de compostos de enxofre. É interessante aliar este resultado em relação a outras técnicas de caracterização, para maior confiabilidade. O ensaio NBR 10505 é um teste bastante usado no setor elétrico para verificar corrosividade dos óleos. Entretanto, trata- se de um teste puramente qualitativo que não deve ser considerado conclusivo para a presença ou não de compostos de enxofre. É interessante aliar este resultado em relação a outras técnicas de caracterização, para maior confiabilidade. Fofana et al. (2010) correlacionaram o teor de água com as propriedades dielétricas dos óleos vegetais e mineral, convertendo as umidades absolutas em relativas de acordo com a Eq. 2. O cálculo da umidade relativa é importante porque óleos de origem mineral e vegetal possuem solubilidades diferentes. Na temperatura de 25 °C óleos de origem vegetal absorvem aproximadamente 1000 ppm de água enquanto o óleo mineral absorve entre 55 e 60 ppm (Oommen & Claiborne, 1999). A umidade relativa do óleo (Wrel) corresponde à quantidade de água dissolvida no óleo em relação à capacidade máxima de umidade que o óleo pode suportar. Figura 1. Wabs - teor de água absoluto do óleo WL(T) - limite de saturação do óleo R. Bras. Eng. Agríc. Ambiental, v.16, n.2, p.229–234, 2012. RESULTADOS E DISCUSSÃO A presença de umidade é um aspecto que merece atenção pois acelera a reação com a celulose do papel isolante, conduzindo-a à degradação. Além disso, também provoca diminuição da eficiência de impregnação do óleo no papel isolante, levando a uma diminuição do ciclo de vida do sistema de isolamento. Desta forma, o teor de água apresentado pelos óleos vegetais comestíveis é um parâmetro que precisa ser melhorado para o funcionamento seguro do equipamento que venha a utilizá-los como líquido dielétrico, independente das umidades relativas. (1) Realizou-se a secagem de óleo de girassol utilizando-se estufa a vácuo (100 mm Hg) a 60 e 80 °C durante seis horas e se constatou que referidas condições não foram eficientes para remoção de umidade a um nível exigido para uso do óleo como isolante elétrico. Obteve-se, como resultado, um óleo com cerca de 700 ppm de água. Também se realizou a secagem em estufa convencional a 100 °C durante seis horas, condições induziram ao primeiro sinal de oxidação do óleo, o ranço. Concluiu-se, portanto, que é preciso investir em métodos alternativos que diminuam o teor de água sem comprometer outras propriedades do óleo como, por exemplo, processos adsortivos para retirada de água (Oliveira et al., 2010; El-Din et al., 2011). Algodão Babaçu Girassol Milho Soja Figura 3. Rigidez dielétrica (A) e fator de perdas (B) dos óleos vegetais antes e após neutralização o óleo de milho que apresentou rigidez dielétrica superior a 40 kV; os demais óleos não apresentaram a rigidez dielétrica mínima recomendada para fluidos dielétricos. O fator de perdas máximo especificado na NBR 15422 (ABNT, 2006c) nas condições ensaiadas, é 4,0%. Como visto na Figura 3B, os óleos de soja e babaçu comerciais não neutralizados não atenderam a este requisito, indicando a presença de umidade e outros produtos de deterioração do óleo. As Figuras 3A e 3B apresentam os gráficos com os valores médios da rigidez diétrica e perda de carga dos óleos vegetais não neutralizados e neutralizados. Observa-se, na Figura 3A, que as cinco espécies de óleos vegetais não neutralizados apresentaram rigidez dielétrica, inferior ao mínimo especificado na NBR 15422, que é 30 kV. Este fato pode estar relacionado ao elevado teor de água, para níveis de isolação elétrica. O máximo teor de água recomendado em norma é 200 ppm. RESULTADOS E DISCUSSÃO Viscosidade dos óleos a 20, 40 e 100°C comparada aos limites estabelecidos na NBR 15422 Fonte: ABNT, 2006c O resultado dos testes de corrosividade dos óleos neutralizados, apresentados na Figura 2, indica que os óleos vegetais não apresentaram comportamento corrosivo, considerando ainda que não houve mudança na coloração das fitas de cobre submetidas ao ensaio. Este comportamento não foi verificado para as fitas de cobre submetidas ao teste na presença do óleo mineral regenerado, classificado como corrosivo. De modo geral, os óleos minerais novos apresentam comportamento não corrosivo quando testados sob as condições ensaiadas de acordo com a NBR 10505 (ABNT, 2006a). Supõe-se que o óleo mineral apresentou comportamento ) T ( W W W L abs rel  (2) (2) onde: Wabs - teor de água absoluto do óleo WL(T) - limite de saturação do óleo Wabs - teor de água absoluto do óleo WL(T) - limite de saturação do óleo R. Bras. Eng. Agríc. Ambiental, v.16, n.2, p.229–234, 2012. 233 Caracterização físico-química e dielétrica de óleos biodegradáveis para transformadores elétricos A. B. Fator de perdas a 100 oC (%) Rigidez dielétrica (Kv) Algodão Babaçu Girassol Milho Soja Figura 3. Rigidez dielétrica (A) e fator de perdas (B) dos óleos vegetais antes e após neutralização As amostras de óleos vegetais comerciais não neutralizados e após neutralização apresentaram teor de água absoluto entre 843 ± 8 e 961 ± 3 ppm; já o óleo mineral apresentou teor de água absoluto de 50 ± 3 ppm. Os valores de Wabs são bem distintos do óleo mineral quando comparados com os de qualquer óleo vegetal avaliado no presente trabalho. Entretanto, as umidades relativas são próximas, independente do óleo ser de origem vegetal ou mineral. Os valores de Wrel dos óleos vegetais variaram entre 84,3±0,8 (óleo de algodão) e 96,1±0,3 (óleo de babaçu) sendo o valor do óleo mineral igual a 90,9±0,3. O valor deste último praticamente é a média entre os valores de menor e maior umidade relativa dos óleos vegetais. Este fato se deve aos limites de saturação dos óleos vegetais e minerais WL(T), na temperatura estudada, que são semelhantes aos das umidades absolutas Wabs, 800 a1000 ppm para os óleos vegetais e 50 a 60 ppm para o óleo mineral. Resultados semelhantes têm sido reportados na literatura (Fofana et al., 2010). RESULTADOS E DISCUSSÃO Tem-se verificado que a rigidez dielétrica de óleos vegetais se mantém acima de 30 kV para concentrações de água abaixo de 500 ppm (Lizhi et al., 2008). Nenhum dos óleos vegetais estudado neste trabalho possui teor de água igual ou menor que o recomendado pela norma. O processo de neutralização também favoreceu a diminuição das perdas dielétricas, o que pode ser constatado na Figura 3B. Acredita-se então, que a melhoria nas propriedades dielétricas pode estar relacionada à remoção de partículas condutoras durante a neutralização. Independentemente do teor de água, umidade relativa dos óleos neutralizados ou não neutralizados serem aproximadamente os mesmos e embora o óleo seja comestível, conduz impurezas para níveis de isolamento elétrico. Como já mencionado, o óleo de babaçu apresentou a maior umidade relativa dentre os óleos avaliados e, consequentemente, menor rigidez dielétrica. O óleo mineral também apresentou baixa rigidez dielétrica. R. Bras. Eng. Agríc. Ambiental, v.16, n.2, p.229–234, 2012. LITERATURA CITADA ABNT - Associação Brasileira de Normas Técnicas. NBR 6869: Determinação de rigidez dielétrica de óleos isolantes (Eletrodos de disco). Rio de Janeiro: ABNT, 1989. 13p. McShane, C. P.; Corkran, J. L.; Harthun, R. A.; Gauger, G. A.; Rapp, K. J.; Howells, E.; US Patent 6.037,537, Cooper Industries, Inc., Houston, TX. Vegetable oil based dielectric coolant. 2000. ABNT - Associação Brasileira de Normas Técnicas. NBR 12133: Líquidos isolantes elétricos - Determinação do fator de perdas dielétricas e da permissividade relativa constante dielétrica). Rio de Janeiro: ABNT, 1991. 13p. Meshram, P. D.; Puri, R. G.; Patil, H. V.; Epoxidation of wild safflower (Carthamus oxyacantha) oil with peroxy acid in presence of strongly acidic cation exchange Resin IR-122 as Catalyst. International Journal of ChemTech Research. v.3, p.1152-1158, 2011. ABNT - Associação Brasileira de Normas Técnicas. NBR 10505: Óleo mineral isolante - determinação de enxofre corrosivo. Rio de Janeiro: ABNT, 2006a. 13p. Oliveira, J. F. G.; Lucena I. L.; Saboya, R. M. A.; Rodrigues, M. L.; Torres, A. E. B. Fernandes, F. A. N.; Cavalcante Jr., C. L.; Parente, J. E. J. S. Biodiesel production from waste coconut oil by esterification with ethanol: The effect of water removal by adsorption. Renewable Energy, v.35, p.2581-2584, 2010. ABNT - Associação Brasileira de Normas Técnicas. NBR 10710: Determinação de água em líquidos isolantes (Método Karl- Fischer). Rio de Janeiro: ABNT, 2006b. 13p. ABNT - Associação Brasileira de Normas Técnicas. NBR 15422: Óleo vegetal isolante para equipamentos elétricos. Rio de Janeiro: ABNT, 2006c. 13p. Oommen, T. V.; Claiborne C. C.; US Patent 5, 949, 017 1999. ABB Power T&D Company Inc. (Raleigh, NC). Electrical transformers containing electrical insulation fluids comprising high oleic acid compositions. Al-Ammar, E. A. Evaluation of seed oils based on statistical breakdown data for their application as insulating fluids in distribution transformers. European Journal of Scientific Research, v.40, p.15-26, 2010. Ribeiro, W. C. Geografia política e gestão internacional dos recursos naturais. Revista Engenharia Sanitária e Ambiental, v.24, p.69-80, 2010. Brock, J.; Nogueira, M. R.; Zakrzevski, C.; Corazza, F. C.; Corazza, M. L.; Oliveira, J. V. Determinação experimental da viscosidade e condutividade térmica de óleos vegetais. Ciência e Tecnologia de Alimentos, v.28, p.564-570. 2008. Shah, Z. H.; Tahir, Q. A. Dielectric properties of vegetable oils. Journal of Scientific Research. v.3, p.481-492, 2011. Shogren R. L.; Petrovic, Z.; Liu, Z.; Erhan, S. Z. Biodegradation behavior of some vegetable oil-based polymersa. Ao CNPq, pelo apoio financeiro. Mariano, A. P.; Bonotto, D. M.; Angelis, D. F.; Pirôllo, M. P. S.; Contiero, J. Biodegradabilidade de óleos diesel comercial e intemperizado. Brazilian Journal of Microbiology, v.39, p.133- 142, 2008. CONCLUSÕES 1. Os óleos de algodão, girassol, milho e soja, possuem viscosidade e densidade adequadas para uso como isolante Na Figura 3A verifica-se que os óleos neutralizados apresentaram melhoria na rigidez dielétrica, com destaque para Claudia R. Silva et al. 234 Ferreira Junior, W. A CPFL cumpre seu papel, Revista P&D: Projetos ambientais. ANEEL, v.1, p.23-24, 2006. em transformador. O óleo de babaçu não possui fluidodinâmica adequada a 20 °C. em transformador. O óleo de babaçu não possui fluidodinâmica adequada a 20 °C. Fofana, I.; Hemmatjou, H. Farzaneh, M.; Low temperature and moisture effects on polarization and depolarization currents of oil paper insulation, Electric Power Systems Research, v.80, p.91-97, 2010. q 2. Os óleos vegetais não apresentaram comportamento corrosivo detectável, com a aplicação da metodologia recomendada pela NBR 10505 (ABNT, 2006a). q 2. Os óleos vegetais não apresentaram comportamento corrosivo detectável, com a aplicação da metodologia recomendada pela NBR 10505 (ABNT, 2006a). 3. Os cinco tipos de óleo vegetal não neutralizados apresentaram rigidez dielétrica inferior à especificada na NBR 15422 (ABNT, 2006c). 3. Os cinco tipos de óleo vegetal não neutralizados apresentaram rigidez dielétrica inferior à especificada na NBR 15422 (ABNT, 2006c). Georgilakis, P. S. Environmental cost of distribution transformer losses. Applied Energy, v.88, p.3146–3155, 2011. Kwofie, A. B.; Yeboah, P. O.; Pwamang, J. Determination of levels of polychlorinated biphenyl in transformers oil from some selected transformers in parts of the Greater Accra R i f Gh Ch h 82 103 106 2011 4. O processo de neutralização favoreceu a diminuição das perdas dielétricas e o aumento da rigidez dielétrica. Dentre os óleos neutralizados, o óleo de milho apresentou rigidez dielétrica superior ao mínimo recomendado para fluidos dielétricos. Region of Ghana, Chemosphere, v.82, p.103–106, 2011. Life, K.; Hilary, I.; Inyang, J. W.; Hilger, H. Aromatic and aliphatic hydrocarbon balance in electric transformer oils. Fuel, v.89, p.3114–3118, 2010. AGRADECIMENTOS Lizhi, H.; Toyoda, K.; Ihara I. Dielectric properties of edible oils and fatty acids as a function of frequency, temperature, moisture and composition, Journal of Food Engineering, v.88, p.151–158, 2008. LITERATURA CITADA Journal of Polymers and the Environment, v.12, p 50-56, 2004. Cannon, G. S.; Honary, L.; US Patent 5.958,851, Soybean based transformer oil and transmission line fluid. 1999. 9p. Carioca, J. O. B.; Correia, R. G.; Hiluy, J. J. F.; Macambra, S. J. Green dielectric oils; problems and perspectives, Journal of Biotechnology, v.1, p.120-128 2010. Sidibé, S. S.; Blin, J.; Vaitilingom, G.; Azoumah, Y. Use of crude filtered vegetable oil as a fuel in diesel engines state of the art: Literature review Renewable and Sustainable. Energy Reviews, v.14, p.2748–2759, 2010. Castelo-Branco, V. N.; Torres, A. G. Capacidade antioxidante total de óleos vegetais comestíveis: determinantes químicos e sua relação com a qualidade dos óleos. Revista de Nutrição, v.24, p.173-187, 2011. Wilhelm, H. M.; Stocco, M. B. C.; Oliveira, J. de; Wilson, U. Gomes Junior, S. B. Investigacao de oleos vegetais como potenciais fluidos de seguranca. Revista Eletricidade Moderna, v.1, p.140-147, 2009. El-Din, M. G.; Hongjing, F.; Wang, N.; Ayala, P. C. Leonidas, P. E.; Przemyslaw, D.; Martin, J. W.; Zubot, W.; Smith, D. W. Naphthenic acids speciation and removal during petroleum-coke adsorption and ozonation of oil sands process-affected water. Science of the Total Environment, v.409, p.5119-5125, 2011. Zhang, G.; Yongnian, N.; Churchill, J.; Kok, S. Authentication of vegetable oils on the basis of their physico-chemical properties with the aid of chemometrics.Talanta, v.70, p.293– 300, 2006. R. Bras. Eng. Agríc. Ambiental, v.16, n.2, p.229–234, 2012.
https://openalex.org/W2000320433
https://europepmc.org/articles/pmc3087315?pdf=render
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Tropomyosin3 overexpression and a potential link to epithelial-mesenchymal transition in human hepatocellular carcinoma
BMC cancer
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7,505
RESEARCH ARTICLE Open Access © 2010 Choi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tropomyosin3 overexpression and a potential link to epithelial-mesenchymal transition in human hepatocellular carcinoma Hye-Sun Choi1,2, Seon-Hee Yim2, Hai-Dong Xu1,2, Seung-Hyun Jung1,2, Seung-Hun Shin1,2, Hae-Jin Hu1,2, Chan-Kwon Jung3, Jong Young Choi4, Yeun-Jun Chung1,2* Abstract Background: Since hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, it is still important to understand hepatocarcinogenesis mechanisms and identify effective markers for tumor progression to improve prognosis. Amplification and overexpression of Tropomyosin3 (TPM3) are frequently observed in HCC, but its biological meanings have not been properly defined. In this study, we aimed to elucidate the roles of TPM3 and related molecular mechanisms. Methods: TPM3-siRNA was transfected into 2 HCC cell lines, HepG2 and SNU-475, which had shown overexpression of TPM3. Knockdown of TPM3 was verified by real-time qRT-PCR and western blotting targeting TPM3. Migration and invasion potentials were examined using transwell membrane assays. Cell growth capacity was examined by colony formation and soft agar assays. Results: Silencing TPM3 resulted in significant suppression of migration and invasion capacities in both HCC cell lines. To elucidate the mechanisms behind suppressed migration and invasiveness, we examined expression levels of Snail and E-cadherin known to be related to epithelial-mesenchymal transition (EMT) after TPM3 knockdown. In the TPM3 knockdown cells, E-cadherin expression was significantly upregulated and Snail downregulated compared with negative control. TPM3 knockdown also inhibited colony formation and anchorage independent growth of HCC cells. Conclusions: Based on our findings, we formulate a hypothesis that overexpression of TPM3 activates Snail mediated EMT, which will repress E-cadherin expression and that it confers migration or invasion potentials to HCC cells during hepatocarcinogenesis. To our knowledge, this is the first evidence that TPM3 gets involved in migration and invasion of HCCs by modifying EMT pathway. Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 * Correspondence: yejun@catholic.ac.kr 1Department of Microbiology, School of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea HCC cell lines HepG2 was obtained from ATCC (American Type Cul- ture Collection, Manassas, VA) and maintained in DMEM (Gibco BLR, Gaithersburg, MD) supplemented with 10% FBS. SNU-739, 423, 449, 886, 475, 878, 387, 398, and 761 were obtained from the Korean cell-line bank (Seoul, Korea) and maintained in RPMI 1640 (Hyclone, Logan, UT) supplemented with 10% FBS at 37°C in humidified air containing 5% CO2. THLE-3 (a human normal liver cell) was purchased from ATCC (Manassas, VA) and main- tained in DMEM supplemented with 10% FBS, 25 mM HEPES buffer and 100 U/ml of penicillin. Quantitative RT-PCR Q Total RNA was extracted from the HCC cell lines using TRIzol (Invitrogen, Carlsbad, CA) according to the man- ufacturer’s instructions. First-strand complementary DNA (cDNA) was synthesized from 5 μg of total RNA using oligo-dT primer and superscript II reverse tran- scriptase (Invitrogen, Carlsbad, CA). To determine the levels of TPM3 messenger RNA (mRNA) expression, real-time qRT-PCR was performed using Mx3000P QPCR System and software MxPro Version 3.00 (Strata- gene, La Jolla, CA). Reaction mixture was composed of 1 × SYBR Green Tbr polymerase Master Mix (FINN- ZYMES, Finland), 0.5 × ROX and 20 pmol of each primer, and 10 ng of cDNA. Primers for TPM3 were 5’- GAGAGGTATGAAGGTTATTCA-3’ for forward and 5’-ATCACCACCTTACGAGCCACC-3’ for reverse. GAPDH was used as internal control. GAPDH primers were designed as 5’-GCGGGGCTCCAGAACATCAT-3’ for forward and as 5’- CCAGCCCCAGCGTCAAGGTG- 3’ for reverse. RNA levels of E-Cadherin and Snail were measured using the following primers according to previous reports [15,16]; for Snail, 5’ -AAG- GATCTCCAGGCTCGAAAG-3’ for forward and 5’- GCTTCGGATGTGCATCTTGA-3’ for reverse; for E-cadherin, 5’-TCGACACCCGATTCAAAGTGG-3’ for forward and 5’- TTCCAGAAACGGAGGCCTGAT -3’ for reverse. The PCR program was as follows: denatura- tion at 95°C for 5 minutes; 40 cycles of 95°C for 30 sec- onds, 60°C for 30 seconds, and 72°C for 40 seconds followed by a 72°C elongation step for 6 minutes. Rela- tive expression quantification was performed by the ΔΔCT method [17]. All the experiments were repeated three times and the mean value of intensity ratios with the SD was plotted for each case. In this study, we explored the biological roles of TPM3 in hepatocarcinogenesis and involved molecular mechanisms by TPM3 knockdown using small interfer- ing RNA (siRNA) in human HCC cell lines. Transfection of TPM3 siRNAs We adopted a forward transfection method. In brief, transfection was performed by adding the mixture of siRNA and the transfection reagent (lipofectamine RNi- MAX, Invitrogen, Carlsbad, CA) onto the cells after the cell seeding. HepG2 and SNU475 cells were seeded at a density of 200,000 and 100,000 cells/well in six-well plates, respectively, and incubated for 24 hours at 37°C with 5% CO2. After 24 hour incubation, HCC cells were transfected with 100 nM siRNAs (2 TPM3 siRNAs and a negative control siRNA) using 1.25 μg/ml lipofecta- mine RNAiMax (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. After 48 hours follow- ing the transfection, HCC cells were harvested and silencing of the TPM3 expression was validated by real- time quantitative RT-PCR (qRT-PCR) and western blotting. Lines of evidence have suggested that non-muscular tropomyosins might be involved in tumor development. TPM3 was reported to be involved in hematopoietic tumorigenesis by forming a TPM3-ALK fusion through (1;2) translocation [8,9]. TPM3 is also known as an inducer of papillary thyroid carcinoma and chronic eosi- nophilic leukemia through a fusion with NTRK1 and PDGFRB [10,11]. In addition, tropomyosin family mem- bers have been reported to be related with tumor cell movement or invasion [12,13]. In Miyado et al.’s obser- vation, the expression level of a low-molecular weight tropomyosin isoform, TM5/TM30nm, was higher in a highly metastatic mouse melanoma cell line than in a low-metastatic one [14]. This evidence suggests that overexpression of TPM3 may contribute to invasion or migration potentials of human malignancies including HCC, but molecular mechanisms behind this has not been explored. Background markers for early diagnosis and accurate prognostication which reflect biological phenomena well. Hepatocellular carcinoma (HCC) is one of the most common human malignancies and the third leading cause of cancer-related death in the world [1]. A num- ber of studies have been suggesting the molecular mechanisms involved in hepatocarcinogenesis such as MAPK, EGFR, p53, Wnt, TGF-B, Ras and Rb pathways [2-5]. However, given that prognosis of the disease remains poor, it is still important to understand hepato- carcinogenesis mechanisms and to identify effective In our recent study which reported the chromosomal alterations in HCC by genome-wide array-CGH analysis, we found that a 1q21.3 locus was recurrently amplified and that a Tropomyosin 3 (TPM3) gene located in this region was coherently overexpressed in primary HCC [6]. This evidence suggests that overexpression of TPM3 may play a role in HCC tumorigenesis. TPM3 is an actin-binding protein present in skeletal and smooth muscle and some non-muscular tissues. In skeletal mus- cle, TPM3 mediates a myosin-actin response to calcium ions and takes part in the stabilization of cytoskeletal * Correspondence: yejun@catholic.ac.kr 1Department of Microbiology, School of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 Page 2 of 11 microfilaments [7]. On the contrary, the function of TPM3 in non-muscular tissues is still obscure. siRNA oligonucleotides We purchased two synthetic double-stranded oligonu- cleotides with the following sequences and introduced them into the pSilencer 3.1-H1 neo siRNA expression vector (Invitrogen, Carlsbad, CA); TPM3 RNAi-1, AGC AUU CUC CUU GUC UAA CUU CAG C: GCU GAA GUU AGA CAA GGA GAA UGC U; TPM3 RNAi-2, UAA CCU UCA UAC CUC UCU CAC UCU C: GAG AGU GAG AGA GGU AUG AAG GUU A. To verify sequence-specific effectiveness of TPM3-RNAi, we used a negative control siRNA (Invitrogen, Carlsbad, CA) that has no significant homology with any known sequences in the human genome. Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 Page 3 of 11 Immunofluorescence staining An independent samples t-test was used to test the sig- nificance of difference between groups and P values < 0.05 were considered significant. Data were analyzed using Stata version 10 software (Stata Corporation, Col- lege Station, TX). The samples of siTPM3 and siNEG transfected cells (SNU-475 and HepG2) were cytocentrifuged onto the slides and immediately fixed with ethyl alcohol for 30 minutes. For immunofluorescence staining, slides were exposed to 0.2% Tween 20 in PBS for 30 minutes and incubated overnight at 4°C with monoclonal antibo- dies against vimentin (1:100, clone V9, DakoCytoma- tion, Glostrup, Denmark) and fibronectin (1:100, clone 568, Novocastra, Newcastle upon Tyne, UK). After thorough washing, cells were incubated with a 1:500 dilution of Alexa Fluor 488-conjugated goat anti- mouse IgG antibody (Invitrogen - Molecular Probes, Eugene, OR) for 30 minutes at room temperature in the dark. Staining of nuclei with diaminophenylindole (Molecular Probes) was also performed. Staining pat- terns were observed by a fluorescence microscope (Carl Zeiss, Axio Imager M1, Oberkochen, Germany) (400×). Western blot analysis transwell membrane was coated with 500 ng/μL of Matrigel (BD Biosciences, San Jose, CA) and incubated for 24 hours at 37°C. Proteins were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membrane was blocked with 5% non-fat dried milk in TBST(20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20, pH 7.5) for 2 hours and incubated overnight with antibodies against TPM3 (1:1,000 dilution; Abnova, Taipei, Taiwan), a-tubulin (1:1,000 dilution; Santa Cruz biotechnology, Santa Cruz, CA), Snail (1:500 dilution; Abcam, Cambridge, UK), E- cadherin (1:1000 dilution; Zymed, San Francisco, CA) at 4°C. After the wash with TBST buffer, membranes were incubated with horseradish peroxidase-conjugated anti mouse IgG secondary antibodies for 1 hour at room temperature and detected by enhanced chemilumines- cence detection system (Amersham-Pharmacia Biotech, Braunschweig, Germany). Suppression of TPM3 expression by siRNA transfection Suppression of TPM3 expression by siRNA transfection For TPM3 knockdown, we transfected two siRNA con- structs into those 8 cell lines which showed TPM3 overexpression compared with the THLE-3 on both mRNA and protein levels; siTPM3-1 and siTPM3-2 targeting exons 1 and 3/4, respectively. As a control, a negative oligonucleotide construct (siNEG) was trans- fected into the same 8 cell lines. As siTPM3-1 showed better knockdown effects between the two siRNA con- structs and the best interfering efficiency was observed in SNU-475 and HepG2 cell lines (data not shown), all the downstream functional analyses were performed using siTPM3-1 (herein after called siTPM3) in these two cell lines. Figure 2 presents real-time qRT-PCR and western blotting results showing the repressed expression of TPM3 induced by transfecting siTPM3. In HepG2, relative TPM3 mRNA expression ratios (siTPM or siNEG/no transfection control) were 1.00 (95% CI 0.76-1.24) and 0.04(95% CI 0.03-0.06) in Colony formation and soft agar assays For the colony formation assay, siRNA-treated and negative control-treated HCC cells (1 × 104) were seeded in 10 cm dishes. Two weeks later, cells were washed with PBS buffer and stained with 0.5% crystal violet in 20% methanol for 20 minutes and the number of colonies was counted. For the soft agar assay, HCC cells were suspended in RPMI1640 containing 0.35% low melting agarose, and plated onto solidified 0.6% agarose containing RPMI1640 in six-well culture plates at a density of 1 × 105 cells per dish. The number and size of colonies were observed 3 weeks after seeding under the microscope (×40). Elevated TPM3 expression in HCC cell lines Elevated TPM3 expression in HCC cell lines We firstly screened baseline TPM3 expression levels in 10 HCC cell lines. In eight out of the 10 HCC cell lines except for SNU-398 and SNU-886, both the mRNA (>1.5 fold) and protein expression levels of TPM3 were found to be increased with respect to the normal liver cell line (THLE-3) (Figure 1). Migration and invasion assays Migration of HCC cells was assayed using the transwell with 8-μm pore filters (Costar, Boston, MA). After filling the lower chamber with complete media, 2 × 104 HCC cells in 0.5 mL serum-free media were loaded onto the upper chamber. After incubation for 12 hours at 37°C, cells that migrated to the bottom surface of the mem- brane were fixed with methanol and stained with 0.5% crystal violet and then subjected to microscopic inspec- tion. Cells on the top surface of the membrane were removed by wiping with a cotton swab. The numbers of cells were counted in five microscopic fields (×200). For the Matrigel invasion assay, the procedures were same as those for the cell migration assay, except that the Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 Page 4 of 11 http://www.biomedcentral.com/1471-2407/10/122 Figure 1 TPM3 expression levels in various human HCC cell lines. One normal human liver cell line (THLE-3) and 10 types of HCC cell lines were examined by TPM3-specific real-time qRT-PCR (top plot) and western blot (bottom plot). Human GAPDH gene was used as internal control for qRT-PCR and alpha-tubulin was used as internal control for western blot analysis. In the top plot, X axis represents cell lines and Y axis relative TPM3 expression ratios (tumor/normal). Error bars represent mean ± standard error of mean. Figure 2 Suppressed TPM3 expression after siTPM3 transfection into HepG2 (A) and SNU-475 (B). TPM3 expression was measured by real-time qRT-PCR (top plots of A and B). X axis represents samples and Y axis relative TPM3 expression ratio (siTPM or siNEG/control). Error bars represent mean ± standard error of mean. Human GAPDH gene was used as internal control. TPM3 expression was also measured by western blot (bottom plots of A and B). TPM3 band intensities of siTPM-treated cells are much weaker than those of siNEG-treated ones and control, while internal control bands are consistent. Alpha-tubulin was used as internal control for western blot analysis. siTPM3, siTPM3 transfected HCC cell line; siNEG, negative oligonucleotide (siNEG) transfected HCC cell line; control, HCC cell line without transfection. Figure 1 TPM3 expression levels in various human HCC cell lines. One normal human liver cell line (THLE-3) and 10 types of HCC cell lines were examined by TPM3-specific real-time qRT-PCR (top plot) and western blot (bottom plot). Migration and invasion assays Human GAPDH gene was used as internal control for qRT-PCR and alpha-tubulin was used as internal control for western blot analysis. In the top plot, X axis represents cell lines and Y axis relative TPM3 expression ratios (tumor/normal). Error bars represent mean ± standard error of mean. Figure 1 TPM3 expression levels in various human HCC cell lines. One normal human liver cell line (THLE-3) and were examined by TPM3-specific real-time qRT-PCR (top plot) and western blot (bottom plot). Human GAPDH gene was for qRT-PCR and alpha-tubulin was used as internal control for western blot analysis. In the top plot, X axis represents c relative TPM3 expression ratios (tumor/normal). Error bars represent mean ± standard error of mean. Figure 2 Suppressed TPM3 expression after siTPM3 transfection into HepG2 (A) and SNU-475 (B). TPM3 expression was measured by real-time qRT-PCR (top plots of A and B). X axis represents samples and Y axis relative TPM3 expression ratio (siTPM or siNEG/control). Error bars represent mean ± standard error of mean. Human GAPDH gene was used as internal control. TPM3 expression was also measured by western blot (bottom plots of A and B). TPM3 band intensities of siTPM-treated cells are much weaker than those of siNEG-treated ones and control, while internal control bands are consistent. Alpha-tubulin was used as internal control for western blot analysis. siTPM3, siTPM3 transfected HCC cell line; siNEG, negative oligonucleotide (siNEG) transfected HCC cell line; control, HCC cell line without transfection. Figure 2 Suppressed TPM3 expression after siTPM3 transfection into HepG2 (A) and SNU-475 (B). TPM3 expression was measured by real-time qRT-PCR (top plots of A and B). X axis represents samples and Y axis relative TPM3 expression ratio (siTPM or siNEG/control). Error bars represent mean ± standard error of mean. Human GAPDH gene was used as internal control. TPM3 expression was also measured by western blot (bottom plots of A and B). TPM3 band intensities of siTPM-treated cells are much weaker than those of siNEG-treated ones and control, while internal control bands are consistent. Alpha-tubulin was used as internal control for western blot analysis. siTPM3, siTPM3 transfected HCC cell line; siNEG, negative oligonucleotide (siNEG) transfected HCC cell line; control, HCC cell line without transfection. Page 5 of 11 Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 Effects of TPM3 silencing on HCC cell migration and invasion Effects of TPM3 silencing on HCC cell migration and invasion siNEG- and siTPM3-transfected cells, respectively (P < 0.0001). In SNU-475, relative TPM3 expression ratios were 0.91(95% CI 0.77-1.05) and 0.07(95% CI 0.06-0.08) in siNEG- and siTPM3-transfected cells, respectively (P < 0.0001). TPM3 protein expression in both siTPM-treated cells is much weaker than those in siNEG-treated ones, while alpha-tubulin bands are consistent (Figure 2A and 2B). In order to explore the potential role of TPM3 on the invasiveness of HCC cells, we performed cell migration and invasion assays using siTPM3-treated HepG2 and SNU-475. Both migration and invasion capacities were found to be profoundly repressed in siTPM3-treated cells (Figure 3). In both cell lines, the numbers of Figure 3 Repressed migration and invasion in TPM3 knockdown HCC cell lines. (A) Migration of siTPM and siNEG transfected HCC cell lines was examined using the Matrigel uncoated transwell membrane. After crystal violet staining, the numbers of colonies in five microscopic fields (X200) were counted. In HepG2, 332.0 in siNEG-treated cells (95% CI 323.0-341.0) versus 29.3 in siTPM3-treated cells (95% CI 25.5-33.1), P < 0.0001; In SNU-475, 359.3(95% CI 350.6-368.1) versus 125.3(95% CI 114.1-136.5), P < 0.0001. (B) Invasion of siTPM and siNEG transfected HCC cell lines was examined using the Matrigel coated transwell membrane. In HepG2, 212.7 in siNEG-treated cells (95% CI 206.4-218.9) versus 43.0 in siTPM3- treated cells (95% CI 36.4-49.6), P < 0.0001; In SNU-475, 162.7(95% CI 156.4-168.9) versus 33.7(28.5-38.8), P < 0.0001. Error bars represent mean ± standard error of mean. * represents P value < 0.05. Figure 3 Repressed migration and invasion in TPM3 knockdown HCC cell lines. (A) Mig migration and invasion in TPM3 knockdown HCC cell lin Figure 3 Repressed migration and invasion in TPM3 knockdown HCC cell lines. (A) Migration of siTPM and siNEG transfected HCC cell lines was examined using the Matrigel uncoated transwell membrane. After crystal violet staining, the numbers of colonies in five microscopic fields (X200) were counted. In HepG2, 332.0 in siNEG-treated cells (95% CI 323.0-341.0) versus 29.3 in siTPM3-treated cells (95% CI 25.5-33.1), P < 0.0001; In SNU-475, 359.3(95% CI 350.6-368.1) versus 125.3(95% CI 114.1-136.5), P < 0.0001. (B) Invasion of siTPM and siNEG transfected HCC cell lines was examined using the Matrigel coated transwell membrane. In HepG2, 212.7 in siNEG-treated cells (95% CI 206.4-218.9) versus 43.0 in siTPM3- treated cells (95% CI 36.4-49.6), P < 0.0001; In SNU-475, 162.7(95% CI 156.4-168.9) versus 33.7(28.5-38.8), P < 0.0001. E-cadherin and Snail expression in TPM3 knockdown HCC cells To explore the potential mechanisms of reduced migration and invasion in TPM3 knockdown HCC cells, we examined the expression patterns of E-cad- herin and Snail, a known factor to repress E-cadherin expression by binding to E-boxes of the E-cadherin promoter in cancers. Before knockdown, we measured the endogenous levels of E-cadherin and Snail in the 10 HCC cell lines and THLE-3 as a reference (Figure 4). Of the eight cell lines showing relative TPM3 over- expression with respect to THLE-3, five cell lines (SNU-387, 423, 475, 739, and HepG2) showed upregu- lated Snail and downregulated E-cadherin levels. Two of the eight cell lines with TPM3 overexpression (SNU-449 and 878) also showed the Snail up- and E- cadherin downregulated pattern, but the endogenous Effects of TPM3 silencing on HCC cell migration and invasion Error bars represent mean ± standard error of mean. * represents P value < 0.05. Figure 3 Repressed migration and invasion in TPM3 knockdown HCC cell lines. (A) Migration of siTPM and siNEG transfected HCC cell lines was examined using the Matrigel uncoated transwell membrane. After crystal violet staining, the numbers of colonies in five microscopic fields (X200) were counted. In HepG2, 332.0 in siNEG-treated cells (95% CI 323.0-341.0) versus 29.3 in siTPM3-treated cells (95% CI 25.5-33.1), P < 0.0001; In SNU-475, 359.3(95% CI 350.6-368.1) versus 125.3(95% CI 114.1-136.5), P < 0.0001. (B) Invasion of siTPM and siNEG transfected HCC cell lines was examined using the Matrigel coated transwell membrane. In HepG2, 212.7 in siNEG-treated cells (95% CI 206.4-218.9) versus 43.0 in siTPM3- treated cells (95% CI 36.4-49.6), P < 0.0001; In SNU-475, 162.7(95% CI 156.4-168.9) versus 33.7(28.5-38.8), P < 0.0001. Error bars represent mean ± standard error of mean. * represents P value < 0.05. Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 Page 6 of 11 Snail levels were lower than that in THLE-3. In case of SNU-761, although it showed TPM3 overexpression, the expression pattern of Snail and E-cadherin was opposite to those of other 7 cell lines. The 2 cell lines without TPM3 overexpression (SNU-398 and 886) did not show Snail upregulation. When we knocked down TPM3 in HepG2 and SNU-475, the expression levels of Snail and E-cadherin became reversed in both cell lines; Snail expression was significantly decreased and E-cadherin was significantly increased compared with the siNEG transfection control on both mRNA and protein levels (Figure 5A and 5B). migrated cells significantly decreased compared with siNEG control (P < 0.0001, Figure 3A). Repression of invasiveness by siTPM3 treatment was also observed. The numbers of the cells that passed through the Matri- gel-coated membrane significantly decreased in siTPM3- treated cells compared with siNEG control (P < 0.0001, Figure 3B). Vimentin and fibronectin expression in TPM3 knockdown HCC cells To further verify the changes of EMT-related pheno- types in TPM3 knockdown cells, we examined the expression of vimentin and fibronectin in cells with and without siTPM3 transfection. In SNU-475, endogenous vimentin and fibronectin were strongly expressed in the cytoplasm of control cells, but both signals decreased in siTPM3-treated cells (Figure 6). Especially, the decrease of vimentin expression after TPM3 knockdown was more noticeable than that of fibronectin. The profile in HepG2 was similar to that in SNU-475, but less Figure 4 Endogenous Snail and E-cadherin expression in 10 HCC cell lines and a normal liver cell line (THLE-3). Endogenous expression levels of Snail and E-cadherin were measured by real-time qRT-PCR before siTPM transfection. Total RNA extraction and qRT-PCR procedure were as described in Materials and Methods. Human GAPDH gene was used as internal control for qRT-PCR. X axis represents cell lines and Y axis represents relative expression ratios of each gene (Cell lines/THLE-3). Error bars represent mean ± standard error of mean. Open bar represents Snail expression and closed bar represents E-cadherin expression. Figure 4 Endogenous Snail and E-cadherin expression in 10 HCC cell lines and a normal liver cell line (THLE-3). Endogenous expression levels of Snail and E-cadherin were measured by real-time qRT-PCR before siTPM transfection. Total RNA extraction and qRT-PCR procedure were as described in Materials and Methods. Human GAPDH gene was used as internal control for qRT-PCR. X axis represents cell lines and Y axis represents relative expression ratios of each gene (Cell lines/THLE-3). Error bars represent mean ± standard error of mean. Open bar represents Snail expression and closed bar represents E-cadherin expression. Choi et al. BMC Cancer 2010, 10:122 Page 7 of 11 Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 http://www.biomedcentral.com/1471-2407/10/122 Figure 5 Changes in E-cadherin and Snail expression after TPM3 knockdown in HepG2 (A) and SNU-475(B). After TPM3 knockdown, E- cadherin and Snail expression levels were measured by real-time qRT-PCR (top plots). * represents P value < 0.05. X axis represents samples and Y axis represents relative expression ratios of E-cadherin and Snail (siTPM/siNEG). Human GAPDH gene was used as internal control for qRT-PCRs. E-cadherin and Snail expression levels were also measured by western blot (bottom plots). Alpha-tubulin was used as internal control for western blot analysis. siTPM3, siTPM3 transfected HCC cell line; siNEG, negative oligonucleotide transfected HCC cell line. Error bars represent mean ± standard error of mean. Inhibited tumor cell growth after TPM3 silencing In addition to the effects on tumor cell migration and invasion, we also assessed the effect of TPM3 knock- down on HCC cell growth (Figure 7). First, we per- formed the colony formation assay. The numbers of colonies in siTPM3 treated cells were significantly reduced compared with those in siNEG treated cells; In HepG2, 685.7(95% CI 663.1-708.2) in siNEG-treated cells versus 108.3(95% CI 104.5-112.1) in siTPM3- treated cells, P < 0.0001; In SNU-475, 190.0(95% CI 176.2-203.8) versus 107.3(95% CI 92.8-121.9), P < 0.0001 (Figure 7A and 7B). We next examined the TPM3 knockdown effect on the anchorage independent growth of HCC cells by the soft agar assay. The numbers and Vimentin and fibronectin expression in TPM3 knockdown HCC cells * represents P value < 0.05. Figure 5 Changes in E-cadherin and Snail expression after TPM3 knockdown in HepG2 (A) and SNU-475(B). After TPM3 knockdown, E- cadherin and Snail expression levels were measured by real-time qRT-PCR (top plots). * represents P value < 0.05. X axis represents samples and Y axis represents relative expression ratios of E-cadherin and Snail (siTPM/siNEG). Human GAPDH gene was used as internal control for qRT-PCRs. E-cadherin and Snail expression levels were also measured by western blot (bottom plots). Alpha-tubulin was used as internal control for western blot analysis. siTPM3, siTPM3 transfected HCC cell line; siNEG, negative oligonucleotide transfected HCC cell line. Error bars represent mean ± standard error of mean. * represents P value < 0.05. Figure 5 Changes in E-cadherin and Snail expression after TPM3 knockdown in HepG2 (A) and SNU-475(B). After TPM3 knockdown, E- cadherin and Snail expression levels were measured by real-time qRT-PCR (top plots). * represents P value < 0.05. X axis represents samples and Y axis represents relative expression ratios of E-cadherin and Snail (siTPM/siNEG). Human GAPDH gene was used as internal control for qRT-PCRs. E-cadherin and Snail expression levels were also measured by western blot (bottom plots). Alpha-tubulin was used as internal control for western blot analysis. siTPM3, siTPM3 transfected HCC cell line; siNEG, negative oligonucleotide transfected HCC cell line. Error bars represent mean ± standard error of mean. * represents P value < 0.05. prominent due to initially weaker vimentin and fibro- nectin signals than those in SNU-475 (data not shown). sizes of anchorage-independent colonies were signi- ficantly lower in siTPM3-treated cells than those in siNEG-treated ones; In HepG2, 212.7 (95% CI 206.4-218.9) in siNEG-treated cells versus 43.0(95% CI 36.4-49.6) in siTPM3-treated cells, P < 0.0001; In SNU-475, 162.7(95% CI 156.4-168.9) versus 33.7(95% CI 28.5-38.8), P < 0.0001 (Figure 7A and 7B). Discussion Previously, we reported a recurrent chromosomal ampli- fication on the 1q21.3 region and related overexpression of the TPM3 gene and suggested its oncogenic potential in hepatocarcinognenesis [6]. Subsequently, we con- ducted this study to elucidate the biological effects of TPM3 overexpression in hepatocarcinogenesis using the RNA interference (RNAi) technology to knockdown the expression of TPM3. We found that TPM3 knockdown Choi et al. BMC Cancer 2010, 10:122 Page 8 of 11 Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 http://www.biomedcentral.com/1471-2407/10/122 Figure 6 Expression profiles of vimentin and fibronectin after TPM3 knockdown in SNU-475. Vimentin (upper boxes) and fibronectin (lower boxes) expression patterns were compared between siTPM3- and siNEG-transfected SNU-475 by immunofluorescence staining (green for vimentin and fibronectin stain; blue for nuclear DAPI stain). siTPM3, siTPM3 transfected SNU-475; siNEG, siNEG transfected SNU-475. ×400 Figure 6 Expression profiles of vimentin and fibronectin after TPM3 knockdown in SNU-475. Vimentin (upper boxes) and fibronectin (lower boxes) expression patterns were compared between siTPM3- and siNEG-transfected SNU-475 by immunofluorescence staining (green for vimentin and fibronectin stain; blue for nuclear DAPI stain). siTPM3, siTPM3 transfected SNU-475; siNEG, siNEG transfected SNU-475. ×400 Figure 6 Expression profiles of vimentin and fibronectin after TPM3 knockdown in SNU-475. Vimentin (upper boxes) and fibronectin (lower boxes) expression patterns were compared between siTPM3- and siNEG-transfected SNU-475 by immunofluorescence staining (green for vimentin and fibronectin stain; blue for nuclear DAPI stain). siTPM3, siTPM3 transfected SNU-475; siNEG, siNEG transfected SNU-475. ×400 vascular invasion and intrahepatic metastasis through losing cell adhesion and increasing cell mobility [16,22,23]. We further examined the expression of Snail in TPM3 knockdown HCC cells showing upregu- lated E-cadherin, because the Snail transcription factor has been known to repress E-cadherin expression by binding to E-boxes in the E-cadherin promoter in can- cers including HCC [17,24,25]. Snail is also known as a key regulatory molecule inducing EMT [17,22,26]. We found that Snail expression was significantly more repressed in siTPM3-treated HCC cell lines than in the untreated cell lines. Although directions of the effect were opposite, there have been studies which reported that overexpression of Snail increases the invasiveness of HCC [17,27,28]. Based on our observa- tions and previous reports, it can be hypothesized that overexpression of TPM3 in the cytoplasm may activate Snail which will subsequently repress E-cadherin profoundly repressed the migration and invasion poten- tials of HCC cells compared with the same cell lines without siTPM3 treatment. Discussion These findings are accordant with the previous reports which suggested that expres- sion of a tropomyosin isoform was higher in highly metastatic mouse tumor cells than in the cells with lower metastatic rate [14]. To explore the mechanisms behind reduced migra- tion and invasion in TPM3 knockdown HCC cells, we examined whether transfection of siTMP3 would affect the levels of E-cadherin expression in HCC cells. Downregulation of E-cadherin expression is one of the well-known hallmarks of tumor metastasis in HCC and an indicator of EMT onset [18-21]. In our study, E- cadherin expression was found to be reversed from low to high through siTPM3 treatment. In previous HCC studies, repressed expression or mutation of E- cadherin was correlated with a histological grade, Page 9 of 11 Choi et al. BMC Cancer 2010, 10:122 Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 http://www.biomedcentral.com/1471-2407/10/122 i i th l d th t thi t T h t i f TPM3 k kd Figure 7 Inhibited tumor cell growth in TPM3 knockdown HepG2 (A) and SNU-475 (B). Top plots of A and B are colony formation assay results. Bottom plots are soft agar assay results. In both the colony formation and anchorage independent growth assays, the number of colonies were counted in siTPM and siNEG plates. siTPM3, siTMP3 transfected HCC cell line; siNEG, negative oligonucleotide (siNEG) transfected HCC cell line. * represents P value < 0.05. Figure 7 Inhibited tumor cell growth in TPM3 knockdown HepG2 (A) and SNU-475 (B). Top plots of A and B are colony formation assay results. Bottom plots are soft agar assay results. In both the colony formation and anchorage independent growth assays, the number of colonies were counted in siTPM and siNEG plates. siTPM3, siTMP3 transfected HCC cell line; siNEG, negative oligonucleotide (siNEG) transfected HCC cell line. * represents P value < 0.05. To see phenotypic consequences of TPM3 knockdown in HCC cells, we examined vimentin and fibronectin expression. Vimentin and fibronectin are the mesenchy- mal cell markers associated with EMT and known to be upregulated in migratory cells [29]. In siTPM3 transfected cells, the expression levels of vimentin and fibronectin decreased compared with the untreated cells. All these results support that TPM3 overexpression could affect migration or invasion potentials through activating EMT. expression in the nucleus and that this event can con- fer migration or invasion potentials to cancer cells dur- ing hepatocarcinogenesis. Author details 1Department of Microbiology, School of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea. 2Integrated Research Center for Genome Polymorphism, School of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea. 3Department of Hospital Pathology, Seoul St Mary’s Hospital, School of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea. 4Department of Internal Medicine, Seoul St Mary’s Hospital, School of Medicine, The Catholic University of Korea, 505 Banpo- dong, Socho-gu, Seoul 137-701, Korea. List of Abbreviations List of Abbreviations (TPM3): tropomyosin3; (HCC): hepatocellular carcinoma; (EMT): epithelial- mesenchymal transition; (siRNA): small interfering RNA; (RNAi): RNA interference. 10. Butti MG, Bongarzone I, Ferraresi G, Mondellini P, Borrello MG, Pierotti MA: A sequence analysis of the genomic regions involved in the rearrangements between TPM3 and NTRK1 genes producing TRK oncogenes in papillary thyroid carcinomas. Genomics 1995, 28:15-24. Discussion Snail activation by TPM3 could be achieved through the direct interaction or activation of TPM3 downstream signaling pathways. However, we could not find the significant positive correlation between endogenous TPM3 and Snail mRNA levels, partly due to the limited number of cell lines we studied. expression in the nucleus and that this event can con- fer migration or invasion potentials to cancer cells dur- ing hepatocarcinogenesis. Snail activation by TPM3 could be achieved through the direct interaction or activation of TPM3 downstream signaling pathways. However, we could not find the significant positive correlation between endogenous TPM3 and Snail mRNA levels, partly due to the limited number of cell lines we studied. Page 10 of 11 Page 10 of 11 Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 Page 10 of 11 Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 Project, Ministry for Health, Welfare &Family Affairs, Republic of Korea (A092258). Project, Ministry for Health, Welfare &Family Affairs, Republic of Korea (A092258). Project, Ministry for Health, Welfare &Family Affairs, Republic of Korea (A092258). We also observed that TPM3 knockdown lowered col- ony formation and anchorage independent growth. In previous observations, upregulation of Snail was found to be associated with tumor cell survival and aggressive behavior of cancer [30,31]. Taken together, it could be suggested that reduced tumor cell growth in siTPM3 treated HCC cells might be due to TPM3 knockdown- related downregulation of Snail. In addition, in our unpublished study, the combined use of TPM3 knock- down and chemotherapeutic agents have been more effective to reduce tumor cell viabilities than the use of chemotherapeutic agents only. This is also coherent with the previous reports suggesting that Snail expres- sion is critical for cancer cells to acquire chemoresis- tence [30,31]. References 1. Parkin DM, Bray F, Ferlay J, Pisani P: Global cancer statistics, 2002. CA Cancer J Clin 2005, 55:74-108. 1. Parkin DM, Bray F, Ferlay J, Pisani P: Global cancer statistics, 2002. CA Cancer J Clin 2005, 55:74-108. 2. Llovet JM, Bruix J: Molecular targeted therapies in hepatocellular carcinoma. Hepatology 2008, 48:1312-1327. 2. Llovet JM, Bruix J: Molecular targeted therapies in hepatocellular carcinoma. Hepatology 2008, 48:1312-1327. 3. El-Serag HB, Rudolph KL: Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007, 132:2557-2576. 3. El-Serag HB, Rudolph KL: Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007, 132:2557-2576. 4. Ueda S, Basaki Y, Yoshie M, Ogawa K, Sakisaka S, Kuwano M, Ono M: PTEN/ Akt signaling through epidermal growth factor receptor is prerequisite for angiogenesis by hepatocellular carcinoma cells that is susceptible to inhibition by gefitinib. Cancer Res 2006, 66:5346-5353. 4. Ueda S, Basaki Y, Yoshie M, Ogawa K, Sakisaka S, Kuwano M, Ono M: PTEN/ Akt signaling through epidermal growth factor receptor is prerequisite for angiogenesis by hepatocellular carcinoma cells that is susceptible to inhibition by gefitinib. Cancer Res 2006, 66:5346-5353. 5. Baek HJ, Lim SC, Kitisin K, Jogunoori W, Tang Y, Marshall MB, Mishra B, Kim TH, Cho KH, Kim SS, Mishra L: Hepatocellular cancer arises from loss of transforming growth factor beta signaling adaptor protein embryonic liver fodrin through abnormal angiogenesis. Hepatology 2008, 48:1128-1137. 5. Baek HJ, Lim SC, Kitisin K, Jogunoori W, Tang Y, Marshall MB, Mishra B, Kim TH, Cho KH, Kim SS, Mishra L: Hepatocellular cancer arises from loss of transforming growth factor beta signaling adaptor protein embryonic liver fodrin through abnormal angiogenesis. Hepatology 2008, 48:1128-1137. Authors’ contributions HSC was involved in the design of this study and execution of most experiments and drafted the manuscript. SHY and HJH participated in the design of this study, statistical analysis, and writing the manuscript. CKJ performed immunofluorescence staining and western blot analysis, HDX, SHJ and SHS assisted experimental procedures such as migration/invasion and cytotoxicity assays. JYC participated in the design of this study and partly contributed to funding. YJC proposed this study, organized the research team, interpreted all the data, and participated in writing the manuscript. There are several limitations in this study. First, we did not examine the direct binding of TPM3 to Snail. TPM3-Snail co-immunoprecipitation or other experi- mental verification of the correlation of these two mole- cules would be necessary. Second, although repression of colony formation and anchorage independent growth was observed in TPM3 knockdown cells, it cannot auto- matically prove the biological consequences of TPM3 overexpression in hepatocarcinogenesis. Larger-scale screening of TPM3 expression profile in invasive pri- mary HCCs and TPM3 overexpression experiment in normal liver cell lines will provide more direct evidence to support its oncogenic potential. Lastly, it is unclear whether the TPM3-Snail pathway is hepatocarcinogen- esis-specific or not. Examining other types of cancers will be required to clarify this possibility. Competing interests h h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 17 September 2009 Accepted: 1 April 2010 Received: 17 September 2009 Accepted: 1 April 2010 Published: 1 April 2010 Conclusion In this study, we demonstrated that TPM3 knockdown profoundly repressed migration and invasion of HCC cell lines. Based on our findings, we formulate a hypoth- esis that overexpression of TPM3 activates Snail expres- sion, which will repress E-cadherin expression and confer migration or invasion potentials to HCC cells during hepatocarcinogenesis. To our knowledge, this is the first evidence that TPM3 gets involved in migration and invasion of HCC by activating Snail mediated EMT pathway. This study will help to understand invasion or metastasis mechanisms in HCC and to define therapeu- tic targets inhibiting them. 6. Kim TM, Yim SH, Shin SH, Xu HD, Jung YC, Park CK, Choi JY, Park WS, Kwon MS, Fiegler H, Carter NP, Rhyu MG, Chung YJ: Clinical implication of recurrent copy number alterations in hepatocellular carcinoma and putative oncogenes in recurrent gains on 1q. Int J Cancer 2008, 123:2808-2815. 7. Pieples K, Arteaga G, Solaro RJ, Grupp I, Lorenz JN, Boivin GP, Jagatheesan G, Labitzke E, DeTombe PP, Konhilas JP, Irving TC, Wieczorek DF: Tropomyosin 3 expression leads to hypercontractility and attenuates myofilament length-dependent Ca(2+) activation. Am J Physiol Heart Circ Physiol 2002, 283:1344-353. 8. Lawrence B, Perez-Atayde A, Hibbard MK, Rubin BP, Dal Cin P, Pinkus JL, Pinkus GS, Xiao S, Yi ES, Fletcher CD, Fletcher JA: TPM3-ALK and TPM4-ALK oncogenes in inflammatory myofibroblastic tumors. Am J Pathol 2000, 157:377-384. 9. Armstrong F, Lamant L, Hieblot C, Delsol G, Touriol C: TPM3-ALK expression induces changes in cytoskeleton organisation and confers higher metastatic capacities than other ALK fusion proteins. Eur J Cancer 2007, 43:640-646. Pre-publication history The pre-publication history for this paper can be accessed here: http://www. biomedcentral.com/1471-2407/10/122/prepub 14. Miyado K, Kimura M, Taniguchi S: Decreased expression of a single tropomyosin isoform, TM5/TM30nm, results in reduction in motility of highly metastatic B16-F10 mouse melanoma cells. Biochem Biophys Res Commun 1996, 225:427-435. doi:10.1186/1471-2407-10-122 Cite this article as: Choi et al.: Tropomyosin3 overexpression and a potential link to epithelial-mesenchymal transition in human hepatocellular carcinoma. BMC Cancer 2010 10:122. 15. Lim SO, Gu JM, Kim MS, Kim HS, Park YN, Park CK, Cho JW, Park YM, Jung G: Epigenetic changes induced by reactive oxygen species in hepatocellular carcinoma: methylation of the E-cadherin promoter. Gastroenterology 2008, 135:2128-2140. 16. Miyoshi A, Kitajima Y, Kido S, Shimonishi T, Matsuyama S, Kitahara K, Miyazaki K: Snail accelerates cancer invasion by upregulating MMP expression and is associated with poor prognosis of hepatocellular carcinoma. Br J Cancer 2005, 92:252-258. 17. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-ΔΔC(T)) Method. Methods 2001, 25:402-408. 18. Huber MA, Kraut N, Beug H: Molecular requirements for epithelial- mesenchymal transition during tumor progression. Curr Opin Cell Biol 2005, 17:548-558. 19. Du GS, Wang JM, Lu JX, Li Q, Ma CQ, Du JT, Zou SQ: Expression of P- aPKC-iota, E-Cadherin, and beta-Catenin Related to Invasion and Metastasis in Hepatocellular Carcinoma. Ann Surg Oncol 2009, 16:1578-1586. 20. Soyama A, Eguchi S, Takatsuki M, Kawashita Y, Hidaka M, Tokai H, Nagayoshi S, Mochizuki S, Matsumoto S, Hamasaki K, Tajima Y, Kanematsu T: Significance of the serum level of soluble E-cadherin in patients with HCC. Hepatogastroenterology 2008, 55:1390-1393. 21. Zhai B, Yan HX, Liu SQ, Chen L, Wu MC, Wang HY: Reduced expression of E-cadherin/catenin complex in hepatocellular carcinomas. World J Gastroenterol 2008, 14:5665-5673. 22. Miyoshi A, Kitajima Y, Sumi K, Sato K, Hagiwara A, Koga Y, Miyazaki K: Snail and SIP1 increase cancer invasion by upregulating MMP family in hepatocellular carcinoma cells. Br J Cancer 2004, 90:1265-1273. 23. Hu CT, Wu JR, Chang TY, Cheng CC, Wu WS: The transcriptional factor Snail simultaneously triggers cell cycle arrest and migration of human hepatoma HepG2. J Biomed Sci 2008, 15:343-355. p p 24. Barrallo-Gimeno A, Nieto MA: The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 2005, 132:3151-3161. p 25. Acknowledgements This project is supported by FG08-11-06 of the 21C Frontier Functional Human Genome Project from the Ministry of Education, Science and Technology in Korea, and a grant of the Korea Healthcare technology R&D 11. Rosati R, La Starza R, Luciano L, Gorello P, Matteucci C, Pierini V, Romoli S, Crescenzi B, Rotoli B, Martelli MF, Pane F, Mecucci C: TPM3/PDGFRB fusion 11. Rosati R, La Starza R, Luciano L, Gorello P, Matteucci C, Pierini V, Romoli S, Crescenzi B, Rotoli B, Martelli MF, Pane F, Mecucci C: TPM3/PDGFRB fusion Page 11 of 11 Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 Choi et al. BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 31. 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BMC Cancer 2010, 10:122 http://www.biomedcentral.com/1471-2407/10/122 Pre-publication history Pon YL, Zhou HY, Cheung AN, Ngan HY, Wong AS: p70 S6 kinase promotes epithelial to mesenchymal transition through snail induction in ovarian cancer cells. Cancer Res 2008, 68:6524-6532. 26. Thiery JP: Epithelial-mesenchymal transitions in development and pathologies. Curr Opin Cell Biol 2003, 15:740-746. 27. Murata K, Sakamoto A: Impairment of clathrin-mediated endocytosis via cytoskeletal change by epithelial to fibroblastoid conversion in HepG2 cells: a possible mechanism of des-gamma-carboxy prothrombin production in hepatocellular carcinoma. Int J Oncol 2008, 33:1149-1155. 28. Sugimachi K, Tanaka S, Kameyama T, Taguchi K, Aishima S, Shimada M, Sugimachi K, Tsuneyoshi M: Transcriptional repressor snail and progression of human hepatocellular carcinoma. Clin Cancer Res 2003, 9:2657-2664. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: 29. 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A new species of Cotesia Cameron (Hymenoptera, Braconidae, Microgastrinae) reared from the hickory horned devil, Citheronia regalis, and luna moth, Actias luna, in east Texas
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Keywords Lepidoptera, parasitism, Saturniidae A new species of Cotesia Cameron (Hymenoptera, Braconidae, Microgastrinae) reared from the hickory horned devil, Citheronia regalis, and luna moth, Actias luna, in east Texas James B. Whitfield1, Robert J. Nuelle Jr.2, Robert J. Nuelle III2 1 Department of Entomology, 320 Morrill Hall, University of Illinois, Urbana, IL 61801 USA 2 Research Associate, Entomology, Sam Houston State Natural History Collections Huntsville, TX 77340 USA Corresponding author: James B. Whitfield (jwhitfie@life.illinois.edu) c editor: M. Sharkey  |  Received 5 February 2018  |  Accepted 14 February 2018  |  Published 27 February 2018 Academic editor: M. Sharkey  |  Received 5 February 2018  |  Accepted 14 February 2018  |  Published 27 February 2018 http://zoobank.org/F7725957-DDDA-4937-828E-CE6BBAC90ECF http://zoobank.org/F7725957-DDDA-4937-828E-CE6BBAC90ECF Citation: Whitfield JB, Nuelle Jr RJ, Nuelle III RJ (2018) A new species of Cotesia Cameron (Hymenoptera, Braconidae, Microgastrinae) reared from the hickory horned devil, Citheronia regalis, and luna moth, Actias luna, in east Texas. ZooKeys 740: 35–44. https://doi.org/10.3897/zookeys.740.242226 Abstract The braconid wasp parasitoid Cotesia nuellorum Whitfield, new species, is described from specimens reared from a caterpillar of the hickory horned devil, Citheronia regalis (F.), and from a caterpillar of the luna moth, Actias luna (L.), in eastern Texas. The species is diagnosed with respect to other species of Co­ tesia recorded from North American Saturniidae, and details of its biology are provided. A new species of ZooKeys 740: 35–44 (2018) doi: 10.3897/zookeys.740.24226 http://zookeys.pensoft.net A new species of ZooKeys 740: 35–44 (2018) doi: 10.3897/zookeys.740.24226 http://zookeys.pensoft.net A new species of ZooKeys 740: 35–44 (2018) doi: 10.3897/zookeys.740.24226 http://zookeys.pensoft.net meron (Hymenoptera, B RESEARCH ARTICLE Copyright James B. Whitfield et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Cotesia Cameron is a common and diverse genus of microgastrine Braconidae that largely specializes in parasitizing exposed larvae of macrolepidopteran moths and but­ terflies (Whitfield et al. 2018). It is one of the larger genera of Microgastrinae in terms of currently described species worldwide; its highest species richness lies in temperate zones, and it is relatively ubiquitous in terrestrial habitats where caterpillars occur. Copyright James B. Whitfield et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 36 James B. Whitfield et al. / ZooKeys 740: 35–44 (2018) Currently, four species in this genus are recorded from saturniid caterpillars in North America (Marsh 1979; Tuskes et al. 1996) – C. anisotae (Muesebeck), C. electrae (Viereck), C. hemileucae (Riley) and C. teleae (Muesebeck). Tuskes et al. (1996) provide a table of these associations along with records of other Nearctic saturniid parasitoids. Recently, two of the authors (RJNJr and RJNIII) collected a batch of larvae of the hickory horned devil, Citheronia regalis (F.), in eastern Texas, and one of these larvae yielded a brood of Cotesia wasps (see below). While Cotesia teleae has been previously recorded as a parasitoid of C. regalis especially in the northeastern U. S. where the moth is now relatively rare (in addition to its more usual host Antheraea polyphemus (Cramer)), the Texas material appears to belong to a new species, described below. It is possible that at least some previous records of C. teleae from C. regalis actually belong instead to the new species, but we have been unable to confirm this. The geographical location of the Texas record places it far from the northeastern U. S., near the south­ western limit of the range of C. regalis, so it is not surprising if the parasitoid commu­ nity is different in this ecologically distinct area. Subsequently, Cotesia specimens reared by Richard S. Peigler from larvae of the luna moth, Actias luna, from the same area were found to be apparently conspecific, and are also included in our definition of the new species. i Below, JBW describes the new species of Cotesia, diagnosing it versus other species of Cotesia known to attack North American saturniids, and RJNJr and RJNIII provide discussion concerning its discovery and field biology. Materials and methods 3), surrounded by clear, luteous liquid, a large number of white, parasitoid cocoons and a few emerged wasp larvae. p The caterpillar host and 18 cocoons were immediately preserved together in 100 % ethyl alcohol. The other 30 cocoons were placed in a sealed container at room tempera­ ture for 5 days. During this period many of the wasps eclosed, and the sealed container was placed in the freezer for 3 days to kill all specimens. These specimens were placed in 100 % Ethyl alcohol. The authors believe that this larva had been parasitized prior to capture, as none of the other larvae captured and reared with this specimen were likewise parasitized. One other more mature caterpillar, captured in the same area on the same day, completed its development successfully. It pupated 7 to 10 days after this parasitic incident occurred. A third specimen, captured in a different location, two weeks later, also completed its larval development and pupated successfully.h The reared Cotesia specimens from both C. regalis and from A. luna were compared to specimens of the described species of Cotesia known to attack Nearctic saturniid cat­ erpillars (C. anisotae (Muesebeck), C. electrae (Viereck), C. hemileucae (Riley), C. teleae (Muesebeck)). All of these species were treated by Muesebeck in his (1920) revision of Apanteles (as then circumscribed), except C. teleae, which he described later (Muesebeck 1926). C. teleae in particular has been recorded to attack Citheronia regal is in the north­ eastern U.S., (Tuskes et al. 1996, see also Table 1), although it is most commonly recov­ ered from the Polyphemus moth caterpillar, A. polyphemus. In both cases the parasitoids attack the earlier instars, and not the last instar larvae. The two species of Cotesia resem­ ble each other in general appearance, but differ in the various features outlined below in the description. It remains to be seen whether Cotesia reared in other parts of North America from C. regalis are indeed C. teleae or sometimes belong to the new species described here. A molecular study of the complex of Cotesia species attacking Neartic saturniids is likely to reveal additional new species. The genus as a whole has proven taxonomically challenging except when ecological and/or molecular data are available to aid in species separation. Materials and methods During October of 2014, RJNJr and RJNIII collected three caterpillars of Citheronia regalis (Fig. 1) in larval form on small American sweetgum (Liquidambar styraciflua L.) trees in the Sam Houston National Forest near Stubblefield Lake Park, Walker County, Texas. One of the specimens was a 2nd or 3rd instar caterpillar which subsequently died after about 13 days during the emergence of the braconid parasitoids described below. The parasitoid emergence was not observed by the authors, but the cocoons were saved and some were allowed to produce adult wasps. The original host, some larval parasi­ toids, cocoons, and adult parasitoids were saved for further study. It was later noted that Richard S. Peigler had collected larvae of the luna moth, Ac­ tias luna (Fig. 2), in the same area, same month, but two years earlier, and recovered par­ asitoids that appeared similar in adult and cocoon appearance to those from C. regalis. We examined these as part of the material described below. pp pp We examined these as part of the material described below.h The three caterpillars of C. regalis were raised on leaves of American sweetgum, Liquidambar styraciflua L., which were changed daily. The caterpillars were housed sep­ arately in well-ventilated plastic containers. The food plant was harvested daily, cleaned and the stems were trimmed under water to ensure a well-hydrated food source. Enclo­ sures were cleaned daily. The caterpillars varied in size, with two appearing to be nearly mature larvae and the third appearing to be much younger. After 13 days, the smallest A new species of Cotesia Cameron (Hymenoptera, Braconidae, Microgastrinae)... 37 Figures 1, 2. 1 Larva of the hickory horned devil, Citheronia regalis 2 Larva of the luna moth, Ac­ tias luna. Photo in 1 by Clayton Bownds, used with permission, photo in 2 by Richard S. Peigler, used with permission. Figures 1, 2. 1 Larva of the hickory horned devil, Citheronia regalis 2 Larva of the luna moth, Ac­ tias luna. Photo in 1 by Clayton Bownds, used with permission, photo in 2 by Richard S. Peigler, used with permission. James B. Whitfield et al. / ZooKeys 740: 35–44 (2018) 38 of the 3 caterpillars stopped eating, as if it was preparing to molt. The following morn­ ing the caterpillar was found lying on the floor of its enclosure (Fig. Materials and methods stigma spun singly A virginiensis Dryocampa rubicunda electrae Agapema anona white dark brown variable black A. galbina spun singly A. homogena Automeris io Coloradia doris C. pandora Hemileuca burnsi H. chinatiensis H. eglanterina H. electra H. grotei H. hera H. nevadensis H. oliviae H. tricolor hemileucae Automeris io white yellowish mostly yellowish mostly yellowish Hemileuca maia spun singly H. slosseri nuellorum Citheronia regalis white dark brown mostly yellowish mostly yellowish Actias luna spun singly teleae Antheraea polyphemus white yellowish mostly black variable Citheronia regalis spun singly The description of the new Cotesia species presented below generally follows the terminology and format used in Fernandez-Triana et al. (2014) and uses primarily terms adopted by the Hymenoptera Anatomy Ontology (Yoder et al. 2010). Materials and methods A small table of described differences among the species attack­ ing saturniids in North America is provided (Table 1), but there are no guarantees that the color characters listed will prove to be stable especially across broad geographic areas. It is interesting that Actias luna is a commonly reared and widespread species that has not been officially recorded to yield Cotesia parasitoids before at any locality, al­ though Peigler (1994) suggests that C. teleae might have been the species Fiske and Thompson (1909) found to attack earlier instar larvae of A. luna in experiments. The Cotesia from Peigler's rearing described here were tentatively previously identified as C. schizurae (Ashmead) (Peigler 2013), but that species has light buff-colored cocoons spun together in parallel rows, and attacks notodontids of the genus Schizura. Possibly in nature this is an unusual association, and only occurred because Actias lar­ vae co-occurred with C. regalis on sweetgum in this habitat. It remains to be seen if further rearings of the two host caterpillar species in east Texas continue to both yield C. nuellorum. A new species of Cotesia Cameron (Hymenoptera, Braconidae, Microgastrinae)... 39 Table 1. Recorded hosts, cocoon types, and several color traits putatively varying between described species of Cotesia known to attack saturniid larvae in North America, including the new species described here. All of the species in this list are similar in having relatively smoother sculpturing on the propodeum and anterior metaso­ mal tergites than is typical. *host are from Tuskes et al. (1996). Cotesia species Recorded hosts* Cocoons Tegulae Fore and mid coxae Hind femur anisotae Anisota senatoria deep buff blackish black black A. stigma spun singly A virginiensis Dryocampa rubicunda electrae Agapema anona white dark brown variable black A. galbina spun singly A. homogena Automeris io Coloradia doris C. pandora Hemileuca burnsi H. chinatiensis H. eglanterina H. electra H. grotei H. hera H. nevadensis H. oliviae H. tricolor hemileucae Automeris io white yellowish mostly yellowish mostly yellowish Hemileuca maia spun singly H. slosseri nuellorum Citheronia regalis white dark brown mostly yellowish mostly yellowish Actias luna spun singly teleae Antheraea polyphemus white yellowish mostly black variable Citheronia regalis spun singly The description of the ne Cotesia species presented belo generall follo s the Cotesia species Recorded hosts* Cocoons Tegulae Fore and mid coxae Hind femur anisotae Anisota senatoria deep buff blackish black black A. Taxonomy Cotesia nuellorum Whitfield, sp. n. http://zoobank.org/1DEC4342-CBC6-444E-A0AF-6057B804C131 Figs 3–7 Type locality. The original habitat is located within the Sam Houston National For­ est, Walker County, Texas, near Stubblefield Lake Recreational area 338 feet AMSL James B. Whitfield et al. / ZooKeys 740: 35–44 (2018) James B. Whitfield et al. / ZooKeys 740: 35–44 (2018) 40 Figure 3. Larva of C. regalis with emerged larvae and cocoons of C. nuellorum, in rearing container. Photo by R. J. Nuelle, Jr. Figure 3. Larva of C. regalis with emerged larvae and cocoons of C. nuellorum, in rearing container. Photo by R. J. Nuelle, Jr. (Lat: 30.524930 Lon: -95.622750 Accuracy: 10 m). This area is described as Piney­ woods: Pine Forest or Plantation, according to the Texas Parks and Wildlife; Texas Ecosystem Analytical Mapper (TPWD T.E.A.M.) application. It is in a managed Na­ tional Forest and is subject to occasional fire events. This successional area contains sweetgum, hickory, oak and various conifers as dominant trees. Many of the deciduous trees are relatively short (less than 6 feet tall) near the borders of roads and trails, and the generally open forest floor is thus highly convenient for sampling caterpillars. gl g g Holotype. Female deposited in USNM. TEXAS: Walker Co., Sam Houston Na­ tional Forest, nr. Stubblefield Lake, 30.524930, -95.622750, October 2014, 100 m. elev., coll. R. J. Nuelle, Jr. and R. J. Nuelle, III, ex larva Citheronia regalis on sweetgum. Paratypes. 4 females, 1 male with same data as holotype, plus 26 females, 7 males (deposited in CNC, INHS, SHSU, TAMUIC, UWIM (Laramie)) from: TEXAS: Walker Co., Sam Houston National Forest, Stubblefield Lake, ex. larva Actias luna on sweetgum, em. 21-22-X-2012, coll. R. S. Peigler. Description. Female. Body color: body mostly dark except palps, portions of legs (see below) and ventral portions of anterior laterotergites. Antenna color: scape black, Description. Female. Body color: body mostly dark except palps, portions of legs (see below) and ventral portions of anterior laterotergites. Antenna color: scape black, A new species of Cotesia Cameron (Hymenoptera, Braconidae, Microgastrinae)... 41 pedicel dark brown, flagellum dark brown/black. Coxae color (pro- , meso, metacoxa): honey yellow; honey-yellow; black proximally, shading to medium brown distally. Femora color (pro-, meso-, metafemur): honey-yellow; honey-yellow; honey-yellow with smoky spot dorsally near distal end. Tibiae color (pro-, meso-, metatibia): hon­ ey-yellow; honey-yellow; honey-yellow with darkened extreme distal end. Taxonomy Tegula and humeral complex color: tegula dark brown translucent, humeral complex dark brown translucent (both slightly more translucent and paler in males). Pterostigma color: dark greyish brown, with indistinct paler junction with C+SC. Fore wing veins color: partially pigmented (a few veins may be dark but most are pale – see figure for pattern). Antenna length/body length: antenna approximately as long as body (head to apex of metasoma). Body in lateral view: not distinctly flattened dorso–ventrally. Body length (head to apex of metasoma): 2.0–2.2 mm. Fore wing length: 2.2–2.4 mm. Ocular– ocellar line/posterior ocellus diameter: 2.3–2.5. Interocellar distance/posterior ocellus diameter: 2.0–2.2. Antennal flagellomerus 2 length/width: 2.9–3.1. Antennal flagel­ lomerus 14 length/width: 1.9–2.1. Length of flagellomerus 2/length of flagellomerus 14: 2.1–2.3. Metafemur length/width: 3.2–3.3. Metatibia inner spur length/metabasi­ tarsus length: 0.4–0.5, about 10% longer than outer spur. Anteromesoscutum: mostly with shallow, dense punctures (separated by less than 2.0 × maximum diameter), but with polished and virtually punctureless strip near scutoscutellar sulcus. Mesoscutellar disc: mostly punctured but sometimes indistinctly so. Number of pits in scutoscutellar sulcus: 9 or 10. Propodeal carinae: strong medial longitudinal carina, vague hints of a transverse carina both otherwise rugose, especially medially and anteriorly. Medioter­ gite 1 length/width at posterior margin: 0.9–1.1. Mediotergite 1 shape: barrel-shaped, broadest in posterior 0.2. Mediotergite 1 sculpture: mostly sculptured, albeit shal­ lowly, otherwise shiny, especially anteriorly. Mediotergite 2 width at posterior margin/ length: 2.0–2.2. Mediotergite 2 sculpture: punctate/rugose, but shinier and smoother laterally. Outer margin of hypopygium: evenly sclerotized, posterior margin reach­ ing tip of metasoma and forming a shallow even convex curve. Ovipositor thickness: evenly narrowing towards tip. Ovipositor sheaths exerted but visible portion shorter than hypopygium length. Length of fore wing veins r/2RS: 1.1–1.2. Length of fore wing veins 2RS/2M: 1.1–1.3. Length of fore wing veins 2M/(RS+M)b: 0.9–1.0. Pter­ ostigma length/width: 3.1–3.5. Point of insertion of vein r in pterostigma: at roughly half way point length of pterostigma. Angle of vein r with fore wing anterior margin: nearly perpendicular, slightly inclined towards fore wing apex. Shape of junction of veins r and 2RS in fore wing: distinctly but not strongly angled. Male. As female but with slightly darker legs, more polished tergites and sometimes more translucent and paler tegulae. Body size usually about 10 % smaller than female. Male. As female but with slightly darker legs, more polished tergites and sometimes more translucent and paler tegulae. Acknowledgments The Nuelles would like to thank Jerry Cook, Ph. D., Sam Houston State Natural His­ tory Collections and William B. Godwin, Ph.D. ,Curator, Sam Houston State Natural History Collections for their ongoing support of our research projects. We would also like to thank Richard S. Peigler, Ph. D., Department of Biology, University of the Incarnate Word, for all of his guidance on rearing saturniid larvae and his mentoring on the importance of parasitoid research, and also providing the photo of, and Cotesia specimens from, A. luna; Daniel J. Bennett, Ph. D., Department of Biology, Stephen F. Austin State University, for his assistance at the start of this process, and Mr. Clayton Bownds, Texas Master Naturalist, for his wonderful photographs of C. regalis prior to the emergence of the parasitoids. JBW would like to thank M. Jared Thomas, Illinois Natural History Survey, for his assistance with the habitus photos of the parasitoids. Taxonomy Body size usually about 10 % smaller than female. p g y y Molecular data. None yet recorded. A broad sample of Cotesia reared from various larger Nearctic saturniids would be useful to clarify how distinct the parasitoid species are both in terms of host specificity and in terms of geographic distribution. In Costa Rica, where the diversity of Saturniidae is higher, the host specificity, at least to host genus, appears high (Smith et al. 2008; Janzen and Hallwachs 2017). James B. Whitfield et al. / ZooKeys 740: 35–44 (2018) James B. Whitfield et al. / ZooKeys 740: 35–44 (2018) 42 J fi y ( ) Figures 4–7. 4 Lateral habitus photo of Cotesia nuellorum female 5 Dorsal habitus photo of Cotesia nuellorum female 6 lateral view of posterior end of metasoma of C. nuellorum, showing hypopygium and ovipositor sheaths 7 Wings of C. nuellorum female. Figures 4–7. 4 Lateral habitus photo of Cotesia nuellorum female 5 Dorsal habitus photo of Cotesia nuellorum female 6 lateral view of posterior end of metasoma of C. nuellorum, showing hypopygium and ovipositor sheaths 7 Wings of C. nuellorum female. Biology/ecology. Gregarious (Fig. 3) on early instar larvae of host. Host: Saturnii­ dae: Ceratocampinae: Citheronia regalis (F.) and Saturniinae: Actias luna (L.). 4th and 5th instar larvae do not appear to serve as hosts, as with some other Cotesia parasitizing large Sphingidae and Saturniidae. Distribution. Known so far only from Texas but likely to be much more widely distributed. Ecologically and/or morphologically similar species. Table 1 provides a compari­ son of the species so far known from saturniids in North America. p Etymology. This species is named by JBW for the original discoverers, Robert J. Nuelle, Jr. and Robert J. Nuelle, III. Etymology. This species is named by JBW for the original discoverers, Robert J. Nuelle, Jr. and Robert J. Nuelle, III. A new species of Cotesia Cameron (Hymenoptera, Braconidae, Microgastrinae)... 43 References Fernandez-Triana JL, Whitfield JB, Rodriguez JJ, Smith MA, Janzen DH, Hallwachs W, Hajib­ abaei M, Burns JM, Solis MA, Brown J, Cardinal S, Goulet H, Hebert PDN (2014) Re­ view of Apanteles (Hymenoptera, Braconidae, Microgastrinae) from Area de Conservacion Guanacaste, Costa Rica, with keys to all described species from Mesoamerica. ZooKeys 383: 1–565. https://doi.org/10.3897/zookeys.383.6418 Fiske WF, Thompson WR (1909) Notes on the parasites of the Saturniidae. Journal of Economic Entomology 2: 450–460. https://doi.org/10.1093/jee/2.6.450 Janzen DH, Hallwachs W (2016) DNA barcoding the Lepidoptera inventory of a large tropical conserved wildland, Area de Conservacion Guanacaste, northwestern Costa Rica. Genome 59: 641–660. https://doi.org/10.1139/gen-2016-0005 Marsh PM (1979) Family Braconidae. In: Krombein KV, Hurd PD, Smith DR, Burks BD (Eds) Catalog of Hymenoptera in America North of Mexico. Vol. I, Symphyta and Apoc­ rita (Parasitica). Smithsonian Institution Press, Washington, DC, 144–313. Muesebeck CFW (1920) A revision of the North American species of ichenumon-flies be­ longing to the genus Apanteles. Proceedings of the United States National Museum 58: 483–576. https://doi.org/10.5479/si.00963801.2349.483 Muesebeck CFW (1926) Descriptions of new reared parasitic Hymenoptera and some notes on synonymy. Proceedings of the United States National Museum 67: 1–18. https://doi. org/10.5479/si.00963801.67-2580.1 Peigler RS (1994) Catalog of parasitoids of Saturniidae of the World. Journal of Research on the Lepidoptera 33: 1–121. Peigler RS (2013) New and reinterpreted observations on Actias luna (Linnaeus, 1758)(Lepi­ doptera: Saturniidae). Nachrichten des entomologischen Vereins Apollo 34 (3): 101–104. Riley CV (1881) Notes on North American Microgasters, with descriptions of new species. Transactions of the Academy of Sciences of St Louis 4: 296–315. James B. Whitfield et al. / ZooKeys 740: 35–44 (2018) 44 Smith MA, Rodriguez JJ, Whitfield JB, Deans, AR, Janzen, DH, Hallwachs W, Hebert PDN (2008) Extreme diversity of tropical parasitoid wasps exposed by iterative integration of natural history, DNA barcoding, morphology, and collections. Proceedings of the National Academy of Sciences 105: 12359–12364. https://doi.org/10.1073/pnas.0805319105 Tuskes PM, Tuttle JP, Collins MM (1996) The Wild Silk Moths of North America: A Natural History of the Saturniidae of the United States and Canada. Comstock Publishing, Ithaca, NY, 250 pp. Viereck HL (1912) Descriptions of five new genera and twenty-six new species of Ichneu­ mon-flies. Proceedings of the United States National Museum 42: 139–153. https://doi. org/10.5479/si.00963801.1888.139 Whitfield JB, Austin AD, Fernandez-Triana JL (2018) Systematics, biology and evolution of microgastrine parasitoid wasps. Annual Review of Entomology 63: 389–406. https://doi. References org/10.1146/annurev-ento-020117-043405 Yoder MJ, Miko I, Seltmann KC, Bertone MA, Deans AR (2010) A gross anatomy ontology for Hymenoptera. PLoS ONE 5(12): e15991. https://doi.org/10.1371/journal.pone.0015991
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A Comparison of the Analysis of Methods for Feature Extraction and Classification by Wavelet Transform in SSVEP BCIs
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A comparison of the analysis of methods for feature extraction and classification by Wavelet transform in SSVEP BCIs *1 Department of Biomedical Engineering, North Tehran Branch, Islamic Azad University, Tehran, Iran. 2 Department of Biomedical Engineering, Central Tehran Branch, Islamic Azad University, Tehran, Iran. z.einalou@srbiau.ac.ir *1 Department of Biomedical Engineering, North Tehran Branch, Islamic Azad University, Tehran, Iran. 2 Department of Biomedical Engineering, Central Tehran Branch, Islamic Azad University, Tehran, Iran. z.einalou@srbiau.ac.ir Abstract: Most of the studies in the field of Brain-Computer Interface (BCI) based on electroencephalography have a wide range of applications. Extracting Steady State Visual Evoked Potential (SSVEP) is regarded as one of the most useful tools in BCI systems. In this study, different methods such as feature extraction with different spectral methods (Shannon entropy, skewness, kurtosis, mean, variance) (bank of filters, narrow-bank IIR filters, and wavelet transform magnitude), feature selection performed by various methods (decision tree, principle component analysis (PCA), t-test, Wilcoxon, Receiver operating characteristic (ROC)), and classification step applying k nearest neighbor (k-NN), perceptron, support vector machines (SVM), Bayesian, multiple layer perceptron (MLP) were compared from the whole stream of signal processing. Through combining such methods, the effective overview of the study indicated the accuracy of classical methods. In addition, the present study relied on a rather new feature selection described by decision tree and PCA, which is used for the BCI-SSVEP systems. Finally, the obtained accuracies were calculated based on the four recorded frequencies representing four directions including right, left, up, and down. Keywords: Brain Computer Interface, Steady State Visual Evoked Potential, Classification, Wavelet. Wavelet. Corresponding author Department of Biomedical Engineering, North Tehran Branch, Islamic Azad University, Tehran, Iran E-mail: z.einalou@srbiau.ac.ir Phone: +98 21 77318896 Fax: +98 21 77318896 1. Introduction The brain-computer interface (BCI) is considered as a possible method for boosting communication and controlling the environments such as amyotrophic lateral sclerosis by which severely disabled people are able to manage their life [1]. BCI aims to create a path between the human brain and an external device such as BCI systems in order to bring human intentions into control signals. A large number of researches have recently focused on an Electro-Encephalography (EEG)-based BCI systems to accomplish the desirable communication. The signals extracted from an EEG signal such as ERP (event-related potentials), ERS (event-related synchronization), and VEP (visual-evoked potential) have been used in many perusals. They have attracted a lot of attention since VEP-based BCI systems enjoy a high information transfer rate (ITR) [2]. Among all these types, BCIs based on the Steady State Visual Evoked Potential (SSVEP) have been more emphasized. As brain responses to a visual stimulus, these significant subsets of VEP-based BCIs include high ITR, high signal-to-noise ratio (SNR), low set-time in train, and optimum steady function [3]. Firman et al. [4] applied the minimum energy combination (MEC) method to detect SSVEP from EEG signals. The method is used when the rapid and accurate recognition is necessary to attain high SNR for BCI systems. They used short segments to delete noises from EEG signals. In [5], the double stimulus frequency was used for eye stimulus in BCI systems, which leads to an increase in the performance of system. Wavelet analysis has been applied to a single EEG channel to extract its features. It was used as a conventional method to analyze EEG in order to detect sun band frequencies [6-7]. Fourier transform was applied to a single channel of EEG signals to discover the phase and amplitude of SSVEP [8, 9]. The main disadvantage of Fourier transform is that it applies the frequency of signal regardless of the time information. When both Wavelet and Fourier transform were put into application, it was possible to reach a better signal extraction level [10]. Fig. 1 shows the block diagram of the stream of the steps applied to the four stages of signal processing module. Fig 1. The block diagram of BCI systems based SSVEPs Fig 1. The block diagram of BCI systems based SSVEPs In this study, a comparative method was adopted for classical methods in three parts of signal processing including feature extraction, feature selection, and classification. 1. Introduction The study was conducted by using five feature extraction methods, spectral approach applying narrow band IIR filters, and wavelet transform computed at evoked frequencies. Further, six feature filters and wavelet transform computed at evoked frequencies, as well as six feature selection methods, and five classifiers were considered in the present study. Furthermore, the performance of each method was analyzed under a five-feature selection including decision tree, Wilcoxon, ROC, Bhattacharyya, and PCA. After combining to be applied on the same database, these methods were reported to describe an interesting comparative approach as follows: 1. When the SSVEP genders a proper response, the frequency domain is applied through feature extraction methods. 2. Non-linear structure is selected for the required SSVEP classification. 2. Non-linear structure is selected for the required SSVEP classification. 3. Channel selection processing is done under three experiments. 3. Channel selection processing is done under three experiments. 3. Channel selection processing is done under three experiments. 4. Statistical calculations are performed through different feature selection methods. 4. Statistical calculations are performed through different feature selection methods. This study was conducted on the database built according to the experimental setup described in the future. This study is a modest contribution to the identification of the best way for SSVEP processing from the feature extraction to the classification methods. To the best of our knowledge, no study has focused on the performance of a decision tree and PCA as feature selection approaches in BCI based on SSVEP. 2.1. Experimental procedure The data were recorded at Brain Science Institute, Laboratory for Advanced Brain Signal Processing. Biosemi Inc. machine recording was used for recording the data built in Netherland. The, 128 active electrodes from four participants were utilized to record the related data. The participants were completely aware of the objectives of this project. In addition, they could identify the light sensitive to the epilepsy disease before recording the final data. Further, the SSVEP stimulation was recorded by reversed white and black checkered (6×6 screen). In the next procedure, the second experiment was performed with a small checkered screen at three stimulus frequencies (8, 14, 28 Hz). The sampling frequency was 256 Hz. Additionally, the participants were asked to sit at the 90 cm distance of a monitor. The SSVEP started and ended 5 seconds and 20 seconds after starting the data, respectively. We had 15 seconds of SSVEP from four batches of participants with three frequencies. Each frequency included five experiments for each participant. The total number of samples was 6370 [11, 12]. 2.2. Preprocessing: Filtering and segmentation; Wavelet transfer Wavelet is used for time-frequency analyses especially for non-stationary signals. It applies two windows to properly perform both extensive and short time-frequency analyses for low and high frequencies respectively [11]. Wavelet is a reliable way to segment the raw signal of EEG. It is useful as it enables the researchers to analyze the signal, considering the aspects of time and frequency [12]. Wavelet decomposes the original signals into two levels: low frequency information (𝑊), and high frequency information (𝑉). It breaks the low frequency information repeatedly, while keeps the high frequency information intact. Finally, it completes a wavelet packet tree [6]. The orthonormal basis 𝑈𝑗,𝑘 𝑛(𝑡) that refers to the nth sub-bound of wavelet packet at jth scale is calculated as 𝑈𝑗,𝑘 𝑛(𝑡) = 2−𝑗 2 𝑢𝑛(2−𝑗 𝑡−𝑘), where 𝑘 is the shift factor [6]. It satisfies with: 𝑢𝑗,0 𝑛(𝑡) = ∑ℎ0(𝑘) 𝑢𝑗−1,𝑘 𝑖 (n is even) 𝑘 (1) 𝑢𝑗,0 𝑛(𝑡) = ∑ℎ1(𝑘) 𝑢𝑗−1,𝑘 𝑗 (n is odd) 𝑘 (2) = ∑ℎ1(𝑘) 𝑢𝑗−1,𝑘 𝑗 (n is odd) 𝑘 (2) (2) The signal is decomposed into 4 levels; ℎ0(𝑘) and ℎ1(𝑘) are two members of the 4th level of filters, is obtained as [6]: The signal is decomposed into 4 levels; ℎ0(𝑘) and ℎ1(𝑘) are two members of the 4th level of filters, is obtained as [6]: ℎ1(𝑘) = (−1)1−𝑘 ℎ0(1 −𝑘) (3) ℎ1(𝑘) = (−1)1−𝑘 ℎ0(1 −𝑘) (3) Decomposing continues repeatedly until the frequency resolution increases [13, 14]. When the scaling and decomposing are finished, the sampling sequence and the Wavelet coefficient are calculated at kth sample and jth level (formerly extracted) as following [6]: Decomposing continues repeatedly until the frequency resolution increases [13, 14]. When the scaling and decomposing are finished, the sampling sequence and the Wavelet coefficient are calculated at kth sample and jth level (formerly extracted) as following [6]: 𝑑𝑗 𝑛(𝑘) = ∑ℎ0(𝑚−2𝑘)𝑑𝑗−1 𝑛 2 (𝑚) (n is even) 𝑚 (4) 𝑑𝑗 𝑛(𝑘) = ∑ℎ1(𝑚−2𝑘)𝑑𝑗−1 (𝑖−1) 2 (𝑚) (n is odd) 𝑚 (5) (4) ) = ∑ℎ1(𝑚−2𝑘)𝑑𝑗−1 (𝑖−1) 2 (𝑚) (n is odd) 𝑚 (5) (5) Finally, it comes to computation of the frequency ranges of the whole subspaces at jth level, where fs is the frequency of sampling [6]. Fig.2 shows the filtering analysis of wavelet decomposition for the three divided steps. Finally, it comes to computation of the frequency ranges of the whole subspaces at jth level, where fs is the frequency of sampling [6]. 2.2. Preprocessing: Filtering and segmentation; Wavelet transfer Fig.2 shows the filtering analysis of wavelet decomposition for the three divided steps. h[n] g[n] ↓ 2 ↓ 2 D1 A1 h[n] g[n] ↓ 2 ↓ 2 D2 A2 h[n] g[n] ↓ 2 ↓ 2 D3 A3 h[n] g[n] ↓ 2 ↓ 2 D4 A4 x[n] Fig. 2. Block diagram of filtering analysis of Wavelet decomposition Fig. 2. Block diagram of filtering analysis of Wavelet decomposition 2.3.1. Entropy Shannon Entropy Shannon is the most famous approach. It sets an absolute limit on the best possible average length of lossless encoding or compression of an information source. The maximum rate happens in the continuous stimulation when the signal has Gaussian distribution. For the dis-continuous stimulation, if p(x) is probability density. The maximum value happens in dis-continuous form when the distribution is monotonous. In practice, for calculating Entropy of the signal, it is possible to first earn the spectrum of probability density according to the repetition of values in different ranges of slope of a considered signal. Then it is the time to enjoy the contributions of the theorems above [16]. 2.4.2. PCA (Principles Components Analysis) PCA is a well-recognized way to reduce the dimensions of data when there is a huge volume of data for classification. It is used to achieve a linear transform from the main complex of data [20]. At the end of the procedure, Bhattacharyya, t-test, ROC, and Wilcoxon were other feature selection methods used in this study. Finally, the results were collected in a table in order to find the best feature selection method. 2.4.1. Decision Tree An amazing way to select the superior data for an analysis is to make a decision tree. Decision trees are produced by algorithms that identify various ways of splitting a data set into branch-like segments. These segments form an inverted decision tree that originates with a root node at the top of the tree. It is a set of nodes which contact to each other through their branches going down to the root when they come to leaf notes [19]. The starting point is the root node, located at the top of the tree according to the agreement; indexes are located in decision nodes and each result is put at one branch. Each branch connects to another decision node or the final leaf node [19]. Here each branch divides into two other branches. Finally, we selected the data at the top of nodes. 2.3.2. Skewness and Kurtosis A fundamental task in many statistical analyses is to characterize the location and variability of a data set. Further characterizations of the data include skewness and kurtosis [17]. The former is a measure of symmetry, or more precisely, the lack of symmetry. A distribution or data set is symmetric if it looks the same to the left and right of the center point [17]. The latter is a measure of whether the data is heavy-tailed or light-tailed relative to a normal distribution. That is, data sets with high kurtosis tend to have heavy tails or outliers. Data sets with low kurtosis tend to have light tails or lack of outliers. A uniform distribution would be the extreme case [17]. Skewness is a standard to find the symmetry or asymmetry in distribution function. If the distribution function is symmetrical, Skewness measure is zero and if the distribution function is asymmetrical, Skewness measure is positive to the higher values and negative to the lower values, and it is defined as below [17]. 𝛾1 = 𝜇3 𝜎3 (6) (6) Kurtosis is the standard to recognize the sharpness of the curve at maximum value, and it defines the normal distribution in the following [18] 𝛾1 = 𝜇4 𝜎4 (7) 𝛾1 = 𝜇4 𝜎4 (7) 2.5. Classifiers After extracting data via Wavelet and selecting the best of them, we had to use the classifications to classify them. We applied k-NN (k-Nearest Neighbor), Perceptron, MLP, SVM (Support Vector Machine) and Bayes. 2.5.1. MLP classifier Multilayer Perceptron (MLP) is put to artificial neural networks with at least three nodes except for the input nodes. Here, nodes are considered as neurons which apply a nonlinear function. MLP uses the backpropagation for its training, which is a supervised method of training [20, 21]. MLP learns 𝑓(. ): 𝑅m →𝑅o where m is the number of dimensions of input and o is the number of dimensions of output. It can learn nonlinear approximation known as hidden layers. Fig. 3 shows the MLP that possesses a hidden layer with scalar outputs [22]. Fig. 3. One hidden layer MLP [22] Fig. 3. One hidden layer MLP [22] Fig. 3. One hidden layer MLP [22] 2.5.2. SVM Classifier It is a supervised machine-learning which is applied in classification of data sets. Training data divides into two groups and SVM makes a model to assign the new given data to one of two groups. It divides these two as far as possible from each other so that it could rise the resolution of groups [23]. 3. Results The diagram below illustrates the arrangement of this study. Fig. 4. The proposed diagram of the whole work of this study Fig. 4. The proposed diagram of the whole work of this study Fig. 4. The proposed diagram of the whole work of this study First, the SSVEP signals from Mat lab were broken into 2-second spans. Then, the discrete Wavelet transform was used for these 2-second spans. In addition, the Wavelet coefficients were decomposed. The stimulus frequencies were 8, 14, 28 Hz and accordingly Wavelet decomposition continued until every stimulus frequency was separately put in each single band. Using a two-time analysis of Wavelet decomposition level could lead us to the slight point according to the range of EEG signal, which is between 0-40 Hz. It is worth noting that the Wavelet was decomposed for four times to reach the stimulus frequencies of 8, 14, and 28 Hz at each band separately. After decomposing signals by Wavelet, we were allowed to extract the features from the decomposed signals including Entropy Shannon, Skewness, Kurtosis, mean, power, and variance. Further, the features were normalized, and the decision tree, PCA, ROC, and statistics methods were used for selecting the optimum data. The result of the decision tree, according to the higher rank, was in accordance to variables input (features) and outputs (labels). Ten features were selected by using the decision tree as show in Table 1. By considering the decision tree presented in Tables 1-3, Entropy Shannon of first channel was selected as the best feature The other methods and their selected features are shown in Tables 2-5. Table 1. The selected features by Decision tree Priorities selection Features s 1 Entropy Shannon of first channel 2 Variance of first channel 3 Minimum of second channel 4 Power for the second channel 5 Maximum of the first channel 6 Kurtosis of first channel 7 Skewness of first channel 8 Mean of first channel 9 Entropy of second channel 10 Skewness of second channel Table 2. Rank-scale normal in comparison with other frequencies Wilcoxon ROC Bhattacharyya t-test Score Number of feature Score Number of feature Score Number of feature Rate of p-value Number of feature 0.2794 179 0.277778 179 Inf 1025 3.44 × 10−13 2086 1 0.2794 243 0.277778 243 0.018852 1026 3.35 × 10−13 2405 2 0.2791 134 0.2775 134 0.018852 1027 3.35 × 10−13 2165 3 0.2788 129 0.277222 129 7.04 × 10−7 1 3.22×10−13 2421 4 0.2788 148 0.277222 148 7.04 × 10−7 2 3.22 × 10−13 2169 5 Table 2. Rank-scale normal in comparison with other frequencies Table 3. Rank-scale 8Hz in comparison with other frequencies Wilcoxon ROC Bhattacharyya t-test Score Number of feature Score Number of feature Score Number of feature Rate of p-value Number of feature 0.8495 1026 0.4505 1026 Inf 1025 2.6 × 10−13 2185 1 0.6925 2100 0.2935 2100 Inf 1027 2.29 × 10−13 2161 2 0.655 2099 0.29 1025 0.004 1026 4.8433 × 10−14 2240 3 0.645 2098 0.26 1027 2.52 × 10−7 2 2.2095 × 10−13 2384 4 0.6435 2097 0.6435 2097 2.52 × 10−7 3 2.133 × 10−13 2138 5 Table 4. Rank-scale 14Hz in comparison with other frequencies Wilcoxon ROC Bhattacharyya t-test Score Number of feature Score Number of feature Score Number of feature Rate of p-value Number of feature 0.801 1027 0.49 1025 Inf 1025 3.27 × 10−13 2331 1 0.6735 226 0.402 1027 Inf 1026 2.49 × 10−13 2058 2 0.6735 418 0.28 1026 0.004087 1027 2.16 × 10−13 2413 3 0.6735 482 0.2745 226 2.52 × 10−7 1 2.1 × 10−13 2327 4 0.673 275 0.2745 418 2.52 × 10−7 2 2.304 × 10−13 2137 5 Table 5. Rank-scale 28Hz in comparison with other frequencies Wilcoxon ROC Bhattacharyya t-test Score Number of feature Score Number of feature Score Number of feature Rate of p-value Number of feature 0 849 1025 0 48 1026 Inf 1025 3 48 × 10−13 2405 1 Table 3. The other methods and their selected features are shown in Tables 2-5. Rank-scale 8Hz in comparison with other frequencies Wilcoxon ROC Bhattacharyya t-test Score Number of feature Score Number of feature Score Number of feature Rate of p-value Number of feature 0.8495 1026 0.4505 1026 Inf 1025 2.6 × 10−13 2185 1 0.6925 2100 0.2935 2100 Inf 1027 2.29 × 10−13 2161 2 0.655 2099 0.29 1025 0.004 1026 4.8433 × 10−14 2240 3 0.645 2098 0.26 1027 2.52 × 10−7 2 2.2095 × 10−13 2384 4 0.6435 2097 0.6435 2097 2.52 × 10−7 3 2.133 × 10−13 2138 5 Table 3. Rank-scale 8Hz in comparison with other frequencies 0.645 2098 0.26 1027 2.52 × 10−7 2 2.2095 × 10−13 2384 4 0.6435 2097 0.6435 2097 2.52 × 10−7 3 2.133 × 10−13 2138 5 Table 4. Rank-scale 14Hz in comparison with other frequencies Wilcoxon ROC Bhattacharyya t-test Score Number of feature Score Number of feature Score Number of feature Rate of p-value Number of feature 0.801 1027 0.49 1025 Inf 1025 3.27 × 10−13 2331 1 0.6735 226 0.402 1027 Inf 1026 2.49 × 10−13 2058 2 0.6735 418 0.28 1026 0.004087 1027 2.16 × 10−13 2413 3 0.6735 482 0.2745 226 2.52 × 10−7 1 2.1 × 10−13 2327 4 0.673 275 0.2745 418 2.52 × 10−7 2 2.304 × 10−13 2137 5 Table 5. Rank-scale 28Hz in comparison with other frequencies Wilcoxon ROC Bhattacharyya t-test Score Number of feature Score Number of feature Score Number of feature Rate of p-value Number of feature 0.849 1025 0.48 1026 Inf 1025 3.48 × 10−13 2405 1 0.664 2319 0.46 1027 Inf 1026 2.9 × 10−13 2141 2 0.6475 2315 0.45 1025 Inf 1027 2.8 × 10−13 2180 3 Table 4. Rank-scale 14Hz in comparison with other frequencies Table 4. Rank-scale 14Hz in comparison with other frequencies Wilcoxon ROC Bhattacharyya t-test Score Number of feature Score Number of feature Score Number of feature Rate of p-value Number of feature 0.801 1027 0.49 1025 Inf 1025 3.27 × 10−13 2331 1 0.6735 226 0.402 1027 Inf 1026 2.49 × 10−13 2058 2 0.6735 418 0.28 1026 0.004087 1027 2.16 × 10−13 2413 3 0.6735 482 0.2745 226 2.52 × 10−7 1 2.1 × 10−13 2327 4 0.673 275 0.2745 418 2.52 × 10−7 2 2.304 × 10−13 2137 5 Table 5. Rank-scale 28Hz in comparison with other frequencies Table 5. The other methods and their selected features are shown in Tables 2-5. Rank-scale 28Hz in comparison with other frequencies Wilcoxon ROC Bhattacharyya t-test Score Number of feature Score Number of feature Score Number of feature Rate of p-value Number of feature 0.849 1025 0.48 1026 Inf 1025 3.48 × 10−13 2405 1 0.664 2319 0.46 1027 Inf 1026 2.9 × 10−13 2141 2 0.6475 2315 0.45 1025 Inf 1027 2.8 × 10−13 2180 3 0.631 2353 0.277 367 2.52 × 10−7 1 2.6322 × 10−13 2421 4 0.6295 2318 0.277 385 2.52 × 10−7 2 2.47 × 10−13 2169 5 After using all of the above methods, the selected features were divided into testing and training groups. Testing and training ratios were 20% to 80%, respectively. The k-NN, MLP, SVM, and Bayes classifiers were learned on the training group, while the testing group was applied to the learned classifiers. The classifications were of a 4-class nature, among which three were related to the frequencies of 8, 14, and 48 Hz. Another frequency was related to a normal frequency. 𝑘 is from 3 to 9 for k-NN classifier and the classifier was used for each number. There were five and six hidden layers of neurons for MLP classifier. The test was performed 100 times, and accordingly the accuracy was reported. The results of all classifiers with each featured method are reported in Table 6, along with their accuracy. In addition, k = 7 for k-NN and n = 6 (the number of neurons in hidden layer) for MLP had the best accuracy. In addition, k = 7 for k-NN and n = 6 (the number of neurons in hidden layer) for MLP had the best accuracy. Table 6 shows that k-NN gained the highest accuracy among the four classifiers (91.39%) and then SVM allocated the second rate of accuracy (89.34%) with the PCA feature selection method. Further, the features selected by PCA and those given to classifiers had higher accuracy. Furthermore, the accuracy of MLP was lower for each feature selection. Finally, Bhattacharyya and Wilcoxon had weaker results compared to other feature selections methods. Table6. The other methods and their selected features are shown in Tables 2-5. The results of feature selection methods and applied classifications Feature selection method Classifiers Accuracy t-test MLP 1.23 ± 73.44 KNN 2.58 ± 75.9 SVM 79.62 ± 3.25 Bayes 83.32 ± 3.41 Bhattacharyya MLP 4.52 ± 63.9 KNN 5.01 ± 68.9 SVM 65.11 ± 3.24 Bayes 73.01 ± 3.23 Wilcoxon MLP 69.21 ± 2.01 KNN 75.25 ± 1.08 SVM 76.98 ± 3.14 Bayes 77.45 ± 2.12 ROC MLP 73.46 ± 2.31 KNN 77.21 ± 1.25 SVM 75.32 ± 1.37 Bayes 79.91 ∓2.15 PCA MLP 59.55 ± 5.75 KNN 91.39 ± 0.85 SVM 89.34 ± 1.05 Bayes 72.54 ± 1.65 Decision Tree MLP 2.03 ± 63.12 KNN 2.71 ± 71.15 SVM 78.70 ± 0.18 Bayes 79.13 ± 0.12 4. Discussion 4. Discussion Comparing the results of the present study with those in other studies is difficult due to EEG (exclusively SSVEP) database, number of participants, type of Wavelet transform, decomposition level, type of classifiers, and feature extraction methods. However, the results were compared in the present study with other related ones. The information related to the database, as a pure SSVEP from EEG, was used to obtain the robust SSVEP, which was completely reliable for BCI systems and clinical applications. In addition, it allowed the researchers to reach the highest levels of accuracy and efficiency and signal-to-noise. Complexity and time-consuming calculations are considered as the most frequent problems regarding non-linear classifies [11]. A large number of studies reported such difficulties and created over-fitting problems [24]. Thus, in the present study, the linear classifications and feature extractions were implemented to obtain the highest accuracy and efficiency. The results demonstrated the highest accuracy, when linear methods are used for both classifiers and feature extractions, compared to other recent studies. However, some cases with lower accuracy were reported in comparison with the present work. In 2006, some studies applying SVM [25] reached an accuracy of 53.98%-56.07%, and some obtained 87.5% when they improved the level of decomposition up to seven [26]. Furthermore, an accuracy of 81.48% was obtained in some studies when they decomposed Wavelet up to five levels [27] and they used k- NN. In addition, some attained an accuracy of 65.90% by using SVM. In 2011, some obtained the 85.4% accuracy by using SVM classifier [28]. It is worth noting that non-linear classifiers were difficult to be calculated due to over-fitting and time calculation in the present study [29]. 5. Conclusion The present study aimed to determine the suitable features for classifying the frequency of 8, 14, and 28 Hz, and one normal class. Further, it answered the question whether the accuracy of SSVEP extraction could be optimized by ranking the features, and using PCA and decision tree methods. The highest level of accuracy was obtained (91.39%). Furthermore, the highest accuracy was reported by using PCA method with k-NN classifier. Unlike other conducted studies, the present research was prioritized due to its classification of all processes such as signal extraction methods, feature extractions, feature selection methods and classifications, as well as its accuracy, which is shown to be the possible value. In addition, PCA, decision tree, and t-test were better than Bhattacharyya and Wilcoxon. The results of Bayes and SVM had better performance that those in k-NN and MLP. Finally, MLP had the weakest result. Funding information This work did not receive any grant from funding agencies in the public, commercial, or not-for-profit sectors. Compliance with ethical standards Compliance with ethical standards Conflict of interest The authors declare that they have no conflict ofinterest. Ethical approval All procedures performed in studies involving humanparticipants were in accordance with the ethical standards of the Brain Science Institute, Laboratory for Advanced Brain Signal Processing and its later amendments or comparable ethical standards. Informed consent Informed consent was obtained from all individualparticipants included in the study. Informed consent Informed consent was obtained from all individualparticipants included in the study. [7] Ahirwal MK, Kumar A, Singh GK. Suri JS. Sub-band classification of decomposed single event-related potential co-variants for multi-class brain–computer interface: a qualitative and quantitative approach. IET Science, Measurement & Technology 2016; 10 (4): 355-363. References [1] kelly SP, Lalor EC, Reilly RB, Foxe JJ. 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Respiratory hazard assessment of combined exposure to complete gasoline exhaust and respirable volcanic ash in a multicellular human lung model at the air-liquid interface
Environmental pollution
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a r t i c l e i n f o Article history: Received 1 December 2017 Received in revised form 29 January 2018 Accepted 31 January 2018 Available online 16 February 2018 Communities resident in urban areas located near active volcanoes can experience volcanic ash expo- sures during, and following, an eruption, in addition to sustained exposures to high concentrations of anthropogenic air pollutants (e.g., vehicle exhaust emissions). Inhalation of anthropogenic pollution is known to cause the onset of, or exacerbate, respiratory and cardiovascular diseases. It is further postulated similar exposure to volcanic ash can also affect such disease states. Understanding of the impact of combined exposure of volcanic ash and anthropogenic pollution to human health, however, remains limited. The aim of this study was to assess the biological impact of combined exposure to respirable volcanic ash (from Soufriere Hills volcano (SHV), Montserrat and Chaiten volcano (ChV), Chile; representing different magmatic compositions and eruption styles) and freshly-generated complete exhaust from a gasoline vehicle. A multicellular human lung model (an epithelial cell-layer composed of A549 alveolar type II-like cells complemented with human blood monocyte-derived macrophages and dendritic cells cultured at the air-liquid interface) was exposed to diluted exhaust (1:10) continuously for 6 h, followed by immediate exposure to the ash as a dry powder (0.54 ± 0.19 mg/cm2 and 0.39 ± 0.09 mg/cm2 for SHV and ChV ash, respectively). After an 18 h incubation, cells were exposed again for 6 h to diluted exhaust, and a final 18 h incubation (at 37 C and 5% CO2). Cell cultures were then assessed for cytotoxic, oxidative stress and (pro-)inflammatory responses. Results indicate that, at all tested (sub-lethal) concentrations, co-exposures with both ash samples induced no significant expression of genes associated with oxidative stress (HMOX1, NQO1) or production of (pro-)inflammatory markers (IL-1b, IL-8, TNF-a) at the gene and protein levels. In summary, consid- ering the employed experimental conditions, combined exposure of volcanic ash and gasoline vehicle exhaust has a limited short-term biological impact to an advanced lung cell in vitro model. © 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Results indicate that, at all tested (sub-lethal) concentrations, co-exposures with both ash samples induced no significant expression of genes associated with oxidative stress (HMOX1, NQO1) or production of (pro-)inflammatory markers (IL-1b, IL-8, TNF-a) at the gene and protein levels. * This paper has been recommended for acceptance by David Carpenter. * Corresponding author. Institute of Hazard, Risk & Resilience, Department of Earth Sciences, Durham University, Science Labs, Durham, DH1 3LE, United Kingdom. ** Corresponding author. BioNanomaterials group, Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700, Fribourg, Switzerland. E-mail addresses: ines.tomasek@durham.ac.uk (I. Tomasek), claire.horwell@durham.ac.uk (C.J. Horwell), christoph.bisig@unifr.ch (C. Bisig), ddamby@usgs.gov (D.E. Damby), pierre.comte@bfh.ch (P. Comte), jan.czerwinski@bfh.ch (J. Czerwinski), alke.fink@unifr.ch (A. Petri-Fink), m.j.d.clift@swansea.ac.uk (M.J.D. Clift), barbara. drasler@unifr.ch (B. Drasler), barbara.rothen@unifr.ch (B. Rothen-Rutishauser). Respiratory hazard assessment of combined exposure to complete gasoline exhaust and respirable volcanic ash in a multicellular human lung model at the air-liquid interface* Ines Tomasek a, b, *, Claire J. Horwell a, Christoph Bisig b, David E. Damby c, Pierre Comte d, Jan Czerwinski d, Alke Petri-Fink b, e, Martin J.D. Clift f, Barbara Drasler b, Barbara Rothen-Rutishauser b, ** Ines Tomasek a, b, *, Claire J. Horwell a, Christoph Bisig b, David E. Damby c, Pierr Jan Czerwinski d, Alke Petri-Fink b, e, Martin J.D. Clift f, Barbara Drasler b, Barbara Rothen-Rutishauser b, ** a Institute of Hazard, Risk & Resilience, Department of Earth Sciences, Durham University, Science Labs, Durham, DH1 3LE, United Kingdom b BioNanomaterials group, Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700, Fribourg, Switzerland c Volcano Science Center, United States Geological Survey, Menlo Park, CA, 94025, United States d Laboratory for IC-Engines and Exhaust Emission Control, Bern University for Applied Sciences, Gwerdtstrasse 25, 2560, Nidau, Switzerland e Ch i t D t t U i it f F ib Ch i d M 1700 F ib S it l d a Institute of Hazard, Risk & Resilience, Department of Earth Sciences, Durham University, Science Labs, Durham, DH1 3LE, United Kingdom b BioNanomaterials group, Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700, Fribourg, Switzerland c Volcano Science Center, United States Geological Survey, Menlo Park, CA, 94025, United States d Laboratory for IC-Engines and Exhaust Emission Control, Bern University for Applied Sciences, Gwerdtstrasse 25, 2560, Nidau, Switzerlan e Chemistry Department, University of Fribourg, Chemin des Musee, 1700, Fribourg, Switzerland f In Vitro Toxicology Group, Swansea University Medical School, Singleton Park Campus, Swansea, SA2 8PP, Wales, United Kingdom stitute of Hazard, Risk & Resilience, Department of Earth Sciences, Durham University, Science Labs, Durham, DH1 3LE, United Kingdom oNanomaterials group, Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700, Fribourg, Switzerland olcano Science Center, United States Geological Survey, Menlo Park, CA, 94025, United States g y aboratory for IC-Engines and Exhaust Emission Control, Bern University for Applied Sciences, Gwerdtstrasse 25, 2560, Nidau, Switzerlan hemistry Department, University of Fribourg, Chemin des Musee, 1700, Fribourg, Switzerland y p , y f g, , , g, f In Vitro Toxicology Group, Swansea University Medical School, Singleton Park Campus, Swansea, SA2 8PP, Wales, United Kingdom Environmental Pollution 238 (2018) 977e987 Environmental Pollution 238 (2018) 977e987 Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/envpol journal homepage: www.elsevier.com/locate/envpol Respiratory hazard assessment of combined exposure to complete gasoline exhaust and respirable volcanic ash in a multicellular human lung model at the air-liquid interface* https://doi.org/10.1016/j.envpol.2018.01.115 0269-7491/© 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.envpol.2018.01.115 * This paper has been recommended for acceptance by David Carpenter. 1. Introduction polycyclic aromatic hydrocarbons) onto volcanic ash, potentially altering the ash surface chemical properties and affecting its po- tential toxicity. Hence, the use of ‘complete’ exhaust is a critical next step in deducing the hazard posed to populations exposed to volcanic emissions and pollutants within the air. Communities resident in urban areas located near active vol- canoes can experience volcanic ash exposures during, and following, an eruption, in addition to sustained exposures to high concentrations of anthropogenic air pollutants (e.g., vehicle exhaust emissions). Furthermore, ash can be transported over great dis- tances (e.g., as occurred during the 2010 Eyjafjallaj€okull eruption, Iceland (Gudmundsson et al., 2012)) and may reach distant urban areas, thereby having various impacts on human lives and liveli- hoods, including potential negative effects upon human health (Barsotti et al., 2010; Horwell and Baxter, 2006; Baxter & Horwell, 2015). Consequently, governments, public health agencies and scientific communities are voicing concerns about the potential health impacts from concomitant exposure to volcanic and anthropogenic emissions (Loughlin et al., 2012), especially whether this may pose a greater respiratory hazard than inhaling respirable volcanic ash and vehicle emissions separately. Hence, it is impor- tant to understand the potential hazard of concomitant exposure so that civil protection managers and health agencies can make informed decisions on whether to advise that citizens take action to protect themselves during periods of intense exposure to both ur- ban pollution and volcanic ash (McDonald et al., 2017). p Inhalation of urban particulate matter is known to cause the onset of, or exacerbate, respiratory and cardiovascular diseases (Pope et al., 2015; Peters et al., 1997; Seaton et al., 1995). Although diesel engines are generally viewed as greater contributors to engine-derived ambient particulate matter (US EPA, 2004), it has been found that vehicles with gasoline engines, which are still the most popular engine type in some European countries (ACEA, 2017), can also emit substantial quantities of soot-like ultrafine particles (diameter < 100 nm) under certain operating conditions (Mathis et al., 2005; Zhang and McMahon, 2012; Banerjee and Christian, 2017). Vehicles with gasoline direct injection (GDI) technology were found to release up to 1012 particles/km, exceeding those of current diesel vehicles equipped with filters (Platt et al., 2017; Mu~noz et al., 2016; Zhang and McMahon, 2012; Mohr et al., 2006). a r t i c l e i n f o In summary, consid- ering the employed experimental conditions, combined exposure of volcanic ash and gasoline vehicle exhaust has a limited short-term biological impact to an advanced lung cell in vitro model. © 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). source: https://doi. ol.2018.01.115 rs. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). I. Tomasek et al. / Environmental Pollution 238 (2018) 977e987 978 Abbreviations ALI Air-liquid interface CASP7 Caspase-7 gene ChV Chaiten volcano CO Carbon monoxide CO2 Carbon dioxide cRPMI Complete RPMI 1640 cell medium (supplemented with 1% L-Glutamine, 1% Penicillin/Streptomycin and 10% fetal bovine serum) DEP Diesel exhaust particles FAS FAS receptor gene GAPDH Glyceraldehyde-3-phosphate dehydrogenase gene GDI Gasoline direct injection GE Gasoline exhaust HMOX1 Heme oxygenase 1 gene IL-1b Interleukin-1 beta IL1B Interleukin-1 beta gene IL-8 Interleukin 8 IL8 Interleukin 8 gene LDH Lactate dehydrogenase LOI Loss on ignition LSM Laser scanning microscopy MDDC Monocyte-derived dendritic cells MDM Monocyte-derived macrophages MDL Method detection limit NMHC Non-methane hydrocarbons NOx Nitrogen oxide(s) NQO1 NAD(P)H dehydrogenase [quinone] 1 gene PBS Phosphate buffered saline PET Polyethylene terephthalate PN Particle number PSD Particle size distribution QCM Quartz crystal microbalance RNA Ribonucleic acid RT-PCR Reverse-transcription polymerase chain reaction SEM Scanning electron microscopy SHV Soufriere Hills volcano SiO2 Silicon dioxide THC Total hydrocarbons TNF-a Tumor necrosis factor-alpha TNFA Tumor necrosis factor-alpha gene VA Volcanic ash WLTC Worldwide Light-duty Test Cycle XRF X-ray fluorescence 2.2. Volcanic ash sources, preparation and characterisation Every explosive volcanic eruption generates ash, but particle characteristics (e.g., surface area and reactivity, composition, par- ticle size, leachable elements) and, potentially, toxicity will vary according to the magma composition and eruption style as well as post-eruptive factors such as distance from vent (of deposition), weathering, etc. Two volcanic ash samples were used in this study, to represent different magmatic compositions (andesite and rhyo- lite; see Section 3.1) and eruption styles. Both samples were chosen because they were erupted, transported and deposited into relatively-clean atmospheres, away from major sources of pollu- tion. Neither was rained on prior to sampling. In addition, both samples have been well characterised for their physical and chemical properties in previous studies (Horwell et al., 2010, 2013; Horwell, 2007). The major elemental oxide composition of bulk samples was determined by X-ray fluorescence (XRF) (Axios- Advanced PW4400 XRF spectrometer, PANalytical, The Netherlands) on fused beads prepared from ignited ash powders mixed with a fluxing agent in a 1:5 ratio. Compositional data were recalculated to account for loss on ignition (LOI) (the weight dif- ference between unignited and ignited powders), which also pro- vided confirmation of sample freshness with regards to contamination (e.g., with organic material). Volcanic ash samples, from Soufriere Hills volcano, Montserrat, and Chaiten volcano, Chile, were used (to represent different magmatic compositions and eruption styles) in combination with freshly-generated complete gasoline exhaust from a GDI vehicle (containing the particulate, condensed and gaseous fractions). This is the first time that a real, complete exhaust has been used to study combined exposures with volcanic ash. Furthermore, this investi- gation is the first to evaluate and report on whether the toxicity of either volcanic ash or complete gasoline exhaust are altered by co- exposures, as well as whether the ash (magmatic) composition could influence the outcome of combined exposures in vitro. As with our previous study, a multicellular in vitro human lung model, composed of epithelial lung cells (A549 alveolar type II-like cells) and two immune cell types (human blood monocyte-derived macrophages and dendritic cells) (Rothen-Rutishauser et al., 2005, 2008; Blank et al., 2006) cultured at the air-liquid interface (ALI) (Blank et al., 2007) was used. This well-established model has been proven to be suitable for various exposures at the ALI (Fytianos et al., 2016; Müller et al., 2011), thus reflecting, in part, the real- istic physiological conditions of a respiratory exposure. 1. Introduction The effects of emissions, including particulate and gaseous phases (i.e., complete exhaust), produced from gaso- line vehicles with various engine technologies have been studied in recent years and some toxic effects, such as oxidative stress and DNA damage in the lungs, have been reported in in vitro and in vivo studies (Bisig et al., 2015; Mauderly et al., 2014; Reed et al., 2008; McDonald et al., 2007; Lund et al., 2007). A recent first investigation, by the authors, into the potential effects of volcanic ash exposure combined with exhaust particulate (intending to simulate an urban environment) showed that concomitant exposure of cells to respirable volcanic ash and stan- dardized diesel exhaust particles (DEP (NIST SRM 2975)) can pro- mote a heightened (pro-)inflammatory response in vitro (Tomasek et al., 2016). However, the understanding of the respiratory hazard which may result from these combined exposures still remains limited, especially since this first study only considered DEP and not complete exhaust (i.e., the additional gaseous component). Exhaust is a complex mixture that contains particles but, also, condensed and gaseous fractions. These phases can impact lung health (e.g., Reed et al., 2008), but could also interact directly with the ash. This interaction may result in the adsorption of inorganic gases, such as CO2, CO and NOx, and volatile organic compounds (e.g., linear and It has been shown that exposure to respirable volcanic ash can exacerbate pre-existing respiratory diseases, such as asthma and bronchitis (Baxter et al.,1981,1983), and suppress immune function (Monick et al., 2013). Even though there is a high variability in discrete results of in vitro and in vivo toxicology assessments for volcanic ash (reviewed by Baxter et al., 2014, Hillman et al., 2012, and Horwell and Baxter, 2006), a general view from the studies, to date, is that ash is a low toxicity particle, but various studies have shown that ash can provoke inflammatory reactions in the lungs and (pro-)inflammatory reactions in vitro (Damby et al., 2013, 2016, 2018; Horwell et al., 2013; Lee and Richards, 2004). Discerning the components of volcanic ash responsible for any observed toxicity I. Tomasek et al. / Environmental Pollution 238 (2018) 977e987 979 the adaptation of using CD14þ magnetic beads (CD14 MicroBeads, Miltenyi Biotec, Germany) (Steiner et al., 2012). 2.2. Volcanic ash sources, preparation and characterisation Further, human epithelial airway barrier models have previously accom- panied hazard assessment studies of products of different engines and/or fuels using a sophisticated, well-characterised exhaust exposure system (Bisig et al., 2015, 2016; Steiner et al., 2013a,b; Müller et al., 2012, 2011, 2010). Here, the multicellular model was directly exposed to gasoline emissions, followed by the addition of respirable volcanic ash and a second gasoline emissions exposure. Subsequent analyses of the cell cultures for cytotoxicity, oxidative stress and (pro-)inflammatory response were undertaken. ( g g ) The first ash sample, from the Soufriere Hills volcano (SHV), Montserrat was generated in a dome-collapse event on 12 July 2003 and was collected 4 km from the vent on the day of eruption. The bulk sample's physicochemical characterisation can be found in previous literature under different sample codes, as follows: Soufriere Hills, Montserrat ‘03 in Horwell et al. (2007), Soufriere Hills, Montserrat 2003 in Horwell (2007), Mon12/7/03 in Horwell et al. (2010) and MBA12/7/03 in Horwell et al. (2013). Briefly, the ash is rich in crystalline silica (~12 wt% of the bulk ash is cristoba- lite), potentially the most pathogenic of the minerals found in volcanic ash (Baxter et al., 1999), and is considered fine grained for ash, with ~11.5 vol% of the bulk sample being sub-4 mm diameter (Horwell, 2007). This sample was used in the previous study investigating co-exposures with DEP in the same in vitro multi- cellular model (as MVO12/7/03 in Tomasek et al., 2016). 1. Introduction The monocyte- derived macrophages (MDM) and dendritic cells (MDDC) were added on the apical (1.2 x 104 cell/cm2) and the basal (6.0 x 104 cell/ cm2) side of the insert with a A549 layer on the upper side of the insert, respectively. After 24 h incubation under submerged con- ditions, in complete RPMI 1640 cell medium (cRPMI; Sigma-Aldrich, Switzerland; supplemented with 1% L-Glutamine, 1% Penicillin/ Streptomycin and 10% fetal bovine serum), the triple-cell co-culture was transferred to the ALI (the cRPMI only present on the basal side of the insert) to habituate for a period of 24 h prior to exposures. has been difficult due to compositional variability amongst samples and eruptions. Possible mechanisms identified for ash toxicity involve the presence of reactive surface species, including, but not exclusively, iron, and the corresponding generation of reactive ox- ygen species (Horwell et al., 2003, 2007), and crystalline silica and its potential to activate the NLRP3 inflammasome (Damby et al., 2018; Baxter et al., 1999). The mechanisms resulting in the (pro-) inflammatory response in vitro following combined exposures to ash and DEP (Tomasek et al., 2016) are not yet clear, but may be driven by the individual particle-cell interactions, or possibly particle-particle interactions, which then interact with cells. The aim of the present study was to assess the biological impact of combined exposure to cells of respirable volcanic ash and com- plete vehicle exhaust. A sophisticated in vitro approach, as also used in our previous study (Tomasek et al., 2016), provides a valuable first assessment of potential adverse impacts of such exposures, especially due to a lack of epidemiological studies that consider health effects of ashfall in heavily polluted urban areas. 2.6. Cell culture exposures The exposure setup was used as described previously (Tomasek et al., 2016). Briefly, the ash was loaded into the sample chamber and then pushed through the device by small pulses of air administered using a 10 mL commercial syringe. The ash was dis- charged as a cloud over the cell culture plate located below the delivery tube within a closed nebulisation chamber. Two cell cul- ture inserts from the same set (exposed beforehand to the exhaust or filtered air, see section 2.6) were exposed simultaneously to a single ash sample (SHV or ChV). The chamber was made of poly- styrene and covered with aluminium foil on the inside to avoid particles being electrostatically attracted and sticking to the chamber walls. The quantification of deposited material was monitored by a quartz crystal microbalance (QCM; Stanford Research Systems, USA), also placed within the nebulisation cham- ber next to the wells, thereby allowing for a reliable estimation of the deposited mass. Calculated from the recorded frequency values before and after deposition of material, the DF value (Hz) is con- verted to deposited mass per area (mg/cm2) (Lenz et al., 2009). The two-day cell exposure scenario (Fig. 1) was designed to simulate a real-life situation when volcanic ash is introduced to the urban environment (where people are continuously exposed to urban pollution; e.g., gasoline vehicle emissions), resulting in pol- lutants being concomitantly inhaled. Two sampling time-points were chosen, 24 h and 48 h, to enable observation of the effects after one day of co-exposure (as in the previous study by Tomasek et al., 2016) and, in addition, to account for possible effects following another, repeated exposure to gasoline exhaust. Daily experimental exposure to diluted (1:10) gasoline exhaust for 6 h was chosen according to earlier studies (Bisig et al., 2015, 2016; Steiner et al., 2013a; Steiner et al., 2012). It is difficult to assess whether this represents real life exposures, but could represent exposure of an urban outdoor worker. The cell-delivered volcanic ash doses fall within the range of the lowest and highest doses used in the previous study (Tomasek et al., 2016). In order to achieve equivalent mass exposure for both ash samples, the QCM was monitored during the exposures until a target dose between 0.4 and 0.5 mg/cm2 was reached. 2.1. Cell cultures The second sample was obtained from Chaiten volcano (ChV), in Patagonia, Chile and was deposited from an explosive eruption which occurred on 2 May 2008. The sample was collected 80 km away from the source, in the Patagonian Argentine province of Chubut, on 8 May 2008 (Horwell et al., 2010). This sample contains substantially less crystalline silica (~3 wt% of the bulk ash is cris- tobalite) than the SHV sample, but is similarly fine-grained, with ~12 vol% sub-4 mm material (as Chai_03 in Horwell et al., 2010). In vitro experiments were performed using an established multicellular lung model composed of three cell types mimicking the human alveolar epithelial tissue barrier as previously described (Rothen-Rutishauser et al., 2005, 2008; Blank et al., 2007). Briefly, the model consists of a layer of human alveolar type II-like epithelial cells (A549) cultured on polyethylene terephthalate (PET) membrane 6-well inserts (4.2 cm2 growth area, 3.0 mm pore size; BD FalconTM Cell Culture Inserts, BD Biosciences, USA) at an initial density of 23.8 x 104 cells/cm2. These were grown for 5 days prior to the addition of immune cells to form a co-culture. Human blood monocytes were isolated from buffy coats provided by the Transfusion Blood Bank (Blutspendedienst SRK Bern AG, Switzerland) as described previously (Lehmann et al., 2010) with A Sioutas Cascade Impactor (SKC Inc., USA) and Leland Legacy sample pump (SKC Inc., USA) attached to a gravitational separation chamber were used to isolate a biologically relevant ‘respirable’ sub-sample of the bulk ash for use in the in vitro exposure model, as previously described (Tomasek et al., 2016). Briefly, bulk ash was introduced into the separation chamber by an airstream I. Tomasek et al. / Environmental Pollution 238 (2018) 977e987 980 exposure). established by the pump at a constant flow rate of 5 L/min. Particles above a theoretical spherical aerodynamic diameter of 5 mm sedi- mented while the remaining, smaller particles were sampled by the impactor. Size-fractionated samples were recovered and combined for use in characterisation and toxicity assays. established by the pump at a constant flow rate of 5 L/min. Particles above a theoretical spherical aerodynamic diameter of 5 mm sedi- mented while the remaining, smaller particles were sampled by the impactor. Size-fractionated samples were recovered and combined for use in characterisation and toxicity assays. The diluted exhaust was pumped through the cell culture exposure chamber with a constant flow of 2 L/min. 2.1. Cell cultures In the chamber, the exhaust emissions pass above the cell culture plates and diffuse onto the cell cultures. Simultaneously, in a reference chamber, filtered ambient air supplied under identical conditions served as the negative control. The conditions in both chambers were controlled at 37 C, 85% relative humidity and 5% CO2. Particle size distributions (PSD) of isolated respirable samples were determined by Mie theory with a laser particle analyser (Beckman-Coulter LS 13 320; Coulter Corporation, USA) in water with sonication, with a refractive index set to 1.63 and an absorp- tion coefficient of 0.1 (after Horwell, 2007). Results are presented as the average of three consecutive measurements of the sample. Scanning electron microscopy (SEM) was used to observe particle morphology. Particles were mounted on polycarbonate discs, coated with 30 nm of gold/palladium and imaged on a Hitachi SU- 70 FEG SEM (Hitachi, Ltd., Japan) using the secondary electron detector. 2.3. Volcanic ash exposure system In order to deduce the individual and combined response from the co-culture, respirable ash samples were nebulized as dry powder directly over the cell cultures at the ALI using a dry powder insufflator (Model DP-4; PennCentury Inc., USA). The dry powder insufflator was found suitable for this application in the previous study (Tomasek et al., 2016), where its efficiency in ash adminis- tration to cells was evaluated, representing a more realistic approach when compared to studies using pre-suspended ash in submerged cell culture conditions. 2.5. Exhaust characterisation Characterisation of the exhaust was performed in parallel to the exposure experiments, yielding detailed information on the emis- sion sample that the cells were exposed to. Measurements were taken during the initial exposures (n ¼ 2) and repeated exposures (n ¼ 2) (each 10 WLTC cycles) resulting in 4 distinct datasets. The particle number (PN) was measured in the 1:10 diluted exhaust using an engine exhaust condensation particle counter (Model 3790, TSI Inc., USA). The concentrations of carbon monoxide (CO), carbon dioxide (CO2), total gaseous hydrocarbons (THC), non- methane hydrocarbons (NMHC) and nitrogen oxides (NOx) were measured using a Horiba MEXA-9400H (Horiba, Japan) exhaust gas measuring system in a constant volume sampling tunnel (Horiba CVS-9500 T, Horiba, Japan). CO2 was added to reach 5% for optimal buffering capacity in the cell culture medium. The concentration was controlled with two sensors, right before and after the cham- ber, and adjusted with a flowmeter, if needed. 2.6. Cell culture exposures There is a lack of dosimetry data for inhalation of ash, or exposure data on ambient air concentra- tions following volcanic eruptions, making average exposures difficult to constrain and apply in vitro, but Tomasek et al. (2016) determined their dose range to be a worst-case scenario. There- fore, these doses may not be realistic for personal exposure and could be considered as a particle over-load relative to a real-life exposure. Overall, the chosen exposure scenario may be consid- ered as a short-term, high-level exposure to both pollutants, indi- vidually and when combined. Even though the doses used likely deviate from realistic inhalation exposure, the assessment of cellular responses herein can be seen as a valuable screening of possible (adverse) effects that this specific type of combined exposure may incite which has not previously been considered or investigated. 2.7. Cellular assays and analysis 2.7.3. Quantification of (pro-)inflammatory response The quantity of tumor necrosis factor-alpha (TNF-a), interleukin-8 (IL-8) and interleukin-1 beta (IL-1b) secreted into the culture medium was assessed in the co-culture supernatants collected at 24 h and 48 h time-points by enzyme-linked immu- nosorbent assay (ELISA DuoSet Development Kit, R&D Systems, USA) according to the manufacturer's protocol. The concentrations were determined spectrophotometrically at 450 nm using a microplate reader (Bio-Rad, Switzerland). Analyses were conducted in duplicate for each repetition. The positive assay control was lipopolysaccharide (LPS, from E-coli 055:B5 strain ((Sigma-Aldrich, Germany), 1 mg/mL) applied as 1.2 mL solution in cRPMI in the bottom compartment of the cell culture insert for 24 h. Cell culture supernatants collected at 24 and 48 h time-points (Fig. 1) were analysed for key/relevant bio-markers of pulmonary- related toxicity in vitro (Donaldson et al., 2005). All analyses were performed according to established, standardized protocols as described below. 2.7.2. Lactate dehydrogenase release i i d i d b Cytotoxicity was determined by measuring the release of lactate dehydrogenase (LDH) into the co-culture supernatant, at both 24 h and 48 h time-points, using a LDH Cytotoxicity Detection Kit (Roche Applied Science, Germany) according to the manufacturer's proto- col. The test was conducted in duplicate for each experimental replication. Absorbance was determined at 490 nm using a micro- plate reader (Bio-Rad, Switzerland), with a reference wavelength set at 630 nm. The positive assay control was 100 mL of 0.2% Triton X-100 in PBS, applied for 24 h on the apical side of the cell culture insert. 2.4. Vehicle exhaust exposure system A flex-fuel GDI vehicle with a three-way catalyst was driven on a chassis dynamometer with standard market gasoline (RON 95) and lubrication oil. A dynamic, worldwide light-duty test cycle (WLTC) (UNECE, 2016), representing transient driving in urban, extra- urban, highway and motorway conditions, was driven and repeated for 6 h per day of exposures (10 cycles). The WLTC is the official driving cycle from September 2017 onwards used by the European Union for new vehicle registration (Euro6). The exhaust exposure experiments were performed at the exhaust gas control station of the Bern University of Applied Sci- ences in Nidau, Switzerland, as previously described (Bisig et al., 2015; Müller et al., 2010, 2011; Morin et al., 2008). Briefly, the exhaust was diluted 1:10 in filtered air, based on previous work (Steiner et al., 2013a, 2013b) and to enable comparison with pre- vious gasoline exhaust studies (Bisig et al., 2015, 2016), where it was noted that it represents a highly-polluted site (i.e., a high dose The multicellular lung model was exposed at the ALI to diluted exhaust (1:10) or filtered air (reference chamber) continuously for Fig. 1. Schematic of the cell culture exposures at the ALI in the present study. Culture supernatant was sampled at the 24 h time-point, and both the supernatant and insert membranes were sampled at the 48 h time-point. Cell-cultures were maintained under air-liquid interface (ALI) conditions throughout the entire exposures (initial exhaust, volcanic ash, repeated exhaust). I. Tomasek et al. / Environmental Pollution 238 (2018) 977e987 981 I. Tomasek et al. / Environmental Pollution 238 (2018) 977e987 981 Fig. 1. Schematic of the cell culture exposures at the ALI in the present study. Culture supernatant was sampled at the 24 h time-point, and both the supernatant and insert membranes were sampled at the 48 h time-point. Cell-cultures were maintained under air-liquid interface (ALI) conditions throughout the entire exposures (initial exhaust, volcanic ash, repeated exhaust). PBS (15e30 min, room temperature). Samples were then stained with the F-actin stain Phalloidin-Rhodamine (Thermo Fisher Scien- tific Inc., USA) and the nuclei stain 4’,6-diamidin-2-phenylindole (DAPI; Sigma-Aldrich, Germany) diluted 1:50 and 1:100 in 0.1% BSA in PBS (1e2 h, room temperature), respectively, and then mounted with Glycergel (DAKO Schweiz AG, Switzerland) on mi- croscope slides. 2.4. Vehicle exhaust exposure system Each 48-h exposure scenario (exhaust, ash, repeat exhaust) was conducted with two different sets of the multicellular lung model (i.e., with cells from different passage numbers and monocyte iso- lations), each exposed separately to volcanic ash after the initial exposures, resulting in two replicates (n ¼ 2) per exposure scenario. Two exposure scenarios were conducted over a 4-day period, resulting in 4 experimental replicates in total (n ¼ 4 per ash sam- ple). All data are presented relative to the filtered air (reference) cultures, however, we also included an untreated control (kept in the incubator) to assess the influence of the exposure protocol on cell response (see supplementary information). Collected super- natants were stored at either 4 C or 80 C prior to biochemical assays (section 2.7). Insert membranes were split and one half of each replicate's membrane was used for gene expression analysis (section 2.7.4) whilst the other half was fixed and prepared for fluorescent labelling, as described in section 2.7.1. Images of the cell membranes were acquired via confocal laser scanning microscopy (LSM; Zeiss 710 Confocal Microscope, Carl Zeiss, Switzerland) using a 63x/1.4 NA oil immersion lens. Repre- sentative images were processed using the public domain image analysis software ImageJ (http://rsb.info.nih.gov/ij). 2.4. Vehicle exhaust exposure system 6 h (i.e., the initial exposure), followed by immediate exposure to respirable volcanic ash as described in section 2.3, and then incu- bated (at 37 C and 5% CO2) for 18 h, maintaining the ALI conditions. Subsequently, the supernatants (i.e., cell culture medium) were collected (24 h time-point) from the basal side of the insert and replaced with fresh cell medium. Cells were exposed again for 6 h to diluted exhaust or filtered air (i.e., the repeated exposure), fol- lowed by a final 18 h incubation, maintained under ALI conditions. The supernatants were then collected (48 h time-point) (Fig. 1). Each 48-h exposure scenario (exhaust, ash, repeat exhaust) was conducted with two different sets of the multicellular lung model (i.e., with cells from different passage numbers and monocyte iso- lations), each exposed separately to volcanic ash after the initial exposures, resulting in two replicates (n ¼ 2) per exposure scenario. Two exposure scenarios were conducted over a 4-day period, resulting in 4 experimental replicates in total (n ¼ 4 per ash sam- ple). All data are presented relative to the filtered air (reference) cultures, however, we also included an untreated control (kept in the incubator) to assess the influence of the exposure protocol on cell response (see supplementary information). Collected super- natants were stored at either 4 C or 80 C prior to biochemical assays (section 2.7). Insert membranes were split and one half of each replicate's membrane was used for gene expression analysis (section 2.7.4) whilst the other half was fixed and prepared for fluorescent labelling, as described in section 2.7.1. 6 h (i.e., the initial exposure), followed by immediate exposure to respirable volcanic ash as described in section 2.3, and then incu- bated (at 37 C and 5% CO2) for 18 h, maintaining the ALI conditions. Subsequently, the supernatants (i.e., cell culture medium) were collected (24 h time-point) from the basal side of the insert and replaced with fresh cell medium. Cells were exposed again for 6 h to diluted exhaust or filtered air (i.e., the repeated exposure), fol- lowed by a final 18 h incubation, maintained under ALI conditions. The supernatants were then collected (48 h time-point) (Fig. 1). 3.1.1. Volcanic ash nebulisation The average cell-delivered doses of nebulized ash using the dry powder insufflator, as monitored by a QCM, were 0.54 ± 0.19 mg/ cm2 and 0.39 ± 0.09 mg/cm2 for SHV and ChV ash, respectively (Fig. 3). All statistical analyses were performed using the software Origin (version 9.3, OriginLab Corporation, USA). Statistical signifi- cance was deduced through the use of a one-way analysis of vari- ance (ANOVA), assuming a normal distribution of the datasets. Subsequent Tukey's post hoc tests were conducted to determine statistical significance between different exposures and the refer- ence exposure (filtered air). The alpha value was set at 0.05. 2.7.4. Gene expression analysis 2.7.4. Gene expression analysis Quantification of gene expression at the transcriptional level was performed by real-time reverse-transcription polymerase chain reaction (real-time RT-PCR), as previously described (Bisig et al., 2015). Cell culture membranes sampled at the 48 h time- point were stored in the ribonucleic acid (RNA) protect buffer (RNAprotect® Cell Reagent, Qiagen, Germany; diluted in PBS 1:4 (v/ v)) prior to the analysis. RNA was isolated with a RNeasy plus kit (Qiagen, Germany). Complementary deoxyribonucleic acid (cDNA) was synthetized with the Omniscript RT system (Qiagen, Germany), Oligo-dT primer (Microsynth, Switzerland) and RNasin Inhibitor (Promega, USA). Real-time RT-PCR was performed using SYBR- green (Applied Biosystems, USA) on a 7500 Fast Real-Time PCR system (Applied Biosystems, USA). Relative expression levels were calculated using the 2DDCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the standard (housekeeping) gene and filtered air as the control. The induction of cell death was determined by expression levels of (pro-)apoptotic genes caspase 7 (CASP7) and FAS receptor (FAS). Heme oxygenase 1 (HMOX1) and NAD(P)H dehydrogenase [quinone] 1 (NQO1) genes were used to observe the onset of oxidative stress. For assessment of (pro-)inflammatory responses, expression of pro-interleukin-1 beta (IL1B) and interleukin-8 (IL8) genes were measured. Fig. 2. Volcanic ash characterisation. A) Particle size distribution (PSD) of the isolated respirable fraction of volcanic ash samples determined by a Beckman Coulter LS 13 320 PSD analyser (Coulter Corporation, USA). Data are the mean of n ¼ 3. B) Representative scanning electron microscopy images of volcanic ash samples from Soufriere Hills volcano (SHV) and Chaiten volcano (ChV). Images were collected at 10.0 kV and WD 16 mm. Scale bars are 5 mm. 3.2. Exhaust characterisation The composition of the gaseous fraction, comprising carbon monoxide (CO), total hydrocarbons (THC), non-methane hydro- carbons (NMHC), nitrogen oxides (NOx) and carbon dioxide (CO2), as well as the average count of produced particles are shown in Table 2. 2.8. Data processing and statistical analysis All data in the figures are presented as single values and means derived from gasoline exhaust or filtered air exposures (the initial exposures (n ¼ 2) and repeated exposures (n ¼ 2), over 4 days in total); each exposure was performed with two different sets of the multicellular lung model (n ¼ 2; each exposed separately to vol- canic ash), leading to 4 repetitions (n ¼ 4). 2.7.1. Cell morphology Cell membranes collected at the 48 h time-point were fixed with 4% paraformaldehyde (15 min, room temperature), washed and stored in phosphate buffered saline (PBS). Subsequently, they were permeabilised with 0.2% Triton X-100 (Sigma-Aldrich, Germany) in I. Tomasek et al. / Environmental Pollution 238 (2018) 977e987 982 Fig. 2. Volcanic ash characterisation. A) Particle size distribution (PSD) of the isolated respirable fraction of volcanic ash samples determined by a Beckman Coulter LS 13 320 PSD analyser (Coulter Corporation, USA). Data are the mean of n ¼ 3. B) Representative scanning electron microscopy images of volcanic ash samples from Soufriere Hills volcano (SHV) and Chaiten volcano (ChV). Images were collected at 10.0 kV and WD 16 mm. Scale bars are 5 mm. Table 2 Table 2 Average exhaust composition for the flex-fuel GDI vehicle in the WLTC as measured during the experiments (n ¼ 4). SD ¼ standard deviation, CO ¼ carbon monoxide, THC ¼ total hydrocarbons, NMHC ¼ non-methane hydrocarbons, NOx ¼ nitrogen oxides, CO2 ¼ carbon dioxide. *Note that the CO2 concentration applied to the cell culture chamber was adjusted as necessary to 5% CO2. PN ¼ particle number. All data are shown 1:10 diluted (as applied to the cell cultures). We have found that exposure of cells to gasoline exhaust, alone, does not induce any significant effects on any of the biological endpoints measured. These results are in agreement with research performed by Bisig et al. (2016) using gasoline exhaust, alone, on a multicellular human lung model mimicking the bronchial compartment, under similar experimental conditions. Another study by Bisig et al. (2015), using the same experimental setup but a different car, found that gasoline exhaust induced oxidative stress; however, the particle number measured in the diluted exhaust was up to three orders of magnitude higher than that used by Bisig et al. (2016), and nearly twice as high as the average daily number of particles produced during exposures in the present study (Table 2). Similarly, the concentrations of volatile compounds in Bisig et al. (2015) exceed those measured in Bisig et al. (2016) and the cur- rent study. Bisig et al. (2015) found that filtration of the particulate fraction from the exhaust was not sufficient to eliminate the adverse effects in vitro, confirming the importance of the volatile compounds in GE-induced toxicity. The toxic effects of gasoline exhaust, particularly after particle filtration, have also been observed with in vivo animal studies (Reed et al., 2008; Lund et al., 2007). It was noted, though, that volatile compounds, alone, might react differently with the lung cells than when part of the complete exhaust, where these compounds can adsorb onto the particle surfaces (Steiner et al., 2014, 2016). Exhaust component Unit Mean SD CO ppm 27.71 2.98 THC ppm 6.97 0.44 NMHC ppm 4.82 0.46 NOx ppm 1.76 0.12 CO2 * % 0.98 0.01 PN #/cm3 1.32Eþ05 1.65Eþ04 comparison to the filtered air (Fig. 4b). The positive LDH assay control Triton X-100 showed a significant (p < 0.05) increase in LDH content in culture medium, confirming that the biological model used was responsive for the measured endpoint. Table 1 Table 1 Bulk chemical compositions of the volcanic ash samples used in the study. Results are presented as component weight percent oxide and recalculated to include loss on ignition (LOI) in the final total. Sample SiO2 TiO2 Al2O3 Fe2O3 MnO MgO CaO Na2O K2O P2O5 SO3 LOI Total SHV 61.8 0.5 17.0 6.6 0.2 2.4 6.3 3.7 0.9 0.1 0.1 0.6 100.1 ChV 73.4 0.2 13.9 1.6 0.1 0.4 1.5 4.2 2.9 0.1 0.0 1.1 99.4 Bulk chemical compositions of the volcanic ash samples used in the study. Results are presented as component weight per ignition (LOI) in the final total. ChV 73.4 0.2 13.9 1.6 0.1 0.4 Fig. 3. Deposition of nebulized respirable volcanic ash. Average mass deposition (mg/cm2) of respirable ash from Soufriere Hills volcano (SHV) and Chaiten volcano (ChV) quantified using a QCM, following their dry nebulisation over cell cultures using a dry powder insufflator (DP-4, Penn Century, USA). All data are presented as single values and mean (solid line), n ¼ 4. positive assay control, significantly (p < 0.05) increased the release of IL-8 (Fig. 5a) as well as TNF-a and IL-1b (SI Fig. 2) compared to the filtered air and other cell treatments. Lack of a (pro-)inflammatory response was supported by the findings on a gene level, where the cell exposures did not induce any change in mRNA levels of measured markers, IL8 and IL1B, relative to filtered air (Fig. 5b). In agreement with the protein measurements, none of the treatments induced a detectable upregulation of TNFA (data not shown). In the ChV ash-treated cultures, a slight, yet insignificant (p > 0.05), in- crease in expression of both IL8 and IL1B was observed compared to the filtered air (reference) exposure. Similarly, the expression of investigated genes related to oxidative stress, namely HMOX1 and NQO1, showed no significant (p > 0.05) increase after exposure to VA, GE or GE þ VA (Fig. 6). Combined exposure to gasoline exhaust and ChV ash (GE þ ChV) did, however, result in a slight upregulation of HMOX1 relative to filtered air, although not significant. Fig. 3. Deposition of nebulized respirable volcanic ash. Average mass deposition (mg/cm2) of respirable ash from Soufriere Hills volcano (SHV) and Chaiten volcano (ChV) quantified using a QCM, following their dry nebulisation over cell cultures using a dry powder insufflator (DP-4, Penn Century, USA). Table 1 All data are presented as single values and mean (solid line), n ¼ 4. 3.3. Biological endpoints / Environmental Pollution 238 (2018) 977e987 983 Table 1 Bulk chemical compositions of the volcanic ash samples used in the study Results are presented as component weight percent oxide and recalculated to include loss on 4. Discussion The purpose of the study was to investigate the potential res- piratory hazard of combined exposure to volcanic ash and anthropogenic pollution, through experiments designed to assess the impact to a multicellular lung model of exposure to both vol- canic ash and complete gasoline exhaust. 3.3. Biological endpoints Particle size analysis of isolated respirable fractions showed that 98% by volume and 84% by volume of particles were sub-10 mm diameter, for ash from Soufriere Hills volcano (SHV) and Chaiten volcano (ChV), respectively (Fig. 2a). The SHV sample consisted of 58% by volume particles with size <4 mm, while ChV contained less with 40% by volume. For the assessed biological endpoints (cytotoxicity, oxidative stress and (pro-)inflammatory mediators, including measurements for both protein production and gene expression), no significant (p > 0.05) changes in cell cultures were observed at 24 h or 48 h time-points for any of the experimental exposures, i.e., volcanic ash (VA), gasoline exhaust (GE) and co-exposures (GE þ VA). The response of the untreated cells (i.e., incubator control) was lower in comparison to the cells treated with filtered air, albeit not signifi- cant (SI Fig. 1). To account for the influence of the potentially stress- inducing airflow as well as for the different baseline levels in the various cultures, the comparison of the effects of cell treatments (VA, GE and GE þ VA) was made with the filtered air (reference) exposure. The morphology of the particles from both volcanoes, as observed by SEM, was mostly blocky and angular with varying amounts of sub-micron particles adhering to the surfaces of larger particles (Fig. 2b). This is congruent with previous observations of respirable volcanic ash (e.g., Damby et al., 2016, Lahde et al., 2013, Horwell et al., 2013, Hillman et al., 2012, Le Blond et al., 2010), but may not mirror the morphology of larger ash particles, which can differ according to (magmatic) composition. For all exposure scenarios (VA, GE and GE þ VA) at 48 h, LSM imaging revealed a homogenous and confluent epithelial cell layer with no alteration in cell morphology compared to the filtered air (reference) exposure (Fig. 4a). LDH release by the cells following the exposures, for both time-points, showed limited elevation in Bulk oxide elemental data for samples are listed in Table 1 and indicate magmatic composition of the ash samples. The SHV ash was confirmed to be ‘andesitic’, with an intermediate composition regarding silicon dioxide (SiO2) content (61.8 wt% SiO2), while ChV is ‘rhyolitic’, being comparatively richer in SiO2 (73.4 wt% SiO2). I. Tomasek et al. Table 2 For expression of pro-apoptotic genes FAS and CASP7, none of the exposures showed a statistically significant (p > 0.05) outcome relative to filtered air (Fig. 4c). CASP7 expression was found to be slightly suppressed by VA, GE, and combined exposure to gasoline exhaust and SHV ash (GE þ SHV), while only slightly increased in the combined exposure to gasoline exhaust and ChV ash (GE þ ChV); these changes, however, are still within the observed biological variation. The biological sensitivity of the employed multicellular model has been validated in the past through use of positive particulate controls, e.g., crystalline quartz (DQ12) for a (pro-)inflammatory response (Endes et al., 2014; Chortarea et al., 2015). The lack of adverse effects observed following gasoline exhaust exposure in the present study may be explained by important differences in experimental parameters, including the employed cell lines, the As determined via release of specifically chosen (pro-)inflam- matory mediators, none of the cell exposures induced a significant (p > 0.05) (pro-)inflammatory response (Fig. 5a). In fact, the con- centrations of TNF-a and IL-1b in all measured samples were below the method detection limits (MDL; SI Fig. 1). LPS, which served as a 984 I. Tomasek et al. / Environmental Pollution 238 (2018) 977e987 ing test cycle and vehicle tested and, hence, the lower particle mbers and volatile concentrations (and consequent lower doses) ompared to other studies, and exposure times. In addition, it is wn that the response of cultured cells to exposure may vary, especially over time (Poland et al., 2014). The evidence f studies such as Bisig et al. (2015) suggests that we s conclude that gasoline exhaust is incapable of inciting a response. . Cell morphology and cell viability of the multicellular lung model following combined exposure to gasoline exhaust and volcanic ash. A) Representative es from XY and YZ projections for cultures exposed to filtered air (reference exposure), (filtered air and) Soufriere Hills ash (SHV), (filtered air and) Chaiten ash (C ust (GE), combined exposure to gasoline exhaust and Soufriere Hills ash (GE þ SHV), and combined exposure to gasoline exhaust and Chaiten ash (GE þ ChV ed with Phalloidin-Rhodamine (F-actin cytoskeleton, magenta) and DAPI (cell nuclei, cyan). Scale bars are 20 mm. B) Extracellular LDH levels in the culture mediu 8 h normalized to filtered air (reference) exposure (dashed line). The positive assay control was 0.2% Triton X-100 in PBS. Table 2 C) Amounts of mRNA of pro-apopto tor (FAS) and caspase-7 (CASP7), 48 h post-exposures, normalized to filtered air (reference) exposure (dashed line). All data are presented as single values and mea ; * denotes a significant difference (p < 0.05) between the positive control and the other samples tested. (For interpretation of the references to colour in this figur r is referred to the Web version of this article.) Fig. 4. Cell morphology and cell viability of the multicellular lung model following combined exposure to gasoline exhaust and volcanic ash. A) Representative confocal LSM images from XY and YZ projections for cultures exposed to filtered air (reference exposure), (filtered air and) Soufriere Hills ash (SHV), (filtered air and) Chaiten ash (ChV), gasoline exhaust (GE), combined exposure to gasoline exhaust and Soufriere Hills ash (GE þ SHV), and combined exposure to gasoline exhaust and Chaiten ash (GE þ ChV). Cells were stained with Phalloidin-Rhodamine (F-actin cytoskeleton, magenta) and DAPI (cell nuclei, cyan). Scale bars are 20 mm. B) Extracellular LDH levels in the culture medium after 24 h and 48 h normalized to filtered air (reference) exposure (dashed line). The positive assay control was 0.2% Triton X-100 in PBS. C) Amounts of mRNA of pro-apoptotic genes FAS receptor (FAS) and caspase-7 (CASP7), 48 h post-exposures, normalized to filtered air (reference) exposure (dashed line). All data are presented as single values and mean (solid line), n ¼ 4; * denotes a significant difference (p < 0.05) between the positive control and the other samples tested. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) especially over time (Poland et al., 2014). The evidence from other studies such as Bisig et al. (2015) suggests that we should not conclude that gasoline exhaust is incapable of inciting a biological response. driving test cycle and vehicle tested and, hence, the lower particle numbers and volatile concentrations (and consequent lower doses) as compared to other studies, and exposure times. In addition, it is known that the response of cultured cells to exposure may vary, I. Tomasek et al. / Environmental Pollution 238 (2018) 977e987 985 Fig. 5. Release of (pro-)inflammatory mediators in the multicellular lung model following combined exposure to gasoline exhaust and volcanic ash. Table 2 (2016), who report no upregulation of TNFA or IL8 in response to gasoline exhaust exposure in a bronchial epithelium model, it appears that co-exposures to volcanic ash and gasoline exhaust do not induce inflammation via these pathways, at least at the doses tested here, and using the lung model composed of cells from healthy donors. However, the non-significant increase in IL8 expression in response to ChV ash remains unexplained, especially since this sample has not been used in cytokine assays previously. perhaps not surprising, and could indicate that these combined exposures did not generate either an additive or synergistic response. Due to experimental design, the effect of direct ash- volatile interactions (e.g., volatile adsorption) prior to co-culture exposures was unable to be tested here. The absence of alter- ations in cell morphology, cell viability and oxidative stress state for any combined exposure scenario are in line with our previous in vitro study, which showed limited cytotoxic and oxidative po- tential of SHV ash when exposed concomitantly with DEP (Tomasek et al., 2016). However, the low (pro-)inflammatory response following combined exposures is contrary to the previous findings, where co-exposures of SHV ash and DEP increased release of (pro-) inflammatory mediators TNF-a and IL-8, as well as significantly increased (p < 0.05) IL-1b (Tomasek et al., 2016). In Tomasek et al. (2016) it was hypothesized that the observed IL-8 production was driven by DEP, and that volcanic ash, alone, did not result in sig- nificant production of TNF-a, although it augmented TNF-a pro- duction in the co-exposures. Together with the results from Bisig et al. (2016), who report no upregulation of TNFA or IL8 in response to gasoline exhaust exposure in a bronchial epithelium model, it appears that co-exposures to volcanic ash and gasoline exhaust do not induce inflammation via these pathways, at least at the doses tested here, and using the lung model composed of cells from healthy donors. However, the non-significant increase in IL8 expression in response to ChV ash remains unexplained, especially since this sample has not been used in cytokine assays previously. The similar biological responses to both ash samples in com- bined exposures with gasoline exhaust indicates that, within the parameters of this particular experimental setup, differences in sample composition (e.g., iron content) and mineralogy (e.g., crys- talline silica content) did not affect the biological response to co- exposures. Table 2 A) Interleukin- 8 (IL-8) release in the culture medium after 24 h and 48 h following exposures to filtered air (reference exposure), (filtered air and) Soufriere Hills ash (SHV), (filtered air and) Chaiten ash (ChV), gasoline exhaust (GE), combined exposure to gasoline exhaust and Soufriere Hills ash (GE þ SHV) and combined exposure to gasoline exhaust and Chaiten ash (GE þ ChV). The positive assay control was lipopolysaccharide (LPS; 1 mg/ mL, 24 h). B) Amounts of mRNA of (pro-)inflammation-related genes encoding interleukin-1 beta (IL1B) and IL-8 (IL8), 48 h post-exposures, normalized to filtered air exposure (a dashed line). All data are shown as single values and mean (solid line), n ¼ 4; * denotes significant difference (p < 0.05) between the positive control and the other samples tested. I. Tomasek et al. / Environmen Fig. 6. Oxidative stress response in the multicellular lung model following com- bined exposure to gasoline exhaust and volcanic ash. Amounts of mRNA of oxidative stress responsive genes, heme oxygenase 1 (HMOX1) and NAD(P)H dehydrogenase [quinone] 1 (NQO1), following exposures to (filtered air and) Soufriere Hills ash (SHV), (filtered air and) Chaiten ash (ChV), gasoline exhaust (GE), combined exposure to gasoline exhaust and Soufriere Hills ash (GE þ SHV) and combined exposure to gas- oline exhaust and Chaiten ash (GE þ ChV), normalized to filtered air exposure (dashed line). Data are shown as single values and mean (solid line), n ¼ 4. Pollution 238 (2018) 977 987 985 The two ash types used in this study, erupted from different volcanoes of different magmatic compositions also did not elicit a perhaps not surprising, and could indicate that t exposures did not generate either an additive response. Due to experimental design, the effec volatile interactions (e.g., volatile adsorption) prio exposures was unable to be tested here. The ab ations in cell morphology, cell viability and oxidativ any combined exposure scenario are in line wit in vitro study, which showed limited cytotoxic an tential of SHV ash when exposed concomitantly wit et al., 2016). However, the low (pro-)inflamm following combined exposures is contrary to the pr where co-exposures of SHV ash and DEP increased inflammatory mediators TNF-a and IL-8, as well increased (p < 0.05) IL-1b (Tomasek et al., 2016). Table 2 (2016), who report no upregulation of TNFA or IL8 in response to gasoline exhaust exposure in a bronchial epithelium model, it appears that co-exposures to volcanic ash and gasoline exhaust do not induce inflammation via these pathways, at least at the doses tested here, and using the lung model composed of cells from healthy donors. However, the non-significant increase in IL8 expression in response to ChV ash remains unexplained, especially since this sample has not been used in cytokine assays previously. The similar biological responses to both ash samples in com- bined exposures with gasoline exhaust indicates that, within the parameters of this particular experimental setup, differences in sample composition (e.g., iron content) and mineralogy (e.g., crys- talline silica content) did not affect the biological response to co- exposures. Whilst we chose two fairly different ash samples, vol- canic ash is a heterogeneous dust, the physicochemical character- istics of which can vary considerably, even during a discrete eruption (Damby et al., 2017; Horwell et al., 2013), and samples from different eruptions have shown differences in toxicity when tested comparatively in vitro, previously (Damby et al., 2016; Horwell et al., 2013; Wilson et al., 2000). Hence, a different sam- ple from an individual eruption or a different ash type might incite a different cellular response perhaps not surprising, and could indicate that these combined exposures did not generate either an additive or synergistic response. Due to experimental design, the effect of direct ash- volatile interactions (e.g., volatile adsorption) prior to co-culture exposures was unable to be tested here. The absence of alter- ations in cell morphology, cell viability and oxidative stress state for any combined exposure scenario are in line with our previous in vitro study, which showed limited cytotoxic and oxidative po- tential of SHV ash when exposed concomitantly with DEP (Tomasek et al., 2016). However, the low (pro-)inflammatory response following combined exposures is contrary to the previous findings, where co-exposures of SHV ash and DEP increased release of (pro-) inflammatory mediators TNF-a and IL-8, as well as significantly increased (p < 0.05) IL-1b (Tomasek et al., 2016). In Tomasek et al. (2016) it was hypothesized that the observed IL-8 production was driven by DEP, and that volcanic ash, alone, did not result in sig- nificant production of TNF-a, although it augmented TNF-a pro- duction in the co-exposures. Together with the results from Bisig et al. Table 2 Whilst we chose two fairly different ash samples, vol- canic ash is a heterogeneous dust, the physicochemical character- istics of which can vary considerably, even during a discrete eruption (Damby et al., 2017; Horwell et al., 2013), and samples from different eruptions have shown differences in toxicity when tested comparatively in vitro, previously (Damby et al., 2016; Horwell et al., 2013; Wilson et al., 2000). Hence, a different sam- ple from an individual eruption or a different ash type might incite a different cellular response. Fig. 5. Release of (pro-)inflammatory mediators in the multicellular lung model following combined exposure to gasoline exhaust and volcanic ash. A) Interleukin- 8 (IL-8) release in the culture medium after 24 h and 48 h following exposures to filtered air (reference exposure), (filtered air and) Soufriere Hills ash (SHV), (filtered air and) Chaiten ash (ChV), gasoline exhaust (GE), combined exposure to gasoline exhaust and Soufriere Hills ash (GE þ SHV) and combined exposure to gasoline exhaust and Chaiten ash (GE þ ChV). The positive assay control was lipopolysaccharide (LPS; 1 mg/ mL, 24 h). B) Amounts of mRNA of (pro-)inflammation-related genes encoding interleukin-1 beta (IL1B) and IL-8 (IL8), 48 h post-exposures, normalized to filtered air exposure (a dashed line). All data are shown as single values and mean (solid line), n ¼ 4; * denotes significant difference (p < 0.05) between the positive control and the other samples tested. The two ash types used in this study, erupted from different volcanoes of different magmatic compositions, also did not elicit a significant response in the biological endpoints measured from the multicellular model. Ash samples from these volcanoes have been previously investigated for their toxic potential and also showed limited biological responses (Damby et al., 2016, Horwell et al., 2013, Wilson et al., 2000, Cullen and Searl, 1998 and unpublished data for ChV). The SHV ash has also caused minimal response to the same multicellular model in our previous study (Tomasek et al., 2016). Hence, these data confirm the generally-observed lack of potential of ash to significantly affect healthy lung cell integrity or function including, in this case, initiation of an inflammatory response for the chosen time-points and endpoints. Table 2 In (2016) it was hypothesized that the observed IL-8 driven by DEP, and that volcanic ash, alone, did n nificant production of TNF-a, although it augmen duction in the co-exposures. Together with the re et al. (2016), who report no upregulation of response to gasoline exhaust exposure in a bronc model, it appears that co-exposures to volcanic a exhaust do not induce inflammation via these path the doses tested here, and using the lung model co from healthy donors. However, the non-significan expression in response to ChV ash remains unexpla since this sample has not been used in cytokine as Fig. 5. Release of (pro-)inflammatory mediators in the multicellular lung model following combined exposure to gasoline exhaust and volcanic ash. A) Interleukin- 8 (IL-8) release in the culture medium after 24 h and 48 h following exposures to filtered air (reference exposure), (filtered air and) Soufriere Hills ash (SHV), (filtered air and) Chaiten ash (ChV), gasoline exhaust (GE), combined exposure to gasoline exhaust and Soufriere Hills ash (GE þ SHV) and combined exposure to gasoline exhaust and Chaiten ash (GE þ ChV). The positive assay control was lipopolysaccharide (LPS; 1 mg/ mL, 24 h). B) Amounts of mRNA of (pro-)inflammation-related genes encoding interleukin-1 beta (IL1B) and IL-8 (IL8), 48 h post-exposures, normalized to filtered air exposure (a dashed line). All data are shown as single values and mean (solid line), n ¼ 4; * denotes significant difference (p < 0.05) between the positive control and the other samples tested. Fig. 6. Oxidative stress response in the multicellular lung mo bined exposure to gasoline exhaust and volcanic ash. Amounts stress responsive genes, heme oxygenase 1 (HMOX1) and NAD [quinone] 1 (NQO1), following exposures to (filtered air and) Souf (filtered air and) Chaiten ash (ChV), gasoline exhaust (GE), co gasoline exhaust and Soufriere Hills ash (GE þ SHV) and combin oline exhaust and Chaiten ash (GE þ ChV), normalized to filtered line). Data are shown as single values and mean (solid line), n ¼ I. Tomasek et al. / Environmental Pollution 238 (2018) 977 987 Fig. 6. Oxidative stress response in the multicellular lung model following com- bined exposure to gasoline exhaust and volcanic ash. Table 2 Amounts of mRNA of oxidative stress responsive genes, heme oxygenase 1 (HMOX1) and NAD(P)H dehydrogenase [quinone] 1 (NQO1), following exposures to (filtered air and) Soufriere Hills ash (SHV), (filtered air and) Chaiten ash (ChV), gasoline exhaust (GE), combined exposure to gasoline exhaust and Soufriere Hills ash (GE þ SHV) and combined exposure to gas- oline exhaust and Chaiten ash (GE þ ChV), normalized to filtered air exposure (dashed line). Data are shown as single values and mean (solid line), n ¼ 4. Fig. 6. Oxidative stress response in the multicellular lung model following com- bined exposure to gasoline exhaust and volcanic ash. Amounts of mRNA of oxidative stress responsive genes, heme oxygenase 1 (HMOX1) and NAD(P)H dehydrogenase [quinone] 1 (NQO1), following exposures to (filtered air and) Soufriere Hills ash (SHV), (filtered air and) Chaiten ash (ChV), gasoline exhaust (GE), combined exposure to gasoline exhaust and Soufriere Hills ash (GE þ SHV) and combined exposure to gas- oline exhaust and Chaiten ash (GE þ ChV), normalized to filtered air exposure (dashed line). Data are shown as single values and mean (solid line), n ¼ 4. perhaps not surprising, and could indicate that these combined exposures did not generate either an additive or synergistic response. Due to experimental design, the effect of direct ash- volatile interactions (e.g., volatile adsorption) prior to co-culture exposures was unable to be tested here. The absence of alter- ations in cell morphology, cell viability and oxidative stress state for any combined exposure scenario are in line with our previous in vitro study, which showed limited cytotoxic and oxidative po- tential of SHV ash when exposed concomitantly with DEP (Tomasek et al., 2016). However, the low (pro-)inflammatory response following combined exposures is contrary to the previous findings, where co-exposures of SHV ash and DEP increased release of (pro-) inflammatory mediators TNF-a and IL-8, as well as significantly increased (p < 0.05) IL-1b (Tomasek et al., 2016). In Tomasek et al. (2016) it was hypothesized that the observed IL-8 production was driven by DEP, and that volcanic ash, alone, did not result in sig- nificant production of TNF-a, although it augmented TNF-a pro- duction in the co-exposures. Together with the results from Bisig et al. Table 2 The similar biological responses to both ash samples in com- bined exposures with gasoline exhaust indicates that, within the parameters of this particular experimental setup, differences in sample composition (e.g., iron content) and mineralogy (e.g., crys- talline silica content) did not affect the biological response to co- exposures. Whilst we chose two fairly different ash samples, vol- canic ash is a heterogeneous dust, the physicochemical character- istics of which can vary considerably, even during a discrete eruption (Damby et al., 2017; Horwell et al., 2013), and samples from different eruptions have shown differences in toxicity when tested comparatively in vitro, previously (Damby et al., 2016; Horwell et al., 2013; Wilson et al., 2000). Hence, a different sam- ple from an individual eruption or a different ash type might incite a different cellular response. Given the lack of significant response to gasoline exhaust, alone, and volcanic ash, alone, the finding that the co-exposures did not cause significant adverse effects in the multicellular model is I. Tomasek et al. / Environmental Pollution 238 (2018) 977e987 986 SEM. Thanks to Pierre-Yves Tournigand for graphic design of the manuscript graphical abstract. We are thankful to Kristi Wallace and two anonymous reviewers for their constructive comments on this manuscript. Any use of trade, firm or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. SEM. Thanks to Pierre-Yves Tournigand for graphic design of the manuscript graphical abstract. We are thankful to Kristi Wallace and two anonymous reviewers for their constructive comments on this manuscript. Any use of trade, firm or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. The potential for diesel exhaust, and DEP in particular, to cause adverse respiratory effects is well known (see review by Steiner et al., 2016) while, on the contrary, the toxicity of exhaust from GDI vehicles is still relatively unknown (CCEM, 2016; Mu~noz et al., 2016). Given this, we believe that there is a need to conduct further studies to clarify the hazard posed by combined exposures, particularly with a fuel that generates exhaust of likely greater toxicity (e.g., diesel) (Bisig et al., 2016), which was not possible during the time-frame of the current experiments. Conflict of interest Chortarea, S., Clift, M.J.D., Vanhecke, D., Endes, C., Wick, P., Petri-Fink, A., Rothen- Rutishauser, B., 2015. Repeated exposure to carbon nanotube-based aerosols does not affect the functional properties of a 3D human epithelial airway model. Nanotoxicology 9 (8), 983e993. The authors declare no conflict of interest. The authors are responsible for the content of the manuscript. Cullen, R.T., Searl, A., 1998. Preliminary Toxicological Hazard Assessment of Montserrat Volcanic Ash: In vitro Cytotoxicity. Institute of Occupational Med- icine, Edinburgh, p. 13. P752/200. Consent for publication Damby, D.E., Horwell, C.J., Baxter, P.J., Delmelle, P., Donaldson, K., Dunster, C., Fubini, B., Murphy, F.A., Nattrass, C., Sweeney, S., Tetley, T.D., Tomatis, M., 2013. The respiratory health hazard of tephra from the 2010 Centennial eruption of Merapi with implications for occupational mining of deposits. J. Volcanol. Geoth. Res. 261, 376e387. All authors have read and approved the manuscript for publication. Table 2 Future studies that consider the very complex and variable components of ambient urban air would be prudent, as would additional end- points, such as genotoxicity, that help derive a more comprehensive understanding of the potential hazard. Furthermore, the experi- mental approach in this study, although performed over a two-day period (as opposed to the commonly-used time-point of 24 h), still represents a short-term exposure scenario. Hence, potential chronic effects that such exposures could elucidate, over a pro- longed period, have not been accounted for and need to be investigated. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.envpol.2018.01.115. Funding information Blank, F., Rothen-Rutishauser, B., Gehr, P., 2007. Dendritic cells and macrophages form a transepithelial network against foreign particulate antigens. Am. J. Respir. Cell Mol. Biol. 36, 669e677. This work was supported by the Marie Skłodowska-Curie Ac- tions Initial Training Network (MSCA-ITN) VERTIGO (FP7; grant agreement number 607905), the University of Fribourg Scholarship and the Adolphe Merkle Foundation. Blank, F., Rothen-Rutishauser, B.M., Schurch, S., Gehr, P., 2006. An optimized in vitro model of the respiratory tract wall to study particle cell interactions. J. Aerosol Med. 19 (3), 392e405. CCEM, 2016. GasOMeP. Gasoline vehicle emission control for organic, metallic and particulate non-legislative pollutants. Annu. Act. Rep. 57e59. Ch t S Clift M J D V h k D E d C Wi k P P t i Fi k A R th 5. Conclusion This study provides the first insights into the biological effects caused by exposure to complete gasoline exhaust in the presence or absence of volcanic ash conducting a realistic in vitro hazard assessment. The findings show that combined, and individual, gasoline exhaust and volcanic ash exposure at the ALI has limited adverse biological impact to a multicellular lung model in vitro, considering the employed experimental conditions and biological endpoints measured (cytotoxicity, oxidative stress and (pro-)in- flammatory response at the protein and gene levels). Baxter, P.J., Horwell, C.J., 2015. Impacts of eruptions on human health. In: The Encyclopedia of Volcanoes, second ed., pp. 1035e1047. d d h h Baxter, P.J., Bonadonna, C., Dupree, R., Hards, V.L., Kohn, S.C., Murphy, M.D., Nichols, A., Nicholson, R.A., Norton, G., Searl, A., Sparks, R.S.J., Vickers, B.P., 1999. Cristobalite in volcanic ash of the Soufriere Hills volcano, Montserrat, British West Indies. Science 283, 1142e1145. Baxter, P.J., Ing, R., Falk, H., French, J., Stein, G.F., Bernstein, R.S., Merchant, J.A., Allard, J., 1981. Mount St Helens eruptions, May 18 to June 12, 1980: an overview of the acute health impact. Jama 246 (22), 2585e2589. Baxter, P.J., Ing, R., Falk, H., Plikaytis, B., 1983. Mount St. Helens eruptions: the acute respiratory effects of volcanic ash in a North American community. Arch. En- viron. Health 38, 138e143. More detailed investigation of the potential respiratory hazard following such combined exposures in future eruptive events is necessary, especially considering the complexity of the ambient urban air. Additional biological markers should be studied in further experiments in vitro and a complete diesel exhaust could also be used. Baxter, P.J., Searl, A.S., Cowie, H.A., Jarvis, D., Horwell, C.J., 2014. Evaluating the respiratory health risks of volcanic ash at the eruption of the Soufriere Hills Volcano, Montserrat, 1995 to 2010. Geol. Soc. Lond. Mem. 39 (1), 407e425. i i h ll b i ki i Bisig, C., Roth, M., Müller, L., Comte, P., Heeb, N., Mayer, A., Czerwinski, J., Petri- FINk, A., Rothen-Rutishauser, B., 2016. Hazard identification of exhausts from gasoline-ethanol fuel blends using a multi-cellular human lung model. Environ. Res. 151, 789e796. Bisig, C., Steiner, S., Comte, P., Czerwinski, J., Mayer, A., Petri-Fink, A., Rothen- Rutishauser, B., 2015. Biological effects in lung cells in vitro of exhaust aerosols from a gasoline passenger car with and without particle filter. Emiss. Contr. Sci. Technol. 1, 237e246. References ACEA, 2017. 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Respuesta del crecimiento de Pinus oocarpa Schiede a variables climáticas en el noroeste de Lagunas de Montebello, Chiapas, México
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Respuesta del crecimiento de Pinus oocarpa a variables climáticas en Chiapas, México María I. López-Hernández1, Julián Cerano-Paredes2*, Salvador Valencia-Manzo1, Eladio H. Cornejo-Oviedo1, José Villanueva-Díaz2, Rosalinda Cervantes-Martínez2 & Gerardo Esquivel-Arriaga2 1. Departamento Forestal, Universidad Autónoma Agraria Antonio Narro, Calzada Antonio Narro No 1923, Buenavi CP 25315, Saltillo, Coahuila; mary isa 18@hotmail.com, svalenciam2016@gmail.com, e.cornejo@forestal.org.m 1. Departamento Forestal, Universidad Autónoma Agraria Antonio Narro, Calzada Antonio Narro No 1923, B CP 25315, Saltillo, Coahuila; mary isa 18@hotmail.com, svalenciam2016@gmail.com, e.cornejo@forest 1. Departamento Forestal, Universidad Autónoma Agraria Antonio Narro, Calzada Antonio Narro No 1923, Buenavista, CP 25315, Saltillo, Coahuila; mary_isa_18@hotmail.com, svalenciam2016@gmail.com, e.cornejo@forestal.org.mx 2. Laboratorio de Dendrocronología, INIFAP CENID-RASPA, km 6.5 Margen derecha del canal Sacramento, CP 35140, Gómez Palacio, Durango; cerano.julian@gmail.com,* villanueva.jose@inifap.gob.mx, rosy cervantes23@yahoo.com.mx, esquivel.gerardo@inifap.gob.mx 1. Departamento Forestal, Universidad Autónoma Agraria Antonio Narro, Calzada Antonio Narro No 1923, Buenavista, CP 25315, Saltillo, Coahuila; mary_isa_18@hotmail.com, svalenciam2016@gmail.com, e.cornejo@forestal.org.mx 2. Laboratorio de Dendrocronología, INIFAP CENID-RASPA, km 6.5 Margen derecha del canal Sacramento, CP 35140, Gómez Palacio, Durango; cerano.julian@gmail.com,* villanueva.jose@inifap.gob.mx, rosy_cervantes23@yahoo.com.mx, esquivel.gerardo@inifap.gob.mx * Correspondencia 2. Laboratorio de Dendrocronología, INIFAP CENID-RASPA, km 6.5 Margen derecha del canal Sacramento, CP 35140, Gómez Palacio, Durango; cerano.julian@gmail.com,* villanueva.jose@inifap.gob.mx, rosy_cervantes23@yahoo.com.mx, esquivel.gerardo@inifap.gob.mx * Correspondencia López-Hernández, M. I., Cerano-Paredes, J., Valencia-Manzo, S., Cornejo-Oviedo,E. H., Villanueva-Díaz, J., Cervantes-Martínez, R., & Esquivel-Arriaga, G. (2018). Respuesta del crecimiento de Pinus oocarpa a variables climáticas en Chiapas, México. Revista de Biología Tropical, 66(4), 1580-1596. López-Hernández, M. I., Cerano-Paredes, J., Valencia-Manzo, S., Cornejo-Oviedo,E. H., Villanueva-Díaz, J., Cervantes-Martínez, R., & Esquivel-Arriaga, G. (2018). Respuesta del crecimiento de Pinus oocarpa a variables climáticas en Chiapas, México. Revista de Biología Tropical, 66(4), 1580-1596. Recibido 07-III-2018. Corregido 03-VIII-2018. Aceptado 03-IX-2018. 66(4): 1580-1596, December 2018 1580 al., 2011), Pinus cooperi Blanco, Pinus duran­ gensis Martínez, Pinus lumholtzii Rob. et Fern. (Chavez et al., 2017), Abies religiosa (Cerano et al., 2014) y Pinus hartwegii Lindl. (Villa­ nueva et al., 2015). Para Centroamérica, Has­ tenrath (1963), Johnson (1980) y Szenjner (2011), han realizado estudios analizando los crecimientos de P. oocarpa. interpretar dichos registros, se conoce como dendrocronología (Benito, 2014), que se define como el estudio de los anillos de crecimiento de los árboles para fechar eventos pasados (Stokes & Smiley, 1996). ( y ) El crecimiento de los árboles en regiones tropicales es casi continuo, lo que hace que se dificulte distinguir bandas o anillos de creci­ miento anual; las coníferas en estas latitudes además desarrollan anillos falsos, lo que incre­ menta el problema para definir anillos anuales, a lo cual se atribuye que existan pocos estudios dendrocronológicos es estas regiones (Hast­ enrath, 1963; Johnson, 1980; Szenjner, 2011). Aunque las investigaciones dendrocronológi­ cas en el trópico se remontan a más de 100 años (Worbes, 2002), algunos científicos dudan de la capacidad de los árboles tropicales para formar anillos de crecimiento anual (Turner, 2004). En 1870 se realizó el primer estudio dendrocrono­ lógico en el trópico, estudiando el incremento radial de la teca (Tectona grandis Lf.), en la cual se determinó los ciclos de corta con base en los anillos de crecimiento; de este modo, se estableció un sistema silvícola sostenible (Wor­ bes, 2002). Posteriormente, se estudió Cordia alliodora (Ruíz y Pav.) Oken en América tro­ pical, este estudio fue desarrollado por César Pérez en 1954 (Tschinkel, 1966) quien supuso acertadamente que los anillos eran anuales. En México, P. oocarpa registra una dis­ tribución desde el norte hasta el sur del país (Martínez, 1992). A pesar de su amplia distri­ bución, sólo existen un par de referencias sobre el análisis dendroclimático de esta especie, la primera realizada en la región de Los Tuxtlas, Veracruz (Gutiérrez, 2013) y la segunda en el bosque La Primavera en Jalisco (Villanueva et al., 2018). Estudios dendroclimáticos en el sur del país con base en P. oocarpa, son muy limitados. Esta especie puede representar una alternativa para reconstruir información cli­ mática en zonas del trópico mexicano, donde se carece de datos climáticos instrumentales extensos; por tal motivo, la presente investiga­ ción tiene como objetivos: 1) desarrollar una cronología con base en los anillos de creci­ miento de P. Recibido 07-III-2018. Corregido 03-VIII-2018. Aceptado 03-IX-2018. oocarpa para la porción noroeste de las Lagunas de Montebello, Chiapas, y 2) determinar el potencial dendroclimático de P. oocarpa para reconstruir variables climáticas en esta región del trópico mexicano. Con base en los resultados de los estudios desarrolla­ dos en Centroamérica por Hastenrath (1963), Johnson (1980) y Szenjner (2011) y de México (Gutiérrez, 2013), que reportan problemas en el crecimiento de P. oocarpa para su fechado; en este estudio se plantean dos preguntas de investigación: 1) ¿A pesar de los problemas de crecimiento documentados para esta especie, es factible generar una cronología para esta región del trópico mexicano? y 2) ¿El crecimiento anual de P. oocarpa es sensible a la variabi­ lidad climática que experimenta esta región de México? Recientemente, se han realizado estudios dendrocronológicos con el género Pinus, como el de Johnson (1980) que estudió P. oocarpa en Honduras, no obstante que, reporta la for­ mación de anillos falsos, logró extender la red de cronologías de anillos de árboles para el tró­ pico. Szenjner (2011) estudio P. oocarpa en la región oriental de Guatemala, donde analizó la anatomía del anillo y factores que influyen en el crecimiento de esta especie, logrando exten­ der el conocimiento del clima en esta región. Para México y Centroamérica, se repor­ tan 47 especies de Pinus (Perry, Graham, & Richardson, 1998). En el norte y centro de México se ha explorado el potencial dendrocli­ mático de varias coníferas como Pseudotsuga menziesii (Mirb.) Franco (Villanueva, Fulé, Cerano, Estrada, & Sánchez, 2009; Cerano et Recibido 07-III-2018. Corregido 03-VIII-2018. Aceptado 03-IX-2018. Abstract: Growth response of Pinus oocarpa to climatic variables in Chiapas, Mexico. Dendrochronological studies are used to reconstruct some climatic variables; in México these studies have focused on central and Northern temperate forests where trees present well defined annual rings. Few studies have been carried out in the Southern part of the country where annual ring growth is not easily identified and thus makes cross-dating problematic. We analyzed the dendrochronological potential of Pinus oocarpa Schiede for reconstructing climat­ ic variables in the Northwest portion of Lagunas de Montebello, Chiapas. We used a selective sampling approach and collected 65 increment cores from 34 trees. While our samples showed a high frequency of false rings (8 to 60 %), we were able to date 30 samples from 22 trees (46 %) using standard dendrochronological techniques and developed total chronologies for total ring width, earlywood, and latewood for a period of 91 years (1925-2015). We found a significant influence of mean precipitation and mean maximum and minimum temperature over the annual ring growth of P. oocarpa in the period 1961-2004. Our results show that winter-spring precipitation (January-May) was the most important for the species’ annual ring growth. However, we found the highest correlation between spring (March-May) precipitation and the earlywood chronology (r = 0.719, P < 0.05). The earlywood chronology also showed potential for reconstructing minimum temperatures (March to May) (r = 0.732, P < 0.05), while the latewood chronology had the potential for reconstructing the maximum temperature (September to January) (r = 0.714, P < 0.05). These results showed that P. oocarpa can be used to reconstruct climatic variables in the Mexican tropics. We recommend that new areas with older trees should be explored in order to increase the depth of chronologies and reconstruct climate records several centuries into the past. Key words: growth rings; false rings; earlywood; latewood; dendroclimatic potential. Ante el actual proceso de cambio en las condiciones del clima, existe el interés cientí­ fico a nivel mundial por estudiar la variabili­ dad climática en el pasado, para esto, se han empleado diversos tipos de registros proxy; entre los más importantes se encuentran los sedimentos de lagos, hielo, corales, archi­ vos históricos, análisis de polen y anillos de crecimiento de árboles (Bradley, 1999). Para este último, la disciplina que se encarga de Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. MATERIALES Y MÉTODOS Área de estudio y muestreo: Se localiza en el Ejido Ojo de Agua, La Independencia, Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 1581 1581 INIFAP, Gómez Palacio, Durango, México. Cada muestra fue montada en una base de madera para dar mayor firmeza y resisten­ cia a las muestras. Una vez que se pegaron firmemente, se procedió a realizar un lijado para resaltar las estructuras de crecimiento, utilizando lijas de diferente grano (120-1 200). Se realizó un pre-fechado de los anillos de crecimiento anual mediante gráficos de cre­ cimiento (Skeleton plots) (Stokes & Smiley, 1996). Las muestras de P. oocarpa presentaron alta frecuencia de anillos dobles o falsos, para distinguir estos anillos, se compararon patrones de crecimiento entre muestras del mismo árbol y posteriormente entre árboles, siguiendo las sugerencias de Stokes y Smiley (1996). Rea­ lizado el pre-fechado, se midió el crecimiento total anual, longitud de madera temprana y de madera tardía de cada una de las muestras, empleando un sistema de medición Velmex de 0.001 mm de precisión. Chiapas (16° 09´ 24” N - 91° 45°29” O), a una altitud de 1 527 msnm (Fig. 1) (INEGI-CONA­ GUA, 2007). La región hidrológica la confor­ man los ríos Grijalva-Usumacinta, el origen de las rocas es sedimentario (INEGI, 2005). Los suelos predominantes son litosoles (INEGI, 2007). El clima es templado subhúmedo con una temperatura media de 12 a 18 °C y una pre­ cipitación anual de 1 500 mm (García, 1998). La vegetación presente es secundaria arbustiva, bosque de pino-encino, bosque mesófilo de montaña y bosque de pino (Rzedowski, 1978). Chiapas (16° 09´ 24” N - 91° 45°29” O), a una altitud de 1 527 msnm (Fig. 1) (INEGI-CONA­ GUA, 2007). La región hidrológica la confor­ man los ríos Grijalva-Usumacinta, el origen de las rocas es sedimentario (INEGI, 2005). Los suelos predominantes son litosoles (INEGI, 2007). El clima es templado subhúmedo con una temperatura media de 12 a 18 °C y una pre­ cipitación anual de 1 500 mm (García, 1998). La vegetación presente es secundaria arbustiva, bosque de pino-encino, bosque mesófilo de montaña y bosque de pino (Rzedowski, 1978). INIFAP, Gómez Palacio, Durango, México. Cada muestra fue montada en una base de madera para dar mayor firmeza y resisten­ cia a las muestras. MATERIALES Y MÉTODOS Una vez que se pegaron firmemente, se procedió a realizar un lijado para resaltar las estructuras de crecimiento, utilizando lijas de diferente grano (120-1 200). Durante septiembre y noviembre 2016, se tomaron muestras de P. oocarpa empleando un muestreo selectivo. Se seleccionaron 34 árbo­ les de los especímenes más longevos, libres de daños mecánicos, plagas y enfermedades. A cada árbol seleccionado, se le extrajo dos virutas (núcleos de crecimiento), en su mayoría a 1.30 m sobre el nivel del suelo con taladro de Pressler, se obtuvo un total de 65 núcleos de crecimiento (incrementos radiales). Análisis estadístico: La calidad del fechado de las series obtenidas (anillo total, madera temprana y madera tardía), se veri­ ficó con el programa COFECHA (Holmes, 1983), el cual estandariza cada serie y analiza Análisis de laboratorio: Las muestras se prepararon para su análisis en el Laborato­ rio de Dendrocronología del CENID-RASPA, Fig. 1. Área de muestreo de Pinus oocarpa en el Ejido Ojo de Agua, La Independencia, Chiapas, y las estaciones climatológicas consideradas para el análisis dendroclimático. Fig. 1. Sampling area of Pinus oocarpa at the Ejido Ojo de Agua, La Independencia, Chiapas and climatological stations considered for the dendroclimate analyzis. Fig. 1. Área de muestreo de Pinus oocarpa en el Ejido Ojo de Agua, La Independencia, Chiapas, y las estaciones climatológicas consideradas para el análisis dendroclimático. Fig. 1. Sampling area of Pinus oocarpa at the Ejido Ojo de Agua, La Independencia, Chiapas and climatological stations considered for the dendroclimate analyzis. Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 1582 y alta frecuencia y registrar los mejores resul­ tados al relacionarla con variables climáticas. estadísticamente el fechado al correlacionar sucesivamente segmentos de 50 años con tras­ lapes de 25 años, compara cada serie individual con todas las muestras y con la serie promedio; con base en este procedimiento, se identifican errores en el fechado atribuidos a la formación de anillos falsos o la no formación de anillos (anillos ausentes) en años particulares. El potencial de P. oocarpa para registrar la variabilidad climática regional, se determinó mediante un análisis de función de respuesta, para lo cual, se relacionaron los datos promedio mensuales regionales (44 años: 1961-2004), tanto de precipitación (estaciones Comitán, Margaritas, Santa Elena y Tziscao) como de temperaturas máximas y mínimas (estaciones Comitán y Santa Elena) (Cuadro 1, Fig. MATERIALES Y MÉTODOS 1) con los índices de la cronología estándar de ancho de anillo, madera temprana y madera tardía. La información climática de estas estaciones se tomó del programa Extractor Rápido de Infor­ mación Climática (ERIC III) (IMTA, 2009). Fechado cada crecimiento, con el pro­ grama ARSTAN se generaron las cronologías o índices de anillo total, madera temprana y madera tardía. Se aplicó la mejor curva de ajus­ te (exponencial negativa, lineal, entre otras) a cada serie de crecimiento (Cook, 1987), para remover la varianza debido a factores biológi­ cos (competencia y liberación) y geométricos (el área de fuste se incrementa con la edad y el crecimiento anual tiende a disminuir al distribuirse en una mayor superficie) no rela­ cionados con el clima y maximizar la varianza debida a factores ambientales (factores climá­ ticos y atmosféricos) que afectan la población. Al dividir el valor del ancho del anillo entre el valor correspondiente de la curva, se obtuvo el índice de crecimiento para cada año. Final­ mente, al promediar los índices anuales de las series individuales, se generó la cronología del sitio. El programa ARSTAN produce tres cronologías, estándar, residual y arstan, para el presente estudio, se trabajó con la cronología estándar por conservar la variación de media En el programa Excel, se corrieron análisis de correlación de Pearson entre la cronología estándar de anillo total, madera temprana y tar­ día con las variables climáticas. Se definieron los meses con mayor correlación y el periodo estacional que más influye en el crecimiento de P. oocarpa. Estos análisis se verificaron y validaron estadísticamente, utilizando el pro­ grama STATISTICA Kernel Release 5.5 (Stat Soft Inc., 2000). Finalmente, con este mismo programa, se corrieron modelos de regresión entre las variables climáticas (precipitación, temperatura máxima y mínima) y los índices de anillo, con el fin de definir el potencial de la especie para reconstruir variables climáticas CUADRO 1 Estaciones meteorológicas empleadas para el análisis dendroclimátologico de Pinus oocarpa, en el Ejido Ojo de Agua, La Independencia, Chiapas TABLE 1 Climatological stations used for the dendroclimate analysis of Pinus oocarpa, at the Ejido Ojo de Agua, La Independencia, Chiapas Estaciones climáticas Longitud (Oeste) Latitud (Norte) Altitud (msnm) Periodo de datos Lugar Municipio Comitán Comitán 92.117 16.25 1 596 1961-2004 Margaritas Margaritas 91.975 16.311 1 512 1962-2004 Santa Elena Las Margaritas 91.967 16.317 560 1966-1990 Tziscao Trinitaria 91.633 16.1 1 475 1977-1996 CUADRO 1 CUADRO 1 Estaciones meteorológicas empleadas para el análisis dendroclimátologico de Pinus oocarpa, en el Ejido Ojo de Agua, La Independencia, Chiapas TABLE 1 Climatological stations used for the dendroclimate analysis of Pinus oocarpa, at the Ejido Ojo de Agua, La Independencia, Chiapas Climatological stations used for the dendroclimate analysis of Pinus oocarpa, at the Ejido Ojo de Agua, La Independencia, Chiapas Estaciones climáticas Longitud (Oeste) Latitud (Norte) Altitud (msnm) Periodo de datos Lugar Municipio Comitán Comitán 92.117 16.25 1 596 1961-2004 Margaritas Margaritas 91.975 16.311 1 512 1962-2004 Santa Elena Las Margaritas 91.967 16.317 560 1966-1990 Tziscao Trinitaria 91.633 16.1 1 475 1977-1996 1583 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 y determinar aquellas que mejor explicaran la variabilidad interanual. presenta P. oocarpa y que dificulta su fechado, se logró determinar un patrón de crecimiento entre muestras. Los resultados de COFECHA indicaron una correlación altamente significa­ tiva entre series (r = 0.50; P < 0.01) (Cuadro 2). Se lograron generar cronologías de anillo total, madera temprana y madera tardía para los últimos 91 años (1925-2015). RESULTADOS Anillos de crecimiento de P. oocarpa: De un total de 65 muestras de 34 árboles de P. oocarpa, después de un riguroso reconocimien­ to de cada anillo e identificación de problemas de crecimiento (anillos falsos y ausentes), se lograron fechar 30 muestras (46 %) de 22 árboles. El resto de las muestras (54 %) no se logró fechar por presentar alto porcentaje de anillos falsos y períodos de supresión. Para las 30 muestras datadas, se determinó un alto porcentaje de anillos falsos (8 a 60 %); donde 1991 y 1997 sobresalieron con 57 % y 52 %, respectivamente (Fig. 2). Respuesta del crecimiento a la preci­ pitación: Los análisis de correlación entre la cronología estándar y los datos climáticos de la precipitación regional (1961-2004), indican que la precipitación promedio mensual presen­ ta correlaciones positivas con las cronologías de anillo total, madera temprana y madera tardía, excepto el mes julio, que registró una relación negativa con la cronología de made­ ra tardía (r = -0.0071) (Fig. 4). Los meses de marzo, abril, mayo, noviembre y diciembre, registran correlaciones significativas (p < 0.05) con las cronologías de anillo total y de madera temprana de P. oocarpa, y los meses de abril, mayo y diciembre con la cronología de madera tardía (p < 0.05; Fig. 4). Los años 1981, 1984, 1985, 1997 y 1999, son un ejemplo de los anillos falsos que regis­ tra el crecimiento de P. oocarpa en esta región del trópico mexicano (Fig. 3). Sin embargo, algunas muestras para años específicos no registran problemas de formación de anillos falsos, un ejemplo es el año 1994, que repre­ senta un crecimiento normal, condición atípica en la especie para esta región del país (Fig. 3). A pesar de los problemas de crecimiento que Al considerar la precipitación acumulada, se observan correlaciones significativas (P < 0.05) para las cronologías de anillo total, madera Fig. 2. Porcentaje de anillos falsos en la mayoría de los años de crecimiento de Pinus oocarpa. El menor porcentaje de anillos falsos al inició del gráfico, obedece al tamaño de muestra y no a una menor formación de anillos falsos. Fig. 2. False ring percentages in the majority of Pinus oocarpa growth years. The lowest percentage at the beginning of the figure is due to the sample depth and not to lower ring false formation. Fig. 2. RESULTADOS Porcentaje de anillos falsos en la mayoría de los años de crecimiento de Pinus oocarpa. El menor porcentaje de anillos falsos al inició del gráfico, obedece al tamaño de muestra y no a una menor formación de anillos falsos. Fig. 2. False ring percentages in the majority of Pinus oocarpa growth years. The lowest percentage at the beginning of the figure is due to the sample depth and not to lower ring false formation. Fig. 2. Porcentaje de anillos falsos en la mayoría de los años de crecimiento de Pinus oocarpa. El menor porcentaje de anillos falsos al inició del gráfico, obedece al tamaño de muestra y no a una menor formación de anillos falsos. Fig. 2. False ring percentages in the majority of Pinus oocarpa growth years. The lowest percentage at the beginning of the figure is due to the sample depth and not to lower ring false formation. Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 1584 mas de precipitación y temparatura para diferentes años (izquierda) y crecimientos a que muestran la formación característica de anillos falsos en la mayoría de los años com Fig. 3. Climogramas de precipitación y temparatura para diferentes años (izquierda) y crecimientos anuales de Pinus oocarpa (derecha), que muestran la formación característica de anillos falsos en la mayoría de los años como respuesta a una disminución en las condiciones de humedad e incremento en temperatura al inicio de la estación de crecimiento (derecha). La barra en color gris indica el período febrero-marzo con constante disminución en la precipitación e incremento en la temperatura. Fig. 3. Precipitation and temperature climograms for different years (left) and annual growths of Pinus oocarpa (right) that show the characteristic false rings formation in the most years as a response to a decrease in humidity conditions and an increase in temperature at the beginning of the growing season (right). The bar in gray indicates the period February-March with constant decrease in precipitation and increase in temperature. Fig. 3. Climogramas de precipitación y temparatura para diferentes años (izquierda) y crecimientos anuales de Pinus oocarpa (derecha), que muestran la formación característica de anillos falsos en la mayoría de los años como respuesta a una disminución en las condiciones de humedad e incremento en temperatura al inicio de la estación de crecimiento (derecha). RESULTADOS La barra en color gris indica el período febrero-marzo con constante disminución en la precipitación e incremento en la temperatura. Fig. 3. Climogramas de precipitación y temparatura para diferentes años (izquierda) y crecimientos anuales de Pinus oocarpa (derecha), que muestran la formación característica de anillos falsos en la mayoría de los años como respuesta a una disminución en las condiciones de humedad e incremento en temperatura al inicio de la estación de crecimiento (derecha). La barra en color gris indica el período febrero-marzo con constante disminución en la precipitación e incremento en la temperatura. p ( ) La barra en color gris indica el período febrero-marzo con constante disminución en la precipitación e incremento en la temperatura. Fig. 3. Precipitation and temperature climograms for different years (left) and annual growths of Pinus oocarpa (right) that show the characteristic false rings formation in the most years as a response to a decrease in humidity conditions and an increase in temperature at the beginning of the growing season (right). The bar in gray indicates the period February-March with constant decrease in precipitation and increase in temperature. Fig. 3. Precipitation and temperature climograms for different years (left) and annual growths of Pinus oocarpa (right) that show the characteristic false rings formation in the most years as a response to a decrease in humidity conditions and an increase in temperature at the beginning of the growing season (right). The bar in gray indicates the period February-March with constant decrease in precipitation and increase in temperature. Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 1585 CUADRO 2 Parámetros estadísticos de cronologías desarrolladas con base en crecimientos de Pinus oocarpa TABLE 2 Sitio No. árboles No. radios Longitud de la serie Correlacióna (r) Sensibilidadb Autor RBSM, Guatemala 21 32 1880-2005 0.57 0.36 Cerano et al. (2008) Pedernal, Guatemala 7 18 1811-2010 0.44 0.23 Szejner (2011) La Primavera, Jalisco 41 35 1850-2014 0.50 0.32 Villanueva et al. (2018) Ojo de Agua, Chiapas 22 30 1925-2015 0.50 0.28 Trabajo actual RBSM: Reserva de la Biosfera Sierra las Minas RBSM: Reserva de la Biosfera Sierra las Minas. a Representa la correlación promedio entre todas las series de crecimiento y la cronología promedio. b b La Sensibilidad media determina la diferencia relativa en el ancho de los anillos de un año al siguiente. b La Sensibilidad media determina la diferencia relativa en el ancho de los anillos de un año al siguiente. Fig. 4. Coeficiente de correlación entre la precipitación promedio mensual y acumulada del período 1961 a 2004 y los índices de anillo total, madera temprana y madera tardía de Pinus oocarpa. Las barras en color negro representan una correlación significativa (P < 0.05). Fig. 4. Correlation coefficient between monthly average precipitation and accumulative from 1961 to 2004, and total annual ring, earlywood and latewood indexes of Pinus oocarpa. The black bars represent significant correlations (P < 0.05). b La Sensibilidad media determina la diferencia relativa en el ancho de los anillos de un año al siguiente. Fig. 4. Coeficiente de correlación entre la precipitación promedio mensual y acumulada del período 1961 a 2004 y los índices de anillo total, madera temprana y madera tardía de Pinus oocarpa. Las barras en color negro representan una correlación significativa (P < 0.05). Fig 4 Correlation coefficient between monthly average precipitation and accumulative from 1961 to 2004 and total annual Fig. 4. Coeficiente de correlación entre la precipitación promedio mensual y acumulada del período 1961 a 2004 y los índices de anillo total, madera temprana y madera tardía de Pinus oocarpa. Las barras en color negro representan una correlación significativa (P < 0.05). g ( ) Fig. 4. Correlation coefficient between monthly average precipitation and accumulative from 1961 to 2004, and total annual ring, earlywood and latewood indexes of Pinus oocarpa. The black bars represent significant correlations (P < 0.05). Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 1586 temprana, y madera tardía con la precipitación enero-abril, enero-mayo y enero-junio. TABLE 2 Sin embargo, la mayor correlación se determinó con el período enero-mayo para las tres cronologías (Fig. 4). La cronología de madera temprana registra la más alta correlación con la lluvia esta­ cional enero-mayo (invierno-primavera) (Fig. 4). indica potencial para reconstruir la variabilidad de la precipitación de la estación de primave­ ra, considerando como variable independiente la cronología de madera temprana, serie que registra el mayor coeficiente de correlación y por lo tanto explica la mayor variabilidad de la lluvia estacional (Cuadro 3, Fig. 6). Al relacionar únicamente los índices de anillo con la precipitación acumulada de los meses con mayor correlación (marzo-mayo), se observó la mayor relación significativa (P < 0.05) con las cronologías de anillo total, made­ ra temprana y madera tardía, con asociaciones de 0.70, 0.71 y 0.53, respectivamente (Fig. 5). Correlaciones que superan a las observadas para el período enero-mayo. El análisis de regresión Respuesta del crecimiento a la tem­ peratura: La relación entre las cronologías de anillo total, madera temprana y tardía con los datos climáticos promedio mensual de temperatura mínima y máxima, indicó que la temperatura mínima mensual, presenta corre­ laciones positivas con las cronologías de anillo total, madera temprana y madera tardía (Fig. 7). p y g p y ( g ) Fig. 5. Relación entre la precipitación estacional marzo-mayo y el índice de anillo total, madera temprana y madera tardía de Pinus oocarpa. Fig. 5. Relationship between March-May seasonal precipitation and total annual ring width, earlywood and latewood indexes of Pinus oocarpa Fig. 5. Relación entre la precipitación estacional marzo-mayo y el índice de anillo total, madera temprana y madera tardía de Pinus oocarpa. Fig. 5. Relationship between March-May seasonal precipitation and total annual ring width, earlywood and latewood indexes of Pinus oocarpa. Fig. 5. Relación entre la precipitación estacional marzo-mayo y el índice de anillo total, madera temprana y madera tardía de Pinus oocarpa. Fig. 5. Relationship between March-May seasonal precipitation and total annual ring width, earlywood and latewood indexes of Pinus oocarpa. Fig. 5. Relación entre la precipitación estacional marzo-mayo y el índice de anillo total, madera temprana y madera tardía de Pinus oocarpa. Fig. 5. Relationship between March-May seasonal precipitation and total annual ring width, earlywood and latewood indexes of Pinus oocarpa. Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 15 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. TABLE 2 66(4): 1580-1596, December 2018 1587 Diagramas de dispersión de los mejores modelos de regresión entre variables climáticas y las cronologías de mader ramas de dispersión de los mejores modelos de regresión entre variables climáticas y las cronologías de madera madera tardía. Fig. 6. Diagramas de dispersión de los mejores modelos de regresión entre variables climáticas y las cronologías de madera temprana y madera tardía. Fig. 6. Scatter plots for the best regression models for climate variables and earlywood and latewood chronologies. La temperatura mínima registra correlaciones significativas (P < 0.05) con las cronologías de anillo total y madera temprana para todos los meses del año, mientras que la cronología de madera tardía, presenta correlación significati­ va (P < 0.05) para los meses de febrero, marzo, abril, mayo, noviembre y diciembre (Fig. 7). Al considerar la temperatura mínima pro­ medio por períodos, se determinó que todos los meses presentan correlaciones significativas (P < 0.05) con las cronologías de anillo total, madera temprana y madera tardía. Sin embar­ go, el periodo estacional marzo-mayo es el que influye de manera más importante (P < 0.05) Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 1588 CUADRO 3 Modelos de regresión lineal que indican la relación significativa (P < 0.05) de los índices de crecimiento con las variables climáticas Lineal regression models that indicate a significant relationship (P < 0.05) between ring growth indexes and climate variables Variable climática Modelo Parámetros estimados Error estándar Cuadrado medio R2 Pr > F Precipitación Intercepto -78.2108 43.61544 98467.50 0.49 0.080 Anillo Total 278.8658 45.38311 < 0.000 Intercepto -65.9745 39.62307 103576.6 0.52 0.103 M. Temprana 265.7699 41.09875 < 0.000 Intercepto 9.1847 46.08078 56267.53 0.28 0.843 M. Tardía 192.5270 49.30295 < 0.000 T. mínima Intercepto 8.112183 1.010391 46.1967 0.50 < 0.000 Anillo Total 5.939023 1.028413 < 0.000 Intercepto 8.322355 0.899073 50.0373 0.54 < 0.000 M. Temprana 5.723840 0.912698 < 0.000 Intercepto 9.757985 1.143384 26.2018 0.28 < 0.000 M. Tardía 4.372182 1.199855 < 0.000 T. máxima Intercepto 21.99617 0.702143 12.4319 0.35 < 0.000 Anillo Total 3.08091 0.714667 < 0.000 Intercepto 22.32631 0.665659 11.4641 0.33 < 0.000 M. Temprana 2.73975 0.675747 < 0.000 Intercepto 21.49784 0.507707 13.74353 0.51 < 0.000 M. Tardía 3.16651 0.532782 < 0.000 Los parámetros indicados en negritas representan los mejores modelos. Bold parameters indicate the best models. Los parámetros indicados en negritas representan los mejores modelos. Bold parameters indicate the best models. TABLE 2 Coeficiente de correlación entre la temperatura promedio máxima y mínima mensual (1961 a 2004) y el índice de anillo total, madera temprana y madera tardía de Pinus oocarpa. Las barras en color negro representan una correlación significativa (P < 0.05). Fig. 7. Correlation coefficients between monthly average minimum and maximum temperature from 1961 to 2004, and total annual ring width, earlywood and latewood indexes of Pinus oocarpa. Black bars represent a significant correlation (P < 0.05). Fig. 7. Coeficiente de correlación entre la temperatura promedio máxima y mínima mensual (1961 a 2004) y e anillo total, madera temprana y madera tardía de Pinus oocarpa. Las barras en color negro representan una significativa (P < 0.05). g ( ) Fig. 7. Correlation coefficients between monthly average minimum and maximum temperature from 1961 to 2004, and total annual ring width, earlywood and latewood indexes of Pinus oocarpa. Black bars represent a significant correlation (P < 0.05). TABLE 2 Los parámetros indicados en negritas representan los mejores modelos. Bold parameters indicate the best models. significativas (P > 0.05), por el contrario, para algunos meses (febrero y marzo) son nega­ tivas. En contraste, la cronología de madera tardía presentan correlaciones significativas (P < 0.05), con los meses de enero y marzo, y las mayores correlaciones (P < 0.05) se registran en noviembre y diciembre. en el crecimiento de P. oocarpa para las tres cronologías, con valores de 0.70, 0.73 y 0.53, respectivamente (Fig. 8). La mayor correla­ ción se obtuvo con la cronología de madera temprana, por lo que se consideró como varia­ ble independiente en el modelo de regresión con fines de reconstrucción de esta variable (Cuadro 3, Fig. 6). Al relacionar la temperatura máxima pro­ medio de septiembre y octubre con las crono­ logías de anillo total y madera temprana, se determinó una relación significativa (P < 0.05) de 0.59 y 0.57, respectivamente (Fig. 8). Sin embargo, la mayor correlación se observó entre la temperatura máxima septiembre-enero y la cronología de madera tardía r = 0.71 (P < 0.05) (Fig. 8). Existe una importante influencia de la temperatura en el crecimiento de P. oocarpa en Para el caso de la temperatura máxima mensual, se determinó una relación significa­ tiva (p < 0.05) con las cronologías de anillo total, madera temprana y madera tardía de forma consistente para los meses de julio, sep­ tiembre y octubre (Fig. 7). Las correlaciones entre la temperatura máxima con las crono­ logías de anillo total y madera temprana para los meses de enero-mayo y diciembre no son Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 1589 Fig. 7. Coeficiente de correlación entre la temperatura promedio máxima y mínima mensual (1961 a 2004) y el índice de anillo total, madera temprana y madera tardía de Pinus oocarpa. Las barras en color negro representan una correlación significativa (P < 0.05). Fig. 7. Correlation coefficients between monthly average minimum and maximum temperature from 1961 to 2004, and total annual ring width, earlywood and latewood indexes of Pinus oocarpa. Black bars represent a significant correlation (P < 0.05). g 7 Coeficiente de correlación entre la temperatura promedio máxima y mínima mensual (1961 a 2004) y el Fig. 7. DISCUSIÓN Best correlations between seasonal minimum temperature, from March to May, and the total annual ring, earlywood and latewood indexes, as well as, between seasonal maximum temperature, from September to October, and the total annual ring and earlywood; and between seasonal maximum temperatures, from September to January, with the latewood index. & Bravo, 2008). El crecimiento radial de P. oocarpa muestra estructuras anatómicas como madera temprana y madera tardía; así mismo, anomalías en la densidad de la madera cono­ cidas como anillos falsos que pueden relacio­ narse con eventos hidro-meteorológicos (Fig. 3). Por ejemplo, la disponibilidad de humedad invernal, favorece el crecimiento inicial de P. oocarpa (Fig. 4), una disminución de hume­ dad y altas temperaturas durante los meses de febrero y marzo propician un cese en la actividad cambial del árbol, dando paso a la formación de una banda de crecimiento falsa (Fig. 3). Como lo sugiere Vaganov, Hughes y Shashkin (2006), la actividad secundaria del crecimiento de P. oocarpa en el área de estudio parece desencadenarse con las lluvias que se & Bravo, 2008). El crecimiento radial de P. oocarpa muestra estructuras anatómicas como madera temprana y madera tardía; así mismo, anomalías en la densidad de la madera cono­ cidas como anillos falsos que pueden relacio­ narse con eventos hidro-meteorológicos (Fig. 3). Por ejemplo, la disponibilidad de humedad invernal, favorece el crecimiento inicial de P. oocarpa (Fig. 4), una disminución de hume­ dad y altas temperaturas durante los meses de febrero y marzo propician un cese en la actividad cambial del árbol, dando paso a la formación de una banda de crecimiento falsa (Fig. 3). Como lo sugiere Vaganov, Hughes y Shashkin (2006), la actividad secundaria del crecimiento de P. oocarpa en el área de estudio parece desencadenarse con las lluvias que se presentan al final de la primavera e inicio del verano. La Zona de Convergencia Intertropical (ZCIT) está relacionada con el inicio de la tem­ porada de lluvias en estas latitudes, producien­ do precipitaciones durante el verano (Curtis, 2002). Esta condición está asociada de manera positiva con el crecimiento de la madera tem­ prana de P. oocarpa (Fig. 4), caracterizada por grandes traqueidas, paredes celulares delgadas y gran lumen. Para realizar un análisis dendroclimático, la identificación correcta del inicio y final del anillo de crecimiento anual es un paso fun­ damental para lograr una datación confiable (Stokes & Smiley, 1996; Stahle, 1999). DISCUSIÓN esta región de Chiapas. La temperatura mínima juega un papel importante en el crecimiento de la madera temprana y la temperatura máxima en el desarrollo de la madera tardía. Las cro­ nologías generadas con base en los anillos de crecimiento de P. oocarpa presentan potencial para reconstruir la temperatura mínima marzo- mayo con base en la cronología de madera temprana y la temperatura máxima septiembre- enero con base en la cronología de madera tardía (Cuadro 3, Fig. 6). Anillos de crecimiento de P. oocarpa: Los anillos de crecimiento anual de los árboles están influenciado por varios factores ambien­ tales que interactúan entre sí, como radiación solar, temperatura, precipitación, contenido de nutrientes del suelo, entre otros. Dependien­ do de las condiciones ambientales y de las especies, uno o más de estos factores pueden limitar el crecimiento (Fritts, 1976; Bogino Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 1590 Fig. 8. Gráficos que muestran las mejores correlaciones entre la temperatura mínima estacional marzo-mayo con el índice de anillo total, madera temprana y madera tardía. Así mismo, la temperatura máxima estacional septiembre-octubre con el anillo total y la madera temprana y septiembre-enero con el índice de la madera tardía. Fig. 8. Best correlations between seasonal minimum temperature, from March to May, and the total annual ring, earlywood and latewood indexes, as well as, between seasonal maximum temperature, from September to October, and the total annual ring and earlywood; and between seasonal maximum temperatures, from September to January, with the latewood index. Fig. 8. Gráficos que muestran las mejores correlaciones entre la temperatura mínima estacional marzo-mayo con el índice de anillo total, madera temprana y madera tardía. Así mismo, la temperatura máxima estacional septiembre-octubre con el anillo total y la madera temprana y septiembre-enero con el índice de la madera tardía. Fig. 8. Best correlations between seasonal minimum temperature, from March to May, and the total annual ring, earlywood and latewood indexes, as well as, between seasonal maximum temperature, from September to October, and the total annual ring and earlywood; and between seasonal maximum temperatures, from September to January, with the latewood index. Fig. 8. DISCUSIÓN La comparación de los patrones de crecimiento con base en el fechado cruzado empleando Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 1591 gráficos de crecimiento o “skeleton plots”, permitieron identificar y discriminar problemas de crecimiento. Esta técnica trata no solo de la sincronización, es útil para reconocer anillos falsos, perdidos y permite corregir y asignar el año exacto a cada anillo, es el principio más importante en dendrocronología (Fritts, 1976; Cook, 1985; Stokes & Smiley, 1996). La datación cruzada permite estar seguros sobre la fecha de cada anillo de crecimiento en muestras diferentes. Adicionalmente, se empleó el programa COFECHA que considera la posible existencia de anillos falsos mediante la falta de una buena correlación entre muestras (Holmes, 1983). el primer objetivo y se da respuesta a la primera pregunta de investigación. Respuesta del crecimiento a la preci­ pitación: Los análisis de correlación entre la cronología y los datos climáticos de la preci­ pitación regional (1961-2004), indican que la precipitación presenta correlaciones positivas con el crecimiento de P. oocarpa. Sin embargo, los meses del período estacional invierno- primavera indican una mayor influencia en el crecimiento. Una respuesta similar del cre­ cimiento de P. oocarpa a la lluvia estacional invierno-primavera, se reportó para el bosque La Primavera en Jalisco (Villanueva et al., 2018). Sin embargo, la respuesta de P. oocarpa es diferente en regiones de Guatemala, donde la mayor influencia en crecimiento ocurre con la precipitación primavera-verano (Cerano et al., 2008; Szejner, 2011). La dificultad para lograr un fechado correcto de los crecimientos anuales de P. oocarpa atribuido a la formación de anillos falsos y períodos de supresión, ha sido reporta­ do en estudios dendroclimáticos desarrollados en Centroamérica (Hastenrath, 1963; Szejner, 2011). El fechado de 46 % de las muestras de P. oocarpa en este estudio, corrobora lo planteado por Villalba (1990), Lara y Villalba (1993) y Neira (1995), quienes han documentado, que el porcentaje de muestras fechadas, puede variar de 43 a 90 %. A pesar de la amplia distribución de esta especie son pocos los trabajos que han estudia­ do la influencia del clima en su crecimiento. Hastenrath (1963) reportó el primer estudio de anillos en P. oocarpa en El Salvador, en la búsqueda de relacionar la variabilidad de la lluvia y el crecimiento, encontró una relación positiva entre ambas variables. DISCUSIÓN Huante, Rincón y Swetnam (1991) reportan una correlación significativa del incremento radial anual de Abies religiosa con la tempe­ ratura media y mínima de enero y febrero. Cerano et al. (2014) encontraron una relación significativa (P < 0.05) de la temperatura media y mínima de enero y febrero con el crecimien­ to anual de Abies religiosa. Cerano, Rivera, Estrada, Trucios y Ríos (2012) documentaron que los crecimientos de la madera temprana de Pinus cooperi, responde de manera signi­ ficativa (P > 0.05) a la temperatura mínima de enero, febrero, marzo y abril. Un estudio reciente ha reportado una influencia signifi­ cativa (P < 0.05) de la temperatura mínima de enero y febrero en el crecimiento de Pinus cooperi y Pinus durangensis, y una respuesta negativa con la temperatura máxima (Chávez et al., 2017). Respuesta del crecimiento a la tem­ peratura: La relación entre las cronologías y las temperaturas, indican una influencia significativa tanto de la temperatura máxima como mínima en el crecimiento de P. oocarpa. La temperatura mínima de primavera (marzo- mayo) es el período con mayor influencia (P < 0.05) en el crecimiento de P. oocarpa, lo cual se atribuye, a que, en este período, la tempera­ tura mínima se ubica en un rango que permite la actividad fotosintética, ya que temperaturas cercanas a 5 °C o por debajo de ésta, propician que los estomas se cierren interrumpiendo el intercambio de gases (Barceló, Nicolás, Sabater, & Sánchez, 2001). Por otra parte, el proceso de evapotranspiración se incrementa con mayores temperaturas abatiendo en menor tiempo la disponibilidad hídrica en el suelo y en consecuencia se reduce el crecimiento. Las cronologías generadas con base en los anillos de crecimiento de P. oocarpa tie­ nen potencial como una fuente “proxy” para reconstruir variables climáticas, como la pre­ cipitación y la temperatura mínima de prima­ vera (marzo-mayo) con base a la cronología de madera temprana, y temperatura máxima septiembre-enero con base en la cronología de madera tardía. Estos resultados cubren el segundo objetivo de esta investigación y sus­ tentan la segunda pregunta de investigación. Las correlaciones significativas (P < 0.05) de noviembre, diciembre, enero y marzo con la cronología de madera tardía, contrario a lo observado para la madera temprana, puede explicar la formación de anillos falsos al inicio del crecimiento (Fig. 3). DISCUSIÓN Johnson (1980) en Copan, Honduras, estudió la anatomía del anillo anual para entender los factores ambien­ tales que influyen en el crecimiento de los árboles. Cerano et al. (2008) en un estudio en Sierra Las Minas, Guatemala, determinaron una influencia significativa de la precipita­ ción mayo-septiembre en el crecimiento de P. oocarpa. Szejner (2011) al este de Guatemala relacionó el crecimiento de P. oocarpa con la precipitación reportando que esta especie res­ ponde de manera significativa (P < 0.001) al periodo mayo-julio (primavera-verano); mien­ tras que para los Tuxtlas, Veracruz, México, Gutiérrez (2013) señala una influencia de la precipitación diciembre-junio en el crecimiento de la madera total de esta especie. A pesar de los problemas de crecimiento que presenta P. oocarpa, los resultados de COFECHA indicaron una correlación significa­ tiva entre series (r = 0.50; P < 0.01), superando los parámetros estadísticos que dicho progra­ ma establece (r = 0.3281, P < 0.01) (Holmes, 1983). Los parámetros estadísticos de esta serie son similares a los reportados para P. oocarpa en el bosque La Primavera, Jalisco (Villanueva et al., 2018) y la Reserva de la Biosfera Sierra de las Minas (RBSM) en Guatemala (Cerano et al., 2008); pero superiores a los parámetros determinados por Szejner (2011) en el sitio Pedernal en Guatemala (Cuadro 2). Estos resul­ tados indican la sensibilidad de P. oocarpa a los cambios ambientales y su potencial para emplearse en el desarrollo de reconstrucciones dendroclimáticas (Fritts, 1976; Delgado, 2000; Grissino-Mayer, 2001). Se logró generar una cronologia para los últimos 91 años (1925- 2015) y con base en estos resultados, se cubre A diferencia de la respuesta del crecimien­ to de P. oocarpa a la lluvia primavera-verano en Centroamérica (Cerano et al., 2008; Szej­ ner, 2011), en el trópico mexicano esta especie Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 1592 oocarpa, caso contrario a la respuesta con la temperatura mínima. responde a la lluvia estacional invierno-prima­ vera similar a otras coníferas distribuidas en el norte, centro y sur de México (Cleaveland et al., 2003; Constante, Villanueva, Cerano, Cornejo, & Valencia, 2009; Villanueva et al., 2009; Santillán et al., 2010; Cerano et al., 2011, 2013, 2014). Una mayor respuesta del crecimiento tem­ prano a la temperatura mínima y menor rela­ ción ante condiciones de mayor temperatura, ha sido documentada en diversos estudios. DISCUSIÓN Esto se puede atribuir a que al registrarse temperaturas por encima de 25 °C producen una fuerte presión al nivel de los estomas, el déficit de presión de vapor se incrementa hasta por arriba de los 2.0 kPa, lo que provoca cierre de estomas y se inhibe el intercambio de gases y en consecuencia el crecimiento radial (Barceló et al., 2001). Esto produce la acumulación de carbohidratos en las paredes de las células aumentando su espesor (Bidwell, 1979; Giménez, Moglia, Hernán­ dez, & Gerez, 2014). La menor correlación del crecimiento temprano con la temperatura máxima, significa que, a una mayor tempera­ tura al inicio de la estación de crecimiento se favorece un menor crecimiento del anillo de P. Esta especie representa una de las primeras coníferas en México en registrar altas correla­ ciones con la variabilidad de las temperaturas. Estos resultados son de gran relevancia en dos aspectos: 1) P. oocarpa representa una de las pocas coníferas con amplia distribución en esta región, por lo tanto, se podrán generar registros extensos en diferentes áreas del trópico, y 2) que, ante el incremento de la temperatura, esta especie constituye una opción potencial para su reconstrucción, analizar su variabilidad inte­ ranual por décadas o siglos y analizar posibles Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 1593 tendencias ante los diversos escenarios de cam­ bio climático. Es importante dar continuidad a este tipo de estudios, detectar áreas con arbola­ do longevo y lograr generar series extensas que cubran al menos los últimos dos siglos, con el objetivo de analizar tendencias y recurrencia de eventos hidro-climáticos extremos. Este trabajo de investigación aporta conocimiento que contribuye a fomentar los estudios dendro­ climáticos en esta región del trópico mexicano. desarrollo de cronologías de anillo total, madera temprana y madera tardía para un período de 91 años (1925-2015). Se encontró una influencia significativa de la precipitación media y de la temperatura media máxima y mínima del período 1961-2004 sobre el crecimiento anual de P. oocar­ pa. Los resultados muestran que la precipitación invierno- primavera (enero-mayo) fue la más importante para el crecimiento del anillo anual de la especie. Sin embargo, la correlación más alta se observó entre la precipitación de primavera (marzo-mayo) y la cronología de la madera temprana (r = 0.719, P < 0.05). REFERENCIAS Un agradecimiento especial a Roberto Gar­ cía Cancino por su apoyo en la logística duran­ te el muestreo de campo, al ejido Ojo de Agua, La Independencia, Chiapas, por permitirnos el acceso y la toma de muestras. Así mismo, gracias a Gerardo López, José Luis Hernán­ dez, Adolfo Cruz, Valdemar Guillen y Maynor Morales por el apoyo en el levantamiento de datos de campo. Este proyecto fue financiado con fondos personales de los autores. Barceló, J., Nicolás, G., Sabater, B., & Sánchez, R. (2001). Fisiología vegetal. Madrid, España: Edicio­ nes Pirámide. Benito, F. G. (2014). Archivos climáticos y paleohidro­ lógicos. Introducción a datos proxy y su análisis. Madrid, España: CSIC- Museo Nacional de Ciencias Naturales. Bidwell, R. G. S. (1979). Fisiología vegetal. México: AGT Editor. Bogino, S. M., & Bravo, F. (2008). Growth response of Pinus pinaster Ait. to climatic variables in central Spanish forests. Annals of Forest Science, 65(5), 506-506. DISCUSIÓN La cronología de la madera temprana también mostró potencial para reconstruir la temperatura mínima (marzo a mayo) (r = 0.732, P < 0.05), mientras que la cronología de madera tardía registra poten­ cial para reconstruir la temperatura máxima (septiembre- enero) (r = 0.714, P < 0.05). Estos resultados muestran que P. oocarpa puede emplearse para reconstruir variables climáticas en los trópicos mexicanos. Se recomienda explo­ rar nuevas áreas con árboles más viejos a fin de aumentar la extensión de las cronologías y reconstruir los registros climáticos varios siglos en el pasado. Declaración de ética: los autores declaran que todos están de acuerdo con esta publica­ ción y que han hecho aportes que justifican su autoría; que no hay conflicto de interés de cualquier tipo; y que han cumplido con todos los requisitos y procedimientos éticos y legales pertinentes. El documento firmado se encuen­ tra en los archivos de la revista. Palabras claves: anillos de crecimiento; anillos falsos; madera temprana; madera tardía; potencial dendroclimático. Palabras claves: anillos de crecimiento; anillos falsos; madera temprana; madera tardía; potencial dendroclimático. Palabras claves: anillos de crecimiento; anillos falsos; madera temprana; madera tardía; potencial dendroclimático. Cerano, P. J., Rivera, G. M., Estrada, A. J., Trucios, C. R., & Ríos, S. J. C. (2012). Análisis dendrocronológico de Pinus cooperi en Durango, México. Agrofaz, 12(3), 81-88. RESUMEN Evaluating crossdating accuracy: a manual and tutorial for the computer program COFECHA. Tree-Ring Research, 57(2), 205-221. Gutiérrez, G. G. (2013). La relación entre crecimiento y clima en árboles tropicales: un estudio dendrocro­ nológico en la selva de los Tuxtlas, Veracruz, México (Tesis Doctoral). UNAM, México. Cerano, P. J., Villanueva, D. J., Valdez, C. R. D., Cornejo, O. E. H., Sánchez, C. I., & Constante, G. V. (2011). 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(2007). Prontuario de la información geográfica municipal de los Estados Unidos Mexicanos. La Independencia, Chiapas. Recuperado de http://www3.inegi.org.mx/ sistemas/mexicocifras.pdf Cook, E. R. (1987). The decomposition of tree-ring series for environmental studies. Tree-Ring Bulletin, 47, 37-59. Curtis, S. (2002). RESUMEN Los estudios dendrocronológicos se utilizan para reconstruir algunas variables climáticas; en México, estos estudios se han centrado en los bosques templados del cen­ tro y del norte, donde los árboles presentan anillos anuales bien definidos. Pocos estudios se han llevado a cabo en la parte sur del país, donde el crecimiento anual del anillo no se identifica fácilmente y, por lo tanto, esto hace que la datación se dificulte. Se analizó el potencial dendro­ cronológico de Pinus oocarpa para la reconstrucción de variables climáticas en la porción noroeste de Lagunas de Montebello, Chiapas. Empleando un muestreo selectivo se recolectaron 65 núcleos de incremento de 34 árboles. Aun­ que las muestras presentaron una alta frecuencia de anillos falsos (8 a 60 %), se logró fechar 30 muestras de 22 árboles (46 %) mediante técnicas dendrocronológicas estándar y el Bradley, R. S. (1999). Paleoclimatology: Reconstructing climates of the Quaternary. (2nd ed.). USA: Acade­ mic Press. Cerano, P. J., Méndez, G. J., Amaro, S. A., Villanueva, D. J., Cervantes, M. R., & Rubio, C. E. (2013). Recons­ trucción de precipitación invierno-primavera con anillos anuales de Pinus douglasiana en la Reserva de la Biósfera de Manantlán, Jalisco. Revista Chapingo Serie Ciencias Forestales y del Ambiente, 29(3), 413- 423. DOI: 10.5154/r.rchscfa.2013.02.007 Cerano, P. J., Rivera, G. M., Estrada, A. J., Trucios, C. R., & Ríos, S. J. C. (2012). Análisis dendrocronológico de Pinus cooperi en Durango, México. Agrofaz, 12(3), 81-88. Rev. Biol. Trop. (Int. J. Trop. Biol. 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Facultad de Agricultura y Zootecnia-Universidad Juárez del Estado de Durango, México. Grissino-Mayer, H. D. (2001). RESUMEN Interannual variability of the bimodal distribution of summertime rainfall over Central America and tropical storm activity in the far-eastern Pacific. Climate Research, 22, 141-146. Johnson, W. C. (1980). Muestreo dendrocronológico de Pinus oocarpa cerca de Copán, Honduras: una nota preliminar. Biotrópica, 12, 315-316. Lara, A., & Villalba, R. (1993). A 3,620-year temperature reconstruction from Fitzroya cupressoides tree rings in southern South America. Science, 260, 1104-1106. Delgado, C. S. (2000). Aplicaciones estadísticas en análisis dendrocronológicos. En F. A. Roig (Ed.), Dendrocro­ nología en América Latina (pp. 79-102). Mendoza, Argentina: Editorial de la Universidad Nacional de Cuyo. Martínez, M. (1992). Los pinos mexicanos (3era ed.). Méxi­ co: Editorial Botas. Fritts, H. C. (1976). Tree-rings and Climate. London - New York - San Francisco: Academic Press. Neira, E. P. (1995). Desarrollo de cronologías para alerce (Fitzroya cupressoides) en las Cordilleras de la Costa Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 1595 Turner, I. M. (2004). The Ecology of trees in the tropical rain forest. Cambridge, UK.: Cambridge University Press. y de los Andes (Tesis de Grado). Universidad Austral de Chile, Valdivia, Chile. Perry, J. P. Jr., Graham, A., & Richardson, M. D. (1998). The history of pines in México and Central America. In M. D. Richardson (Ed.), Ecology and Biogeogra­ phy of Pinus (pp.137-149). Cambridge, UK: Cam­ bridge University Press. Vaganov, E. A., Hughes, M. K., & Shashkin, A. V. (2006). Growth dynamics of tree rings: an image of the past and future environments. Berlin: Springer-Verlag. Villalba, R. (1990). 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One hundred of tree-ring research in the tropics - a brief history and outlook to future cha­ llenges. Dendrochronologia, 20, 217-231. Tschinkel, H. M. (1966). Annual growth rings in Cordia alliodora. Turrialba, 16, 73-80. Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1580-1596, December 2018 1596
https://openalex.org/W3191226521
https://jwcn-eurasipjournals.springeropen.com/track/pdf/10.1186/s13638-021-02038-7
English
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Research on application of GPS-based wireless communication system in highway landslide
EURASIP Journal on wireless communications and networking
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cc-by
6,487
© The Author(s), 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the mate- rial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​ creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. RESEARCH Open Access Research on application of GPS‑based wireless communication system in highway landslide Zhiwen Xiong* *Correspondence: hnlgdxxiongzhiwen@163. com Department of Electrical Engineering, Guangxi Technological College of Machinery and Electricity, Nanning 530007, People’s Republic of China Abstract Machine learning is a branch of the field of artificial intelligence. Deep learning is a complex machine learning algorithm that has unique advantages in image recogni- tion, speech recognition, natural language processing, and industrial process control. Deep learning has It is widely used in the field of wireless communication. Prediction of geological disasters (such as landslides) is currently a difficult problem. Because land- slides are difficult to detect in the early stage, this paper proposes a GPS-based wireless communication continuous detection system and applies it to landslide deformation monitoring to achieve early treatment and prevention. This article introduces the GPS multi-antenna detection system based on deep learning wireless communication, and introduces the time series analysis method and its application. The test results show that the GPS multi-antenna detection system of the wireless communication net- work has great advantages in response time, with high accuracy and small error. The horizontal accuracy is controlled at 0–2 mm and the vertical accuracy is about 1 mm. The analysis method is simple and efficient, and can obtain good results for short-term deformation prediction. Keywords:  Deep learning, Wireless network communication, GPS detection system, Time series analysis, Data processing Xiong J Wireless Com Network (2021) 2021:163 https://doi.org/10.1186/s13638-021-02038-7 Xiong J Wireless Com Network (2021) 2021:163 https://doi.org/10.1186/s13638-021-02038-7 *Correspondence: hnlgdxxiongzhiwen@163. com Department of Electrical Engineering, Guangxi Technological College of Machinery and Electricity, Nanning 530007, People’s Republic of China 1  Introduction At present, frequent natural disasters around the world not only affect people’s property safety, but also seriously affect people’s lives and travel. If monitoring can be effectively carried out, it will be a good thing. Deep learning is a branch of machine learning. Its main principle is to use algorithms that contain complex structures or are composed of multiple nonlinear transformations to multiple processing layers to abstract data at a high level. In wireless networks, GPS technology is developing fastest. As a high-tech modern geodetic survey technology, GPS has become one of the most advanced defor- mation monitoring technologies and has been widely used due to its high-precision, fast, all-weather, and highly automated features. The text is mainly aimed at highway slope deformation. Research and analysis. Xiong J Wireless Com Network (2021) 2021:163 Xiong J Wireless Com Network (2021) 2021:163 Page 2 of 15 Currently, there are two main modes of applying GPS to highway slope deformation detection. The first is the conventional monitoring mode of conventional GPS static rela- tive positioning, and the second is the continuously operating station-type GPS monitor- ing system [1]. In the conventional monitoring mode, multiple GPS receivers are usually used to manually collect data point by point on a regular basis, and perform post-pro- cessing to obtain the deformation of each cycle, so as to predict the mid-to-long-term trend deformation of the slope. If the slope is already in an unstable state, it is neces- sary to use a continuously operating station GPS monitoring system to obtain the slope deformation status in real time [2]. However, the use of continuously operating station- type GPS monitoring systems requires more investment in GPS hardware equipment, and the high price limits the application range of GPS technology. This article adopts the design scheme of GPS multi-antenna monitoring system, and connects multiple antenna arrays to the same receiver by attaching a GPS signal time-sharing connection switch without changing the existing GPS receiver structure. After the algorithm is processed, the deformation law of the deformable body can be obtained [3]. The GPS signal time- sharing connection switch (called GPS multi-antenna switch) is used to switch between multiple antennas. The switching interval can be determined according to the state of the deformable body, ranging from a few seconds to a few hours. 1  Introduction Since the price of the antenna is much lower than that of the receiver, the use of an antenna instead of a GPS receiver greatly reduces the cost of the GPS deformation monitoring system, making GPS technology a broad application prospect in deformation monitoring [4]. This article considers that landslide disasters mostly occur in dangerous or remote areas. In order to ensure personal safety and realize unattended working mode, the wire- less communication network is used to transmit GPS monitoring data in real time and carry out GPS multi-antenna monitoring. To this end, we have done relevant research and developed a wireless communication network system that can quickly and accu- rately monitor slope deformation. 2.1.1  Brief description of GPS As a new generation of precise satellite positioning system, GPS system represents the cutting-edge technology and is the crystallization of the development of contemporary science and technology. Initially, GPS was originally developed for military applications, such as positioning and navigation of military vehicles, aircraft, and ships [5, 6]. Because of its ranging and timing functions, GPS can provide global users with high-precision, all-weather, and large-scale position and time information, which can well meet the mili- tary and civilian positioning and navigation needs. The composition of the GPS system is shown in Fig. 1. As can be seen from Fig. 1, the GPS system is mainly composed of three parts: the space satellite part, the ground monitoring part, and the user receiving part [7]. The space satellite part is mainly composed of 24 satellites distributed in 6 elliptical orbital planes of the earth, including 21 working satellites and 3 standby satellites in orbit. These satellites are 17,700 km away from the earth and the satellite operating cycle Xiong J Wireless Com Network (2021) 2021:163 Page 3 of 15 is 1.58 h. Four satellites are deployed on the plane, and the coverage angle is 55 degrees [8, 9]. In actual use, the GPS system receiver can capture more than 4 satellites. At this time, in order to improve the positioning accuracy, the receiver divides the captured sat- ellites into several groups according to the constellation distribution, with 4 satellites in each group. After calculation and analysis, the group with the smallest error is selected for positioning calculation. ( ) G d i i Master station Satellite system Monitoring station Injection station User positioning device Fig. 1  GPS system composition diagram Master station Satellite system Monitoring station Injection station User positioning device Fig. 1  GPS system composition diagram Monitoring station is 1.58 h. Four satellites are deployed on the plane, and the coverage angle is 55 degrees [8, 9]. In actual use, the GPS system receiver can capture more than 4 satellites. At this time, in order to improve the positioning accuracy, the receiver divides the captured sat- ellites into several groups according to the constellation distribution, with 4 satellites in each group. After calculation and analysis, the group with the smallest error is selected for positioning calculation. (3) User receiving parth The user receiving part is mainly composed of GPS signal receiver [11]. The main func- tion is to collect satellite signals. Through the collection and calculation of parameters such as the satellite orbit, the distance between the satellite and the receiver, the current position information of the user is obtained, including latitude and longitude, altitude, and speed of movement. GPS receiver is mainly composed of antenna and receiver. The receiver is powered by a DC power source inside and outside the machine. Generally, the power is supplied by an external power source and the battery is charged. After the power is turned off, the internal battery powers the memory to ensure data storage. (2) Ground monitoring parth The ground monitoring part is mainly composed of a main control station, 3 ground control stations and 5 global monitoring stations [10]. The main control station is located in Colorado, the United States mainland. The five monitoring stations distrib- uted around the world under the direct control of the master control station are the data collection centers of the GPS system. Among them, the monitoring stations are equipped with receivers that can continuously measure visible satellite data and cesium clocks with precise time measurement. The main function of the monitoring station is to obtain satellite observation data including ionospheric and meteorological data and send it to the main control station. Then the main control station analyzes these data, cal- culates the clock parameters and satellite orbits, and sends the analysis results to three ground control stations. (3) User receiving parth 2.1.2  GPS positioning principle GPS positioning methods are divided into two methods: absolute positioning and rela- tive positioning. The former is used to determine the position of the moving carrier in the earth reference frame in real time. The positioning accuracy is within 100 m. The latter uses multiple machines to determine the mutual relationship between measure- ment stations. After a certain period of observation, the data is processed by data post- processing software, and its relative accuracy reaches nanometer level [12, 13]. (1) Absolute positioning principle (1) Absolute positioning principle Xiong J Wireless Com Network (2021) 2021:163 Page 4 of 15 Page 4 of 15 The basic principle of the so-called absolute positioning is to use only one receiver to observe satellite signals and determine itself independently. The position of the antenna phase center in the coordinate system is called absolute positioning because this position is the only absolute. Positioning can be divided into dynamic absolute positioning and static absolute positioning [13, 14]. The former is mainly used for flying because of its low positioning accuracy. Navigation is required for machines, vehicles, and ships that require less precision. The latter can continuously measure the pseudo-range from the satellite to the observa- tion station and improve the positioning accuracy through data processing, so it can be used for observation or navigation in some fine industries with high accuracy require- ments [15]. GPS observation can get the position of the satellite and the distance from the sat- ellite to the ranging point, and then use the satellite as the center and the distance as the radius to make a spherical surface. If three satellites are observed at the same time, we will get three spherical surfaces. It is the position of the measurement point that is required to be solved. Of course, in the actual measurement, due to the factor of the clock difference, the pseudo range measured by the receiver includes three coordinate component unknowns and one clock difference unknown, so if you want to solve these four unknowns, you must observe at least four satellites to establish The equations are used to settle the station coordinates corresponding to the user’s receiver antenna. 2.1.2  GPS positioning principle Let P be the pseudo-range observation, R be the true distance from the receiver to the satellite, C be the speed of light, and T be the difference in the reception clock, then the observa- tion equation is: (1) ρ = R + c + τ =  (xs −xr)2 +  ys −yp 2 +  zs −zp 2 + c × τ (1) (2) Relative positioning principleh (2) Relative positioning principleh (2) Relative positioning principleh The accuracy of absolute positioning is often inaccurate, which is mainly affected by factors such as satellite orbit errors, clock synchronization errors, and errors generated during propagation in the atmosphere. Although we can eliminate the errors caused by weakening some systems through methods such as mathematical modeling, its position- ing accuracy can only reach meters, which is difficult to meet the needs of high precision [16]. Relative positioning is also called differential positioning. The basic principle is to use multiple receivers to observe GPS satellites simultaneously to determine the mutual relationship between the stations where each receiver is located in the earth coordinate system. Therefore, within a certain distance range, the orbit error of the satellite, the sat- ellite clock error, the receiver clock error, and the refraction errors of the ionosphere and troposphere have a certain correlation with the impact on the observations. Use dif- ferent combinations of these observations for relative positioning., The influence of the above errors can be eliminated or reduced, thereby improving the positioning accuracy. In relative positioning, at least two GPS signal receivers are required, which are respectively set at the two ends of the baseline. One of the endpoints is a known coor- dinate point. The same set of GPS satellites are simultaneously observed, and the differ- ence between the coordinate components between the two points and the baseline are Xiong J Wireless Com Network (2021) 2021:163 Page 5 of 15 measured. Length, the relative position of the baseline endpoint or the baseline vector is calculated, and the exact coordinates of the other point can also be calculated. Rela- tive positioning can also be divided into static and dynamic positioning methods. The static relative positioning method is currently the most accurate of all GPS positioning methods, but the measurement time is relatively long, and generally takes one to three hours [17]. The dynamic relative positioning method is to press one receiver on a mov- ing carrier and install the other receiver at a known point (reference station). The former is called a dynamic GPS signal receiver, while the latter is called a reference GPS signal receiver. These two receivers simultaneously observe a group of GPS satellites in sight, and the reference receiver provides differential correction numbers for the dynamic receiver, which is called GPS differential positioning data [18]. (2) Relative positioning principleh The dynamic receiver uses its own GPS observations and differential correction data from the reference receiver to accurately calculate the user’s 3D coordinates. 2.2  GPS multi‑antenna detection system for wireless network communication (1) GPS one-machine multi-antenna monitoring systemh The GPS one-machine multi-antenna monitoring system aims to give full play to the advantages of GPS measurement technology in automated real-time deforma- tion monitoring and reduce the cost of purchasing GPS receivers. The design idea is: a GPS receiver is connected to multiple antennas, so that each Only GPS antennas are installed on the monitoring points, and no receivers are installed. Multiple monitoring points share a GPS receiver, which can greatly reduce the cost of the monitoring system without reducing the accuracy of conventional GPS measurements. Based on this idea, a GPS multi-antenna control switch can be designed so that one GPS receiver connects to multiple antennas, and these antennas work automatically in sequence by software control. (2) Multi-antenna controllerh The multi-antenna controller includes software and hardware, and is one of the core parts of a multi-antenna system. The hardware part is composed of multi-channel microwave switch and corresponding control circuit, a GPS receiver and corresponding processing chip; the off-state of several signal channels in the microwave switch is con- trolled by the switch control circuit in real time. The software part mainly realizes the functions of controlling the multi-channel working mode, setting the observation time of the measuring point, real-time communication with the GPS receiver and data trans- mission. The newly developed GPS multi-antenna controller, the field computer uses an embedded industrial control computer, and integrates the control circuit board and the dual-frequency GPSOEM board, and is equipped with an LCD liquid crystal display, which can intuitively monitor the situation of the multi-antenna data collection site. The key technical problem to be solved in the hardware part of the GPS multi-antenna controller is the high isolation of the GPS signals of each channel in the microwave switch. The key technical problem to be solved in the software part is to realize real-time precise positioning, so that the positioning accuracy reaches mm level. (3) Design of data transmission system (3) Design of data transmission system (3) Design of data transmission system The data transmission from the GPS antenna to the multi-antenna controller can only be transmitted through a wired medium, so the coaxial cable or optical fiber can be used Xiong J Wireless Com Network (2021) 2021:163 Page 6 of 15 for data transmission. Coaxial cable is only suitable for short-distance data transmis- sion; however, regardless of the distance of optical fiber, the quality and reliability of data transmission are guaranteed, but its cost is relatively high. The coaxial cable consists of a layer of mesh copper conductor and a copper conductor located on the central axis. Compared with the ordinary twisted pair, the coaxial cable has strong anti-interfer- ence ability and good shielding performance, and is often used for connection between devices. If a repeater (signal amplifier) is used, the length of the network connected by the coaxial cable can be increased up to several kilometers. Data transmission from field data to the monitoring center. Since the monitoring site is generally located in a remote mountainous area, the field data received by the receiver adopts wireless transmission mode, GPRS and GSM are both good choices. Here we use the GPRS communication method. The specific method is to connect the GPS multi- antenna controller through the RS-232 serial port on the controller and the GPRS termi- nal RS-232 serial port through a patch cord to transmit the original GPS data to GPRS terminal, and then continuously send to the monitoring center through the terminal wireless mode. (4) Data processing system The data processing system is responsible for the entire process of transmitting, stor- ing, analyzing, calculating and displaying the original data of the receiver. First, the data of the multi-antenna receiver and the reference station are transmitted to the GPRS transmitter through the RS-232 serial port, and then the data is sent to the monitor- ing center through the wireless network long-distance transmission, and the monitor- ing center data processing software classifies and analyzes the multi-site data. Converted into location information. Through the process of comparing the position information of Fig. 2  Data processing flowchart Xiong J Wireless Com Network (2021) 2021:163 Page 7 of 15 the reference station and the measuring point, and time sequence analysis, the predicted deformation trend curve can be generated, as shown in Fig. 2 [19]. 2.3  Inter sequence analysis For a long time, deformation analysis and processing methods have assumed that the observed data are statistically independent or uncorrelated, such as regression analy- sis methods. This kind of statistical method is a static data processing method, which cannot realize dynamic prediction of variables. However, whether it is observation data arranged in time series or observation data arranged in spatial order, there is more or less statistical autocorrelation between the data. With the development of modern sci- ence and technology and the improvement of computer application, various theories and methods have provided a wide range of research methods for deformation analysis and deformation prediction. (1) Definition of stochastic process and time series i A stochastic process is a (family of) random variable that depends on a parameter. For example: the terminal voltage of an electronic component or device due to the random thermal disturbance of internal micro-particles is called thermal noise voltage, and its value at any given moment is a random variable; the temperature at each moment of the day is a random variable, which Sets constitute a random process. The definition of a stochastic process is: let E be a random test and S = (P) be its sample. If for each e ∈ s, there is always a real-valued function X (e, t)Corresponding to this, the function of the parameter t of this family is called a random process, and each function in the family is called a sample function of the random process, and T is the variation range of the parameter t, called a parameter set. Random processes can be divided into continuous random processes and discrete random processes according to whether they are continuous random variables or discrete random variables at any time. The specific value obtained by the random process in the test results is called the "implementation" of the random process, or the sample func- tion, also called the sample observation. Time series are random sequences, that is, random sequences with discrete parameters. (2) Time series modeling method (2) Time series modeling method (2) Time series modeling method The key of time series analysis is to establish an appropriate mathematical model based on a reasonable analysis of observation results. The key of time series analysis is to establish an appropriate mathematical model based on a reasonable analysis of observation results. The general steps for modeling are:h The general steps for modeling are:h The general steps for modeling are:h (1) Preparation stage. The acquisition of initial data requires that the data can accurately and truly reflect the behavioral state of the modeling system. First, the data needs to be analyzed and tested, including the elimination of glitches and compensation data. The zero-mean test requires data preprocessing for sequences that do not meet the stability requirements. The processing methods mainly include differential processing or trend item extraction, and digital signal processing methods can process data flexibly. (1) Preparation stage. The acquisition of initial data requires that the data can accurately and truly reflect the behavioral state of the modeling system. First, the data needs to be analyzed and tested, including the elimination of glitches and compensation data. The zero-mean test requires data preprocessing for sequences that do not meet the stability requirements. The processing methods mainly include differential processing or trend item extraction, and digital signal processing methods can process data flexibly. l (2) Preliminary determination of model structure and category. To determine the struc- ture and category of the model, you need to choose a modeling method. (3) After the structure of the model is determined, the appropriate method for selecting the model parameters should be estimated according to certain principles; then the model suitability test of the model is performed to determine the final appropriate model. (4) Model establishment According to the stationarity formula of the time series, the parsed data is firstly dis- cretized, and then substituted into formula 5 to obtain the parameter mean value, the mean value is substituted into formula 6 to obtain the data variance, and finally substi- tuted into formula 7 for stationarity analysis. After the above series of processing, Judg- ing whether there is a trend of deformation according to the size of the difference. (2) Time series modeling method (3) The mathematical foundation of the model i (3) The mathematical foundation of the model Xiong J Wireless Com Network (2021) 2021:163 Page 8 of 15 Auto-covariance function of random variables: Auto-covariance function of random variables: Auto-covariance function of random variables: (2) D(Xt, Xs) = Cov(Xt, Xs) = E{[Xt −E(Xt)][Xs −E(Xs)]} (2) Autocorrelation function and autocorrelation coefficient of random variables: (3) RX(t, s) = E(Xt, Xs) (3) RX(t, s) = E(Xt, Xs) RX(t, s) = E(Xt, Xs) (3) nitial estimation of model parameters: Initial estimation of model parameters: Initial estimation of model parameters: (4) Xt = φ1Xt−1 + φ2Xt−2 + · · · + φpXt−p + at (4) Test of time series stationarity: According to the definition of stationary time series, the mean and variance of stationary time series are constant; the interval between self- coordinates is related to the breakpoint of this interval. The relevant formula for the test of time series stationarity is as follows: (5) Xi = 1 M M  j=1 Xij (5) Xi = 1 M M  j=1 Xij (6) ˆσ 2 i = 1 M M  j=1  Xj i −Xi 2 (7) rτ(i) = 1 M M−τ  j=1  Xij −Xi  Xi,j+τ −Xi  ˆσ 2 t (5) Xi = 1 M M  j=1 Xij (6) ˆσ 2 i = 1 M M  j=1  Xj i −Xi 2 (7) rτ(i) = 1 M M−τ  j=1  Xij −Xi  Xi,j+τ −Xi  ˆσ 2 t (5) (6) ˆσ 2 i = 1 M M  j=1  Xj i −Xi 2 (6) (7) rτ(i) = 1 M M−τ  j=1  Xij −Xi  Xi,j+τ −Xi  ˆσ 2 t (7) 3.1  Performance test and analysis of GPS detection system for wireless network communication 3.1  Performance test and analysis of GPS detection system for wireless network communication (1) Response time analysis In this article, the WGS84 coordinate system is used, and the origin is the center of mass of the earth. The X axis points to the intersection of the zero-degree meridian plane defined by the BIH and CTP equator, the Y axis and the Z axis, and the X axis constitutes a right-handed coordinate system. The response time of the GPS detection system used for wireless network communication is analyzed, and the result is shown in Fig. 3. It can be seen from Fig. 2 that the response time of the GPS detection system for wireless network communication in this article is faster than the actual shortest time required. The response time of the system determines the performance of the system. Page 9 of 15 Xiong J Wireless Com Network (2021) 2021:163 . 3  System response time analysis Fig. 3  System response time analysis Fig. 3  System response time analysis Xiong J Wireless Com Network (2021) 2021:163 Page 10 of 15 The GPS detection system for wireless network communication in this article has a great advantage in response time. (2) Precision analysish (2) Precision analysish (2) Precision analysish This paper uses GPSensor software to solve the data. At the beginning of the solution, the solution time is set to 15 min. After the data is stable, the solution time is changed to 1 min. After a certain observation, the GPS01 antenna direction X moves 10 mm to the north. Similarly, when the antenna switching controller switched to the channel corre- sponding to the GPS02 antenna, after a period of observation, GPS02 also moved 8 mm northward along X. After continuous testing, the results obtained are compared with the displacement to analyze the accuracy and sensitivity of the system. The results are shown in Fig. 4. It can be seen from Fig. 3 that after analyzing the observation data of GPS01 and GPS02, the observation results show that the accuracy is: the horizontal accuracy is con- trolled at 0–2 mm, and the vertical accuracy is about 1 mm. It can be seen that the GPS detection system for wireless network communication in this article has a good advan- tage in accuracy. (1) Time series analysis 5  Test results of sequence stationarity Xiong J Wireless Com Network (2021) 2021:163 Page 13 of 15 Table 1  Autocorrelation function and partial autocorrelation function values k 0 1 2 3 4 5 Autocorrelation function 1 − 0.405 0.007 − 0.023 0.080 0.057 Partial correlation function – − 0.536 − 0.301 − 0.226 − 0.213 − 0.77 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 Measured value Forecast value Absolute error Fig. 6  Analysis of deformation and settlement detection and prediction results of highway slope Table 1  Autocorrelation function and partial autocorrelation function values Fig. 6  Analysis of deformation and settlement detection and prediction results of highway slope It can be known from Fig. 6 that the monitored deformation value of the highway slope is basically consistent with the predicted value, and the forecast accuracy decreases with the increase of the number of forecast steps. As an efficient method for modern dynamic data processing, intersequence has the advantages of easy modeling, simple calcula- tion, fast prediction and high accuracy. Time series used in deformation monitoring can achieve higher fitting accuracy and prediction effect, especially for short-term predic- tion. Because the accuracy of the time series forecast decreases with the increase of the number of forecast steps, it is suitable for short-term forecasting; if medium- and long- term forecasting is required, the forecast results should be continuously revised using the measured data. (1) Time series analysis Take the GPS detection system of wireless network communication as an example to analyze and predict highway slope deformation, and compare the test results with the actual data results. First perform a time series analysis to determine the stability of the data. There are three ways to judge. 1) Use the unit root test method to check the stability of the data; 1) Use the unit root test method to check the stability of the data; 2) Observe the data line graph. If the line chart is irregular, or returns to a straight line infrequently, it means that the sequence is unstable; 3) Observe the sample autocorrelation function graph. If the sample autocorrelation function does not show an exponential decay trend, it indicates that the sequence is unstable. If the sequence is judged to be non-stationary, it can be segmented into a stationary sequence, but the difference should not be too large, otherwise the revers- ibility of the sequence will be destroyed, and the variance will increase, so the vari- ance can be used to determine whether the difference is too large. This article adopts the third method to establish and analyze the time series analysis model. The result is shown in Fig. 5. The values of the autocorrelation function and part of the autocorrelation function are shown in Table 1. From the combination of Fig. 5 and Table 1, it can be seen that the original sequence diagram shows that the sequence has a clear trend. After two differences, a stable sequence was obtained, the model fitting accuracy was high, and the trend of the model fitting was basically the same as that of the original sequence. The stable sequence data indicated that the obtained slope did not present a risk of deformation. (2) Analysis of application results (2) Analysis of application results The GPS detection system of wireless network communication in this paper is used to predict the deformation of highway slope, and the results are shown in Fig. 6. Page 11 of 15 Page 11 of 15 Xiong J Wireless Com Network (2021) 2021:163 s of monitoring data meter results Fig. 4  Analysis of monitoring data meter results Fig. 4  Analysis of monitoring data meter results Page 12 of 15 Xiong J Wireless Com Network (2021) 2021:163 equence stationarity Fig. 5  Test results of sequence stationarity Fig. References Bensaali, A wireless oxygen saturation and heart rate monitoring and alarming system based on the Qatar early warning scoring system J Emerg Med Trauma Acute Care 2016(2), 155 (2016) 7. S. Alshorman, F.T. Jaber, F. Bensaali, A wireless oxygen saturation and heart rate monitoring and alarming system based on the Qatar early warning scoring system. J. Emerg. Med. Trauma Acute Care 2016(2), 155 (2016) 8. F. Yao, H. Wu, Y. Chen et al., Cluster-based collaborative spectrum sensing for energy harvesting cognitive wireless communication network. IEEE Access PP(99), 1–1 (2017) 9. P. Gupta, Correlating road network information and user mobility information for wireless communication network planning. Infect. Control Hosp. Epidemiol. 15(4), 280 (2017) 10. S. Mondal, S.K. Mohanty, S. Nandi, Energy efficient secure communication architecture for wireless sensor network: energy efficient secure communication architecture for wireless sensor network. Secur. Commun. Netw. 9(16), 3314–3323 (2016) 11. C. Liang, Q. Zhang, J. Ma et al., Research on neural network chaotic encryption algorithm in wireless network secu- rity communication. EURASIP J. Wirel. Commun. Netw. 2019(1), 151 (2019) 12. N. Sood et al., Integrating physics-based wireless propagation models and network protocol design for train com- munication systems. IEEE Trans. Antennas Propag. PP(99), 1–1 (2018) munication systems. IEEE Trans. Antennas Propag. PP(99), 1–1 (2018) 13. S. Gopikrishnan, P. Priakanth, Retracted article: HSDA: hybrid communication for secure data aggregation in wireless sensor network. Wirel. Netw. 22(3), 1–18 (2017) 14. M. Singh, P.M. Khilar, A range free geometric technique for localizat controlled communication range. Wirel. Pers. Commun. 94(3), 1–2 14. M. Singh, P.M. Khilar, A range free geometric technique for localization of wireless sensor network (WSN) based on controlled communication range. Wirel. Pers. Commun. 94(3), 1–27 (2017) 15. S. Bhowmik, C. Giri, A fuzzy communication model of sensor nodes in wireless sensor network. Int. J. Sens. Netw. 21(1), 1–15 (2016) 16. F. Afsana, Md. Asif-Ur-Rahman, M.R. Ahmed et al., An energy conserving routing scheme for wireless body sensor nanonetwork communication. IEEE Access PP(99), 1–1 (2018) 7. R. Costa, J. Lau, P. Portugal et al., Handling real-time communica 17. R. Costa, J. Lau, P. Portugal et al., Handling real-time communi the RT-WiFi approach. J. Commun. Netw. 89(99), 1–15 (2019) 17. R. Costa, J. Lau, P. Portugal et al., Handling real-time communication in infrastructured IEEE 802.11 wireless networks: the RT-WiFi approach. J. Commun. Netw. 89(99), 1–15 (2019) the RT-WiFi approach. J. Commun. Netw. 89(99), 1–15 (2019) 18. Y.-M. Ko, J.-H. Abbreviations Abbreviations WGS84: World geodetic system; GPS: Global positioning system; CTP: Agreement earth pole; CIO: Conventional interna- tional origin. Availability of data and materials Data sharing not applicable to this article as no datasets were generated or analysed during the current study. Competing interests h h d l h p g The authors declare that they have no competing interests in this section. Received: 8 May 2021 Accepted: 2 August 2021 Received: 8 May 2021 Accepted: 2 August 2021 Funding u d g Funded by the Guangxi University Young and Middle-aged Teachers’Research Ability Improvement Project, project num- ber: 2020KY32013; Funded by Scientific Research Project of Guangxi Mechanical and Electrical Vocational and Technical College, project number: 2019YKYZ002. 4  Conclusions This text has carried on the systematic test to the GPS multi-antenna monitoring system of the wireless communication network. The research found that the wireless communi- cation network GPS multi-antenna monitoring system in this paper has great advantages in system response time. In addition, in terms of accuracy, the GPS multi-antenna moni- toring system of the wireless communication network in this article has higher detection accuracy and smaller errors. In addition, this paper conducted a case study. Although the reliability of the application examples was verified, the number of case samples was not comprehensive enough to explain the applicability of the system to complex terrain. Further testing and improvement are needed in the follow-up. In addition, this paper also established a time series model for prediction, and veri- fied the reliability of the model through experiments. Based on the verification results of the examples in this paper, considering the accuracy requirements and costs of landslide safety monitoring, the GPS multi-antenna monitoring system based on wireless com- munication network in this paper can automatically and continuously monitor landslide disasters, and can greatly reduce the overall The cost of the monitoring system is one of Page 14 of 15 Xiong J Wireless Com Network (2021) 2021:163 the ideal technologies for landslide and other geological disaster deformation monitoring, and can provide a certain paradigm role for the future geological disaster technology. the ideal technologies for landslide and other geological disaster deformation monitoring, and can provide a certain paradigm role for the future geological disaster technology. Authors’ contributions ZX carried out the research of GPS wireless communication continuous detection system, participated in time series analysis, established a time series model for prediction, and verified the reliability of the model through experiments and drafted a manuscript. The authors read and approved the final manuscript. References e e e ces 1. C. Àlvarez, J. Díaz, J. Petit et al., High level communication functionalities for wireless sensor networks. Theor. Com- put. Sci. 406(3), 240–247 (2016)i 1. C. Àlvarez, J. Díaz, J. Petit et al., High level communication functionalities for wireless sensor networks. Theor. Com- put. Sci. 406(3), 240–247 (2016)i 2. P. He, T. Fan, Distributed fault-tolerance consensus filtering in wireless sensor networks-Part I: communication failure. Int. J. Sens. Netw. 22(2), 127–142 (2016) 2. P. He, T. Fan, Distributed fault-tolerance consensus filtering in wireless sensor networks-Part I: communication failure. Int. J. Sens. Netw. 22(2), 127–142 (2016)fi 2. P. He, T. Fan, Distributed fault-tolerance co Int. J. Sens. Netw. 22(2), 127–142 (2016) Int. J. Sens. Netw. 22(2), 127–142 (2016)fi Int. J. Sens. Netw. 22(2), 127–142 (2016)fi 3. S. Randhawa, S. Jain, Energy-efficient load balancing scheme for two-tier communication in wireless sensor net- works. J. Supercomput. 74(4), 1–31 (2017) 3. S. Randhawa, S. Jain, Energy-efficient load balancing scheme for two-tier communication in wireless sensor net- works. J. Supercomput. 74(4), 1–31 (2017) 3. S. Randhawa, S. Jain, Energy-efficient load balancing sche works. J. Supercomput. 74(4), 1–31 (2017) works. J. Supercomput. 74(4), 1–31 (2017) p p 4. Y. Kwon, A. Kwasinski, A. Kwasinski, Coordinated energy management in resilient microgrids for wireless communi- cation networks IEEE J Emerg Sel Top Power Electron 4(4) 1158–1173 (2016) 4. Y. Kwon, A. Kwasinski, A. Kwasinski, Coordinated energy management in resilient microgrids for wireless commu cation networks. IEEE J. Emerg. Sel. Top. Power Electron. 4(4), 1158–1173 (2016) 4. Y. Kwon, A. Kwasinski, A. Kwasinski, Coordinated energy management in resilient microgrids for wireless commun cation networks. IEEE J. Emerg. Sel. Top. Power Electron. 4(4), 1158–1173 (2016) 5. N. Wan, G.L. Kan, G.J. Wilson, Addressing location uncertainties in GPS-based activity monitoring: a methodological framework. Trans. GIS 21(4), 764 (2016) 5. N. Wan, G.L. Kan, G.J. Wilson, Addressing location uncertainties in GPS-based activity monitoring: a methodological framework. Trans. GIS 21(4), 764 (2016) 6. S. Rungraengwajiake, P. Supnithi, S. Saito et al., Ionospheric delay gradient monitoring for GBAS by GPS stations near Suvarnabhumi airport, Thailand. Radio Sci. 50(10), 1076–1085 (2016) Suvarnabhumi airport, Thailand. Radio Sci. 50(10), 1076–1085 7. S. Alshorman, F.T. Jaber, F. Bensaali, A wireless oxygen saturation and heart rate monitoring and alarming system 7. S. Alshorman, F.T. Jaber, F. References Giil, H.-K. Song, An improved dual-hop transmission method for high reliability in wireless communica- tion networks. Wirel. Pers. Commun. 97(9), 1–10 (2017) Page 15 of 15 Xiong J Wireless Com Network (2021) 2021:163 Xiong J Wireless Com Network (2021) 2021:163 19. W. Li, Z. Qin, D. Xiaoli, D.W. Jiao, Y. Wentao, Z. Xianyun, GPS based on wireless communication network Antenna monitoring system and its application. J. Earth Sci. Environ. 31(3), 323–326 (2009) 19. W. Li, Z. Qin, D. Xiaoli, D.W. Jiao, Y. Wentao, Z. Xianyun, GPS based on wireless communication network Antenna monitoring system and its application. J. Earth Sci. Environ. 31(3), 323–326 (2009) 19. W. Li, Z. Qin, D. Xiaoli, D.W. Jiao, Y. Wentao, Z. Xianyun, GPS based on wireless communication network Antenna monitoring system and its application. J. Earth Sci. Environ. 31(3), 323–326 (2009) Publisher’s Note Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Música e seus efeitos sobre o cérebro: uma abordagem da neurociência junto a escolares
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Music and its effects on the brain: a neuroscience approach with students Abstract: With the aim to disseminate and popularize neuroscientific knowledge, the POPNEURO project, among its actions, sought to bring information about the effects of music in the brain to schoolchildren in public school system of the city of Uruguaiana-RS. This action was developed in 4 public schools of Uruguaiana during the year 2015 and was constituted of a theoretical explanation followed by a practical activity. To assess the impact of the action, pre and post-activity questionnaires were applied, both of which were composed of objective questions. The choice of this theme to work neuroscience in the school proved to be an effective strategy, allowing working several concepts of neuroscience. The results obtained in this study allow us to affirm that outreach activities fulfilled their role, allowing the scientific dissemination, bringing this science closer to the school, and minimizing the gap between the scientific environment and society. Keywords: Scientificdisclosure. Nervoussystem. Education. Música e seus efeitos sobre o cérebro: uma abordagem da neurociência junto a escolares Franciele Dornelles Casarotto1, Liane da Silva de Vargas², Pâmela B Mello-Carpes³ Resumo: Com objetivo de divulgar e popularizar conhecimentos neurocientíficos, o projeto POPNEURO buscou levar informações sobre os efeitos da música no cérebro a escolares da rede pública de ensino do município de Uruguaiana-RS. Essa ação foi desenvolvida em 4 escolas públicas do município durante o ano de 2015 e constituída de uma explanação teórica seguida por uma atividade prática. Para avaliar o impacto da ação, foram aplicados questionários pré e pós-atividades, ambos compostos por perguntas objetivas. A escolha dessa temática, para trabalhar a neurociência na escola, mostrou-se uma estratégia efetiva, possibilitando trabalhar diversos conceitos de neurociência. Os resultados obtidos neste estudo permitem afirmar que as atividades de extensão cumpriram seu papel, permitindo a divulgação científica, aproximando essa ciência da escola e minimizando o abismo entre o meio científico e a sociedade. Palavras-chave: Divulgação Cientifica. Sistema Nervoso. Educação. Palavras-chave: Divulgação Cientifica. Sistema Nervoso. Educação. p p ² Universidade Federal do Rio Grande do Sul. Doutoranda PPG Ciências Biológicas: Fisiologia. p j 24218441, E-mail: pamelacarpes@unipampa.edu.br 1 Universidade Federal do Pampa. Curso de Fisioterapia. Universidade Federal do Rio Grande do Sul. Doutoranda PPG Ciências Biológicas: Fisiologia. ³ Universidade Federal do Pampa. Professora adjunta. Lab de Neuroquímica. BR 472, km 592, CEP 97500-970, Cx postal 118, Uruguaiana/RS, Telefone: (55) 24218441, E-mail: pamelacarpes@unipampa.edu.br g g ³ Universidade Federal do Pampa. Professora adjunta. Lab de Neuroquímica. BR 472, km 592, CEP 97500-970, Cx postal 118, Uru 24218441, E-mail: pamelacarpes@unipampa.edu.br versidade Federal do Pampa. Curso de Fisioterapia. versidade Federal do Rio Grande do Sul. Doutoranda PPG Ciências Biológicas: Fisiologia. g g do Pampa. Professora adjunta. Lab de Neuroquímica. BR 472, km 592, CEP 97500-970, Cx postal 118, Uruguaiana/RS, Telefone: (55 melacarpes@unipampa.edu.br eral do Pampa. Curso de Fisioterapia. eral do Rio Grande do Sul. Doutoranda PPG Ciências Biológicas: Fisiologia. ² Universidade Federal do Rio Grande do Sul. Doutoranda PPG Ciências Biológicas: Fisiologia. ³ Universidade Federal do Pampa. Professora adjunta. Lab de Neuroquímica. BR 472, km 592, CEP 97500-970, Cx postal 118, Uruguaiana/RS, Telefone: (55) 24218441 E mail: pamelacarpes@unipampa edu br Música e seus efeitos sobre o cérebro: uma abordagem da neurociência junto a escolares Franciele Dornelles Casarotto1, Liane da Silva de Vargas², Pâmela B Mello-Carpes³ Música e seus efeitos sobre o cérebro: uma abordagem da neurociência junto a escolares Franciele Dornelles Casarotto1, Liane da Silva de Vargas², Pâmela B Mello-Carpes³ iversidade Federal do Pampa. Professora adjunta. Lab de Neuroq p p ² Universidade Federal do Rio Grande do Sul. Doutoranda PPG Introdução A neurociência busca compreender o sistema nervoso, sendo um campo que vem avançando em estudos e pesquisas que buscam esclarecimentos sobre o cérebro (LURIA 1981; LUNDY-EKMAN, 2008). Como uma das disciplinas mais dinâmicas e revolucionárias destas primeiras décadas do século 21, a neurociência ganha destaque pelos grandes benefícios oriundos de seus estudos e esclarecimentos, cativando públicos amplos com suas descobertas (LENT, 2010; ALVARENGA, 2012). Caracterizada como uma área interdisciplinar, essa ciência conta com diversas subáreas de estudos (LENT, 2010). Segundo Carvalho (2011), a neurociência cognitiva tem atenção prevalente ao estudo das capacidades mentais mais complexas, tais como a linguagem e a memória, de forma que entender como o cérebro funciona, especialmente no que diz respeito aos aspectos cognitivos, possibilita uma melhor compreensão de como se dá o processo de aprendizagem e, consequentemente, quais fatores podem influenciar positivamente na mesma. De acordo com Ratey (2001), no momento em que aprendemos sobre o funcionamento do cérebro, tornamo-nos ainda mais responsáveis na seleção de escolhas que possam maximizar o processo de aprendizagem. Da mesma forma, tornamo-nos aptos a evitar escolhas que possam prejudicar a mesma, fator este que contribui para otimização da construção do saber (RATEY,2001). A neuroeducação, área interdisciplinar que alia os conhecimentos entre neurociência, educação e psicologia (CONSENZA & GUERRA, 2011) é uma subárea da neurociência que busca potencializar a compressão e a adequação de práticas pedagógicas, fundamentando estratégias didáticas de acordo com o conhecimento já adquirido acerca do funcionamento do cérebro, permitindo o uso adequado de novas ferramentas para o ensino, um exemplo é a música. A música atua sobre o cérebro favorecendo o desenvolvimento cognitivo, linguístico, psicomotor, sócio afetivo e cultural dos envolvidos (BRÉSCIA, 2003; MUSZKAT, 2016). Estudos relacionados à música e a neurociência expandiram conhecimentos das bases neurobiológicas sobre como ocorre o processamento da música no cérebro, tendo como finalidade a compreensão de como a mente percebe, interpreta, apreende e comanda a música, além de buscar desvendar os processos envolvidos na percepção, aprendizagem e cognição musical (MUSZKAT, 2008). La música y sus efectos sobre el cerebro: Una perspectiva de la neurociencia con las escuelas Resumen: Con el fin de difundir y popularizar el conocimiento neurocientífico, el proyecto POPNEURO, entre sus acciones, ha tratado de llevar información sobre los efectos de la música sobre el cerebro a los estudiantes de escuelas públicas de la ciudad de Uruguaiana-RS. Esta acción se desarrolló en cuatro escuelas públicas de la ciudad durante el año 2015 y consistió en una explicación teórica seguida de una actividad práctica. Para evaluar el impacto de la acción fueron aplicados cuestionarios previos y posteriores a las 55 Revista ELO - Diálogos em Extensão Volume 06, número 02 - outubro de 2017 Revista ELO - Diálogos em Extensão Volume 06, número 02 - outubro de 2017 CASAROTTO, F.D. et al. actividades, ambos compuestos por preguntas objetivas. La elección de este tema, para trabajar la neurociencia en la escuela, ha demostrado ser una estrategia efectiva, lo que permite el trabajo de varios conceptos de la neurociencia. Los resultados de este estudio permiten afirmar que las actividades de extensión cumplieran su función, lo que permitirá la divulgación científica, acercándose la escuela de la ciencia y minimizando el espacio entre la comunidad científica y la sociedad. Palabras clave: Divulgación científica. Sistema nervioso. Educación. Palabras clave: Divulgación científica. Sistema nervioso. Educación. 56 Revista ELO - Diálogos em Extensão Volume 06, número 02 - outubro de 2017 Materiais e Métodos A fim de realizar o objetivo proposto foi criada uma ação, inclusa na metodologia do programa de extensão POPNEURO, que busca divulgar e popularizar conhecimentos básicos de neurociência junto à comunidade escolar do município de Uruguaiana – RS. O programa conta com 18 bolsistas e dois voluntários dos cursos de graduação em Educação Física, Enfermagem, Farmácia e Fisioterapia da Universidade Federal do Pampa – Campus Uruguaiana, além de uma aluna de doutorado e três docentes. A ação aqui relatada foi desenvolvida em 4 escolas públicas do município de Uruguaiana-RS durante o ano de 2015, atingindo 121 alunos com idades de 9 a 11 anos, sendo 56 meninos e 57 meninas, e 4 professoras da Educação Básica. A ação foi organizada em dois momentos, que são detalhados a seguir: (i) Explanação teórica: Com auxílio de projetor tipo datashow e utilizando uma apresentação de slides, foram trabalhadas questões como: (i) Explanação teórica: Com auxílio de projetor tipo datashow e utilizando uma apresentação de slides, foram trabalhadas questões como: • O que você sente ao escutar uma música? Falamos da interpretação da linguagem, sensação e percepção sonora; • O que você sente ao escutar uma música? Falamos da interpretação da linguagem, sensação e percepção sonora; • De que forma recebemos e captamos os sons? Falamos desde a captação dos sons por vibrações sonoras, gerada por meio do deslocamento de moléculas de ar, como a captação desses movimentos pelas células que recebem essas vibrações, localizadas em nosso ouvido interno; • Como nosso cérebro traduz essas informações? Elucidamos o trajeto dos estímulos sonoros, desde sua codificação pelos receptores auditivos até os centros em nosso SNC (córtex auditivo do lobo temporal) - essas informações foram demonstradas em conjunto com um vídeo. ; • Como o nosso cérebro percebe o ritmo, melodia e harmonia em uma canção? Explanamos que nosso córtex auditivo possui capacidade de decodificação da altura, timbre e ritmo, gerando informações para todo nosso cérebro por intermédio de circuitos que levam e trazem informações, ativando demais áreas do nosso encéfalo. Posteriormente foi realizada uma discussão para troca de ideias com os alunos, abordando os benefícios pertinentes à experiência musical, como a maior conectividade sináptica, maior ativação de áreas cerebrais que potencializam tanto as funções musicais como nossa capacidade linguística, funções cognitivas como a atenção e memória, assim como nossa linguagem corporal (LUNDY-EKMAN, 2008; MUSZKAT, 2008). Introdução A música desperta emoções complexas, tendo capacidade de ativar diferentes partes e funções do nosso cérebro, como sensopercepção musical e memórias, incluindo redes de recompensa (áreas do cérebro, como a área tegumentar ventral, que levam a liberação de dopamina no córtex pré-frontal, gerando uma sensação de bem-estar), processos sensório-motores e sensações prazerosas em decorrência da ativação do sistema límbico, responsável pela autorregulação emocional, o que pode explicar a riqueza única das emoções musicais (MUSZKAT, 2008; TROST et al, 2011). Esta pode ser um instrumento facilitador para reorganizar funções amplas, com impacto em atividades extramusicais, como a atenção, o planejamento e a memória. Vivenciada e presente em nosso dia a dia, de diversas maneiras, e, presente em diversas atividades coletivas na sociedade humana, a música se transforma em traço exclusivo dos seres humanos, junto à linguagem (MASZKUT, 2008). Diante dos conhecimentos adquiridos acerca dos benefícios da música para o cérebro e sua promissora ação no meio educacional, questiona-se: como esses conhecimentos chegam até a sociedade? Segundo Cavalcanti & Persechini (2011), a ciência precisa ser divulgada de maneira que ela não seja algo que só pode ser entendido por poucos, mas algo que está ao alcance de todos, a fim de que cada cidadão da sociedade possa basear-se na ciência para executar atividades diárias básicas e fazer suas escolhas. A divulgação científica é a janela que suplementa e aproxima a ciência e a sociedade, de maneira que permite uma conversação entre esses meios; nesse sentindo, popularizar a ciência é democratizar o acesso ao conhecimento científico (GERMANO, 2005). Com base nos fatos expostos, o objetivo deste trabalho foi levar informações neurocientíficas sobre os efeitos da música no cérebro à escolares da rede pública de ensino do município de Uruguaiana-RS, bem como avaliar o impacto dessas ações como ferramenta de divulgação e popularização da neurociência. Música e seus efeitos sobre o cérebro: uma abordagem da neurociência junto a escolares (ii) Atividade prática: Foi idealizada uma atividade lúdica e dinâmica abordando os assuntos trabalhados na teoria, como também a aproximação dos conhecimentos teóricos da prática/do dia a dia. Para tal, criou-se uma atividade que foi nomeada: “Ativando memórias”. A atividade buscou mostrar a capacidade de associação da linguagem musical com algo marcante vivenciado ao longo da nossa vida, em formato de jogo com perguntas e respostas. Os participantes foram expostos a diferentes Revista ELO - Diálogos em Extensão Volume 06, número 02 - outubro de 2017 Questões presentes apenas no questionário inicial aplicado aos estudantes: Questões presentes apenas no questionário inicial aplicado aos estudantes: 1. Você concorda que a música faz bem para o cérebro? 2. Você acha que a música afeta nossas emoções? Questões presentes no questionário aplicado às professoras após as açõ 1. Você já tinha conhecimento que a música faz bem para o nosso cérebro? j 2. Você já trouxe alguma atividade com música para seus alunos? j g 3. Você acha que a música pode trazer benefícios para as áreas de linguagem e cálculos matemáticos dos alunos? Fonte: Material produzido pelo programa (POPNEURO, 2015). 58 Revista ELO - Diálogos em Extensão Volume 06, número 02 - outubro de 2017 Materiais e Métodos Para crianças de 10 anos, ao referir a essa música, geralmente, ocorre evocação de memórias do filme “Frozen”, seus personagens, a fatos relacionados a esses ou situações que vivenciaram ouvindo a música; III. No projetor utilizou-se uma representação das possíveis repostas da atividade Para avaliar o impacto desta ação, foram aplicados questionários aos alunos, sendo um antes da atividade e outro após, ambos em forma de perguntas objetivas, com respostas “sim” ou “não”, com exceção de uma questão aberta (quadro 1). Também foi aplicado um questionário aos professores, a fim de verificar sua percepção sobre a ação. Os resultados são apresentados na forma de frequência relativa (%). Quadro 1 - Questões propostas nos questionários. Materiais e Métodos Toda a atividade foi desenvolvida de forma interativa, interrogativa e dialogada, buscou-se fazer com que os escolares fossem membros ativos na construção de seus conhecimentos (figura 1). Figura 1 - Explanação teórica acerca da música e o cérebro para estudantes. Fonte: Material produzido pelo programa (POPNEURO, 2015). Figura 1 - Explanação teórica acerca da música e o cérebro para estudantes. Fonte: Material produzido pelo programa (POPNEURO, 2015). (ii) Atividade prática: Foi idealizada uma atividade lúdica e dinâmica abordando os assuntos trabalhados na teoria, como também a aproximação dos conhecimentos teóricos da prática/do dia a dia. Para tal, criou-se uma atividade que foi nomeada: “Ativando memórias”. A atividade buscou mostrar a capacidade de associação da linguagem musical com algo marcante vivenciado ao longo da nossa vida, em formato de jogo com perguntas e respostas. Os participantes foram expostos a diferentes 57 Revista ELO - Diálogos em Extensão Volume 06, número 02 - outubro de 2017 Revista ELO - Diálogos em Extensão Volume 06, número 02 - outubro de 2017 CASAROTTO, F.D. et al. melodias pré-selecionadas relacionadas a memórias episódicas e/ou semânticas (fatos ou conhecimentos supostamente marcantes de acordo com a sua faixa etária), incluindo filmes/animações marcantes, desenhos animados, jogos e/ou séries. A música era ouvida pelos participantes permitindo um tempo de associação ao fato, logo era discutido com os alunos como e por que relacionamos a música a uma memória prévia, como no exemplo a seguir: melodias pré-selecionadas relacionadas a memórias episódicas e/ou semânticas (fatos ou conhecimentos supostamente marcantes de acordo com a sua faixa etária), incluindo filmes/animações marcantes, desenhos animados, jogos e/ou séries. A música era ouvida pelos participantes permitindo um tempo de associação ao fato, logo era discutido com os alunos como e por que relacionamos a música a uma memória prévia, como no exemplo a seguir: I. Selecionou-se a música “Let It Go - Demi Lovato”; II. Durante a execução da música, foi solicitado aos participantes que relacionassem à qual memória foi remetida. Para crianças de 10 anos, ao referir a essa música, geralmente, ocorre evocação de memórias do filme “Frozen”, seus personagens, a fatos relacionados a esses ou situações que vivenciaram ouvindo a música; II. Durante a execução da música, foi solicitado aos participantes que relacionassem à qual memória foi remetida. Fonte: Material produzido pelo programa (POPNEURO, 2015). Quadro 1 - Questões propostas nos questionários. Quadro 1 Questões propostas nos questionários. Questões presentes apenas no questionário inicial aplicado aos estudantes: Opções de resposta 1. Você gosta de ouvir músicas?2. Você sabe tocar algum instrumento musical? Se sim, qual? ___________ Sim/NãoSim/Não Questões presentes nos questionários aplicados aos estudantes pré e pós-ação: 1. Você concorda que a música faz bem para o cérebro? 2. Você acha que a música afeta nossas emoções? Sim/NãoSim/Não Questões presentes no questionário aplicado às professoras após as ações: 1. Você já tinha conhecimento que a música faz bem para o nosso cérebro? 2. Você já trouxe alguma atividade com música para seus alunos? 3. Você acha que a música pode trazer benefícios para as áreas de linguagem e cálculos matemáticos dos alunos? Sim/NãoSim/NãoSim/Não Resultados e Discussões A escolha da temática “música”, para trabalhar a neurociência na escola, mostrou-se uma estratégia efetiva, pois os estudantes revelaram-se interessados, questionadores e participativos. Além disso, o tema permitiu que diversos conceitos de neurociência fossem trabalhados a partir dele, a citar: receptores sensoriais auditivos, percepção auditiva, áreas cerebrais envolvidas com a interpretação auditiva, memória, cognição e emoção, entre outros. Cavalcanti & Persechinii (2011) relatam que, associar a ciência a situações do cotidiano, torna-a mais fácil de ser compreendida. Nossa ação buscou divulgar a neurociência associando aà uma temática presente no dia a dia dos sujeitos, o que tornou a atividade mais prazerosa e o tema de fácil entendimento. Na avaliação inicial verificamos que 96,6% dos estudantes gostam e possuem o hábito de ouvir música e 56,2% destses tocam ou já tocaram algum tipo de instrumento, como violão (37,3%), flauta (20%), bateria (16%) e/ou outros (26,7%). Percebemos que a ação foi efetiva em aumentar os conhecimentos dos estudantes acerca desta temática. Ao analisar os resultados podemos mensurar a visão dos escolares quantos aos benefícios da música ao cérebro, percebendo que, embora alguns já tivessem uma ideia dos efeitos da música sobre o cérebro, as ações qualificaram seus conhecimentos e ampliaram o percentual de estudantes que consideram que a música faz bem para o cérebro (de 82% para 94,1%) e que ela afeta nossas emoções (de 77% para 95%) (figura 2). Considerando que as atividades realizadas objetivaram popularizar a neurociência, e, com isso, despertar o gosto científico nos escolares, foram criadas atividades interativas, podendo ser visível o seu gosto na participação durante as ações. Para Vargas et al (2014), ações de divulgação da neurociência tornam-se importantes na formação do aluno, uma vez que essas permitem atingir o objetivo de popularizar conceitos científicos, além de promover uma aproximação construtiva do aluno com a universidade. 58 Revista ELO - Diálogos em Extensão Volume 06, número 02 - outubro de 2017 Música e seus efeitos sobre o cérebro: uma abordagem da neurociência junto a escolares Figura 2 - Opinião dos estudantes da Educação Básica sobre os efeitos da música sobre o cérebro antes e após a ação. Fonte: Material produzido pelo programa (POPNEURO 2015) Figura 2 - Opinião dos estudantes da Educação Básica sobre os efeitos da música sobre o cérebro antes e após a ação. l d d l ( ) Fonte: Material produzido pelo programa (POPNEURO, 2015). Fonte de Financiamento O projeto recebeu financiamentos da Pró-Reitoria de Extensão da Universidade Federal do Pampa por meio de editais de fomento à extensão (PROEXT/UNIPAMPA), do Ministério da Educação, mediante Edital PROEXT/MEC 2015, e da Coordenação de Aperfeiçoamento de Pessoal do Ensino Superior (CAPES), pelo Edital Novos Talentos/CAPES e, em cooperação com o British Council (Newton Fund), do Edital de Cooperação Internacional STEM. Resultados e Discussões A neurociência, quando aplicada à educação, visa elucidar os fatores intrínsecos e extrínsecos que interferem o processo educacional, fortalecendo a ligação entre educação e neurociência e agregando conhecimentos importantes tanto para o estudante como para o professor (BARRETA NETA, 2009). Nestse sentido, procuramos avaliar, também, a percepção dos professores das turmas sobre a temática trabalhada junto aos estudantes. Sobre o conhecimento a respeito do tema abordado, 100% dos professores afirmaram que já haviam proporcionado atividades com uso de música aos seus alunos e que sabiam que a música pode estimular áreas cerebrais relacionadas à linguagem e cálculo matemático. Foi possível perceber que os professores aprovaram a ação, visto que atribuíram nota de 9,75 ± 3,12 (considerando uma escala de 0 a 10) à atividade. Conclusão Os resultados obtidos neste estudo permitem afirmar que as atividades de extensão propostas cumpriram seu papel de levar informações neurocientíficas sobre os efeitos da música no cérebro a escolares, tendo um impacto positivo sobre os conhecimentos dos estudantes acerca desta temática. Além disso, as ações permitiram a divulgação e popularização da neurociência, aproximando esta ciência da escola, minimizando o abismo entre o meio científico e a sociedade. O uso de um tema de interesse dos estudantes, como a música, é, portanto, uma estratégia interessante para a divulgação da neurociência na escola. Revista ELO - Diálogos em Extensão Volume 06, número 02 - outubro de 2017 Agradecimentos Os autores agradecem a receptividade da direção, professores e alunos das escolas estaduais Hermeto José Pinto Bermudez, Dr. Roberval Behegaray Azevedo e Cândido Rondon, e da escola 59 Revista ELO - Diálogos em Extensão Volume 06, número 02 - outubro de 2017 Revista ELO - Diálogos em Extensão Volume 06, número 02 - outubro de 2017 CASAROTTO, F.D. et al. municipal Marechal Humberto Castelo Branco, que participaram das ações, bem como aos demais alunos de graduação envolvidos na execução das atividades aqui relatadas. 60 Revista ELO - Diálogos em Extensão Volume 06, número 02 - outubro de 2017 municipal Marechal Humberto Castelo Branco, que participaram das ações, bem como aos demais alunos de graduação envolvidos na execução das atividades aqui relatadas. Referências ALVARENGA, S. P. Contribuição da neurociência no processo de ensino aprendizagem em alunos com para- lisia cerebral. 2012. 39 f. Monografia de conclusão de curso (Especialização)– Faculdade Integrada, Universidade Cândido Mendes, Rio de Janeiro, 2012. BARRETO NETA, L. Formação de professor: de aprendente a ensinante. Construção psicopedagógica,v. 17, n. 15, p. 37-55, 2009. BRÉSCIA, V. L. Educação Musical: bases psicológicas e ação preventiva. São Paulo: Átomo, 2003. CARVALHO, F. A. H. Neurociências e educação: uma articulação necessária na formação docente. Trabalho Educação e Saúde, Rio de Janeiro, v. 8, n. 3, p. 537-550, nov. 2010/ fev. 2011. CAVALCANTI, C.; PERSECHINII, M. P. Museus de ciência e a popularização no Brasil. Field Actions Science Reports, 3, 1-10; 2011. CONSENZA, Ramon .M.; GUERRA, Leonor. B. Neurociência e educação: como o cérebro aprende. Porto Alegre: Artmed, 2011. GERMANO, M. Popularização da ciência como ação cultural libertadora. Em: Universidade Federal de Pernambuco, V Colóquio Internacional Paulo Freire: Desafio à Sociedade Multicultural. pp. 4-12. Recife: UFPE, 2005. LENT, R. Cem bilhões de neurônios?:conceitos fundamentais de neurociência/ Roberto Lent. 2ed.São Paulo: Atheneu, 2010. LUNDY-EKMAN L.; Neurociências: para reabilitação, 3ed, Rio de Janeiro-RJ,Elsevier Lt LURIA, A. R. Fundamentos de Neuropsicologia, RJ, Livros Técnicos e Científicos; tradução de Ricardo Juarez Aranha da edição da Penguin Books (Middlesex, 1973) São Paulo: EDUSP, 1981. MUSZKAT, M. Música, neurociência e desenvolvimento humano. Ministério da cultura e vale apresen- tam, p. 67, 2007 – JAZEN. MUSZKAT. M, Entrevista Transformação pela música. Ciência Hoje pág. 323, vol. 54. Disponível em: <http://cercor.oxfordjournals.org/content/early/2011/12/15/cercor.bhr353.full.pdf+html>. Acesso em: 19 set. 2016. RATEY, John J. O cérebro: um guia para o usuário. Rio de Janeiro: Objetiva, 2001. VARGAS, L. S.; MENEZES, J.; ALVES, N.; SOSA, P.; MELLO-CARPES, P.B. Conhecendo o sistema nervoso: ações de divulgação e popularização da neurociência junto a estudantes da rede pública de educa- ção básica. Revista Ciências e Cognição, v. 19, n. 2, p. 233-241, 2014. Recebido para publicação em 7/1/2017 e aprovado em 12/6/2017. 60 Revista ELO - Diálogos em Extensão Volume 06, número 02 - outubro de 2017 60 Revista ELO - Diálogos em Extensão Volume 06, número 02 - outubro de 2017
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مسؤولية الأطفال غير المميزين التقصيرية في قانون المعاملات المدنية العماني مقارنة بالشريعة الإسلامية
Maǧallaẗ al-ʿulūm al-tarbawiyyaẗ wa-al-dirāsāt al-insāniyyaẗ Silsilaẗ al-ādāb wa-al-ʿulūm al-tarbawiyyaẗ wa-al-insāniyyaẗ wa-al-taṭbīqiyyaẗ
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Dr. Abdullah bin Ali bin Salim Al Shibili Faculty of low - Sahar University - Sultanate of Oman The responsibility of children who are not discriminating in the Omani Civil Procedure law compared to Islamic law Key Words: Tort liability; not discriminated children; Civil Procedure law ISSN: 2617-5908 مسؤولية األطفال غير المميزين التقصيرية في قانون المعامالت المدنية العماني مقارنة بالشريعة اإلسالمية(  ) إعداد مجلة العلوم التربوية والدراسات اإلنسانية /د عبدهللا بن علي بن سالم الشبلي أستاذ القانون المدني ال مساعد بكلية القانون جامعة صحار– سلطنة عمان ISSN: 2617-5908 مسؤولية األطفال غير المميزين التقصيرية في قانون المعامالت المدنية العماني مقارنة بالشريعة اإلسالمية(  ) إعداد مجلة العلوم التربوية والدراسات اإلنسانية /د عبدهللا بن علي بن سالم الشبلي أستاذ القانون المدني ال مساعد بكلية القانون جامعة صحار– سلطنة عمان مجلة العلوم التربوية والدارسات اإلنسانية 312 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م * )-تاريخ تسليم البحث22 /4 / 2222م تاريخ قبوله للنشر02 /5 / 2222 .م :ملخص البحث ال هددد : هدد البحددا الحددإلى الددو اللىددل ليددو طفدد للر امميددإ ردد الططردد ر التقصر ر فى ىإنل اإلج اءات الط نر العطإنى طقإ ن بإلشد رع اإلفديطر و لطد جد تحقرددا الفدد الدد برث ليبحددا تدد االمددي ليددو ام برددإت لالطددلا القإنلنردد ةات الصددي بطلضددل البحددا المددنه : تدد اتبددإ الطددنفا الل صدديى القددإنلنى التحيريددى الطتط دد فددى طجطلل اإلجد اءات البح رد التدى تتلإطد للصد ال دإه ل ل الطلضدل التطدإ ا ليدو جطد الحقدإبا لالبرإندإت لتصدنريفإ لطعإلجتفددإ لتحيريفدإ تحيدري لإفردإ ل ىرقدإ الفددت ي اللتفإ لاللصل الو نتإبا ل تعطرطإت لد ال دإه ل ل الطلضدل طحد البحد ا؛ لهدل طددإ تدد تمبرقددج فددى طجددإ البحددا حددل طفدد للر امميددإ ردد الططردد ر التقصددر ر فددى ىإنل اإلج اءات الط نر العطإنى طقإ ند بإلشد رع اإلفديطر النتداج : فد ت النتدإبا ل لجل تبإر بر طف للر امميإ ر الططرد ر التقصدر ر فدى ىدإنل اإلجد اءات الط نر العطدإنى ط قإ ند بإلشد رع اإلفديطر للدةلا ليإرد النصدل القإنلنرد الطتعيقد بطف للر امميإ ر الططر ر التقصر ر فى ىدإنل اإلجد اءات الط نرد العطدإنى التدى تع جإب ل ليض الةي ى ريحا بإلطض ل التوصيات: رلصدى البإحدا ب أهطرد تللرد الطجتط لت قريج حل ىإنل اإلج اءات ال ط نر تجإه الغر حيإ إ ليو الططتيلدإت العإطد لال إصدد طدد لبددا امميددإ ردد الططردد ر لةلددا طدد ددي نشدد القددلانر الطتعيقدد بطف للر امميإ ر الططر ر التقصر ر فى الصح الطحير للب لفإب التلاصد االجتطإلى الطتنلل :كلمات المفتاحية الطف للر التقصر ر- امميإ ر الططر ر- القإنل الط نى مجلة العلوم التربوية والدارسات اإلنسانية 312 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م Abstract: The purpose of the current research is to examine the responsibility of children who are not discriminated in the Omani civil procedure law compared to Islamic law. In order to achieve the main objective of the research, the research has examined the related literature and legal articles to the research. The research have used the analytical descriptive approach to compare the Omani law and the Islamic Sharyiah. The research have found that this research is the latest in the field of tort liability.The results showed that there is no difference between the responsibility of children who are not discriminating in the Omani civil procedure law compared to Islamic law. As well as the adequacy of the legal provisions on the responsibility of children who are not privileged in the Omani Civil Procedure Code, which is considered to be harmful to the injured. The researchers recommend for the importance of educating the community about the civil procedure law in order to protect public and private property from tampering with non-privileged children by publishing laws concerning the responsibility of non-discriminating children in local newspapers and through various social media. p p g Key Words: Tort liability; not discriminated children; Civil Procedure law law مجلة العلوم التربوية والدارسات اإلنسانية 315 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م مجلة العلوم التربوية والدارسات اإلنسانية 315 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... Abstract: د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م اإلطار العام للبحث: مننا اومننور المسننلم باننا اقاننا نقانونننا أن جميننع أاعنناف البشننر علنن ا ننت أنشننطتام المختلفننةو نمننا ينننت عناننا مننا اقننو نالتصامنناوأ تا ننق صننورا نأدننكا قانونيننة متعنند تحتاج إل مصيد ما التمحيص نالتدقيق نإعماف الفكر للوصنوف إلن رأق قنانوني صنا يكفننل لليميننع اقننوقامأ نمننا بننيا تلننا القيننايا القانونيننة التنني أًننارو جنند قانونيننا عبننر العصننور المختلفننة بننيا مختلننم النندنف مننا يسننم بالمسننلنلية التقصننيرية لعننديم التمييننصأ صوصا الفعل اليار نأاكامنهو ننجنوت تعنويل المينرنر عمنا لحنق بنه منا نرر التعننويل العننا فو نلعننل ذلننا الينندف يعننو إلنن اوسننا الننقق تقننوم عليننه المسنننلنلية التقصيريةأ ااناك ما أسساا عل أسا ادنث اليررو نآ نرنن أسسنو ا علن أسنا ارتكات الفعل الخاطئ أن الخطاو نقد توصل اقااء القنانون إلن أن المسنلنلية التقصنيرية لعديم التمييصأ ترتكص عل ً ًة أركان ن ي: الفعل اليار (الخطنا،و نالينررو نالع قنة السببيةو نتوصلوا إل أن ذلا اليدف يدنر اوف الفعل اليار (الخطا،و نالقق يمثنل عبنر التاريخ اإلنساني اوسا التقليدق للمسنلنلية المدنينة بشنكل عنامو نالمسنلنلية التقصنيرية .لعديم التمييص عل نجه الخصوص نسلطنة عمان كغير ا ما نف العالم لم يغفل اياا المشرَّ ع العماني عا الحديث اوف ( مسلنلية عديم التمييص التقصيريةو ننطا قه المسنلنليةأ إذ تننص المنا071 / 0 ، منا قانون المعام و المدنية العماني الصا ر بالمرسنوم السنلطاني رقنم22 / 2202 م علن أن "كننل إ ننرار بننالغير يلننصم ااعلننه نلننو كننان ويننر مميننص بننالتعويل" نبننالوقو علنن ننقه الما القانونية يتبيا أن ه يشنترط اني قينام المسنلنلية تنواار ً ًنة أركنان تتمثنل اني الفعنل ( اليارو ناليررو نالع قة السببية ما بيا الفعنل نالينررو نأدنارو المنا42 / 2 ، منا ذاو القانون أن "سا التمييص سبع سنيا كاملة" ن و ما يعني أن ما نن السنبع سننيا اانو وير مميصأ ذلا أن المشرع العماني قد ار ب يا سا التمييص ناقا للما آنفة النقكرو نسنا ( الرددأ إذ اد و الما40 / 2 ."،أن "سا الردد إتمام الثامنة عشر ما العمر كما أن المشرع العمناني اسنتخدم مصنطلا "التعنويل" اني انيا أن بعنل القنوانيا استعملت المصطلا المستخدم ما قبل الفقه ن و "اليمان"و نالنقق يععنرَّ با ننه تعنويل عا رر أن إلصام بتعويل عا ررأ كما أن ناك الفاظا أ رى مناا " التعدق" نلفظ "الخطا" ن قا ا ت اي المصطلحاو القانونية اي مياف المسلنلية التقصنيرية لعنديم التميينصأ قنند يفسنا المينناف أمننام اقاناء القننانون إلن طننرت الكثيننر منا التسننا و المنطقيننة مناا: ل است خدام أيا ما تلا المصنطلحاو قند ينل ق إلن نجنو ارنقناو اني الحكنم بنيا تلا القوانيا أم ؟ نأق ما تلا اولفاظ أدمل ما او رى أم أناا بقاو المعن ؟ نما مندى إمكانيننة إقامننة مسننلنلية عننديم التمييننص عننا اعلننه اليننار مننا عنندماا؟ نمننا منندى كفايننة النصوص القانونية اني ميناف المسنلنلية ا لتقصنيرية لعنديم التميينص اني قنانون المعنام و .المدنية اي سلطنة عمان نسلطنة عمان كغير ا ما نف العالم لم يغفل اياا المشرَّ ع العماني عا الحديث اوف ( مسلنلية عديم التمييص التقصيريةو ننطا قه المسنلنليةأ إذ تننص المنا071 / 0 ، منا قانون المعام و المدنية العماني الصا ر بالمرسنوم السنلطاني رقنم22 / 2202 م علن أن "كننل إ ننرار بننالغير يلننصم ااعلننه نلننو كننان ويننر مميننص بننالتعويل" نبننالوقو علنن ننقه الما القانونية يتبيا أن ه يشنترط اني قينام المسنلنلية تنواار ً ًنة أركنان تتمثنل اني الفعنل ( اليارو ناليررو نالع قة السببية ما بيا الفعنل نالينررو نأدنارو المنا42 / 2 ، منا ذاو القانون أن "سا التمييص سبع سنيا كاملة" ن و ما يعني أن ما نن السنبع سننيا اانو وير مميصأ ذلا أن المشرع العماني قد ار ب يا سا التمييص ناقا للما آنفة النقكرو نسنا ( الرددأ إذ اد و الما40 / 2 ."،أن "سا الردد إتمام الثامنة عشر ما العمر كما أن المشرع العمناني اسنتخدم مصنطلا "التعنويل" اني انيا أن بعنل القنوانيا استعملت المصطلا المستخدم ما قبل الفقه ن و "اليمان"و نالنقق يععنرَّ با ننه تعنويل عا رر أن إلصام بتعويل عا ررأ كما أن ناك الفاظا أ رى مناا " التعدق" نلفظ "الخطا" ن قا ا ت اي المصطلحاو القانونية اي مياف المسلنلية التقصنيرية لعنديم التميينصأ قنند يفسنا المينناف أمننام اقاناء القننانون إلن طننرت الكثيننر منا التسننا و المنطقيننة مناا: ل است خدام أيا ما تلا المصنطلحاو قند ينل ق إلن نجنو ارنقناو اني الحكنم بنيا تلا القوانيا أم ؟ نأق ما تلا اولفاظ أدمل ما او رى أم أناا بقاو المعن ؟ نما مندى إمكانيننة إقامننة مسننلنلية عننديم التمييننص عننا اعلننه اليننار مننا عنندماا؟ نمننا منندى كفايننة النصوص القانونية اني ميناف المسنلنلية ا لتقصنيرية لعنديم التميينص اني قنانون المعنام و .المدنية اي سلطنة عمان مجلة العلوم التربوية والدارسات اإلنسانية 312 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... Abstract: ن ره ا ن إل كنقا أليناه إلينهو نيرجنع :أصننل الكلمننة إلنن ً ًننة معننانا نني: نن النفننع ناجتمنناع الشننيءو نالقننو امننا اونف اليننرر بمعننني الاعننصاف نيكننون باليننم نإذا كننان نند النفننع يكننون بننالفتا نانني التنصيننل «مسني الير » أق المرض نا سم اليرر. نما الثاني "اجتماع الشنيء" دنا نر أق ذاو لننبا ن ننر اللحننم الميتمننع تحتاننا ن مننا الثالننث "القننو " اليننرير ن ننو قننوق النننف ايقاف: ا ن ذن رير عل الشيء إذا كان ذا صير نمقاسا نا ن نرير أت بنه نرر . ما ذ ات عيا أن ن كما يقصد باليرر ما النااية اللغوية: كل ما و ند النفنعو نالينرم بمعنن الانصاف نسوء الحاف نما نا أتت المْيرَّ و ن ي المنفعة ( .السيوطي0292 : 94 .، كما يقصد باليرر ما النااية اللغوية: كل ما و ند النفنعو نالينرم بمعنن الانصاف نسوء الحاف نما نا أتت المْيرَّ و ن ي المنفعة ( .السيوطي0292 : 94 .، نما ف ما تقدم يمكا القوف أن.اليرر د النفعو ن و بمعن اوذى أن المكرنه أما ما النااية القانونية اكلمة رر نر و بعد تعريفاو منانا: اوذ ى أن الخسنار التني ا ع ة أ اق ف ة إ ال أ ال ّ ع ل ال ا كما يقصد باليرر ما النااية اللغوية: كل ما و ند النفنعو نالينرم بمعنن الانصاف نسوء الحاف نما نا أتت المْيرَّ و ن ي المنفعة ( .السيوطي0292 : 94 .، نما ف ما تقدم يمكا القوف أن.اليرر د النفعو ن و بمعن اوذى أن المكرنه أما ما النااية القانونية اكلمة رر نر و بعد تعريفاو منانا: اوذ ى أن الخسنار التني تعصي الشّخصْ اي جسمه أن ماله نتيية إ ف تعاقدقّ أن جريمنةو ممّنا يعيينص لنه التمنا التَّعويل بدعوى مدنيّة (القنونو0220 .، كما أن كلمة رر تعني: ا عتداء أن اوذى القق يصي اإلنسان بحق ما اقوقه أن اني مصلحة مشرنعة له سواء كان ذلا الحق أن المصنلحة متعلقنة بسن مة جسنمه أن عاطفتنه أن بماله أن بشراه أن باعتباره نسواء كان قا الحق أن ال مصلحة ذا قيمة مالينة أن لنم يكنا كقلا (أبو السعو و0294 : 220 .، (، نعلنن ذلننا االيننرر مننا الناايننة القانونيننة ننو: اوذى الننقق يصنني الشننخص نتييننة المسا بمصلحة منا مصنالحه المشنرنعة أن انق منا اقوقنه. Abstract: 2 - .التعريم باليررو نالخطاو نالتعدق ما النوااي اللغويةو نالشرعية نالقانونية 2 - .التحقق ما مدى نجو ارن جو رية بيا المسئوليتيا المدنية نالينا ية 4 - تو يا الدنر القانوني القق تقوم به .المحكمة العليا اي سلطنة عمان :أهمية البحث تلطدد هطردد هددةا البحددا للنددج رفدديم الضددلء ليددو طفدد للر امميددإ ردد الططردد ر التقصر ر فى ىإنل الطعإطيت الط نر بفيمن لطإ طقإ ن بإلش رع اإلفيطر :مه البحث ت افت ا الطنفا القإنلنى اللصيى التحيريى: لهل لبإ ل لد طجطللد اإلجد اءات البح ر التى تتلإط للص ال إه ل ل الطلضل التطإ ا ليو جطد الحقدإبا لالبرإندإت لتصددنريفإ لطعإلجتفددإ لتحيريفددإ تحيددري لإفرددإ ل ىرقددإ الفددت ي اللتفددإ لاللصددل الددو نتإبا ل تعطرطإت ل ال إه ل ل الطلضل طح البحا ؛ لهل طإ تد تمبرقدج فدى طجدإ البحا حل طف للر امميدإ رد الططرد ر التقصدر ر فدى ىدإنل اإلجد اءات الط نرد العطإنى طقإ ن بإلش رع اإلفيطر ( كمننا يعننر مصننطف و نآ ننرنن2224 ، اليننرر لغننة بانننه: نند النفننع يقنناف: ننرَّه ييرعه عرَّا ن رر ا: ألحق به مكرن ا أن أذى . ن ره ا ن إل كنقا أليناه إلينهو نيرجنع :أصننل الكلمننة إلنن ً ًننة معننانا نني: نن النفننع ناجتمنناع الشننيءو نالقننو امننا اونف اليننرر بمعننني الاعننصاف نيكننون باليننم نإذا كننان نند النفننع يكننون بننالفتا نانني التنصيننل «مسني الير » أق المرض نا سم اليرر. نما الثاني "اجتماع الشنيء" دنا نر أق ذاو لننبا ن ننر اللحننم الميتمننع تحتاننا ن مننا الثالننث "القننو " اليننرير ن ننو قننوق النننف ايقاف: ا ن ذن رير عل الشيء إذا كان ذا صير نمقاسا نا ن نرير أت بنه نرر . ما ذ ات عيا أن ن كما يقصد باليرر ما النااية اللغوية: كل ما و ند النفنعو نالينرم بمعنن الانصاف نسوء الحاف نما نا أتت المْيرَّ و ن ي المنفعة ( .السيوطي0292 : 94 .، نما ف ما تقدم يمكا القوف أن.اليرر د النفعو ن و بمعن اوذى أن المكرنه أما ما النااية القانونية اكلمة رر نر و بعد تعريفاو منانا: اوذ ى أن الخسنار التني تعصي الشّخصْ اي جسمه أن ماله نتيية إ ف تعاقدقّ أن جريمنةو ممّنا يعيينص لنه التمنا التَّعويل بدعوى مدنيّة (القنونو0220 .، ( كمننا يعننر مصننطف و نآ ننرنن2224 ، اليننرر لغننة بانننه: نند النفننع يقنناف: ننرَّه ييرعه عرَّا ن رر ا: ألحق به مكرن ا أن أذى . Abstract: أمنا منا النااينة الشنرعية ٌاكلمة رر لاا عد تعريفاو نمناا: أن يعد ٌ لْ عل وير ه ررا بمنا ينتفنع نو بنه ( ابنا رج و2220 : 217 .، ( كما يعر منوااي0227 : 27 / 0 ، الينرر اقانا باننه "اإل ن ف ." بمصلحة مشرنعة للنف أن الغير تعديا أن تعسفا أن إ ما نبناء عل ما تم عر ه ما ن ف التعريفناو السنابقة للينرر منا الننوااي اللغوينة نالقانونية نالفقاية يمكا القوف أن تلا التعريفاو أكدو عل أن الينرر ركنا أسنا منا أركننان المسننلنلية المدنيننة بصننور عامننة نانني مينناف المسننلنلية التقصننيرية علنن نجننه الخصننوصأ إذ جننداف أن نن انني ادننتراط نجننو هأ ون المسننلنلية تعننني التصامننا بننالتعويلو نالتعننويل يقنندر بقنندر اليننرر نبانتفا ننه تنتفنني الم سننلنليةو ن يظننل محنن للتعويل ن تكون لمدعي المسلنلية مصلحة اي إقامة الدعوى. مجلة العلوم التربوية والدارسات اإلنسانية 312 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م ي م نالقانونية نالفقاية يمكا القوف أن تلا التعريفاو أكدو عل أن الينرر ركنا أسنا منا أركننان المسننلنلية المدنيننة بصننور عامننة نانني مينناف المسننلنلية التقصننيرية علنن نجننه الخصننوصأ إذ جننداف أن نن انني ادننتراط نجننو هأ ون المسننلنلية تعننني التصامننا بننالتعويلو نالتعننويل يقنندر بقنندر اليننرر نبانتفا ننه تنتفنني الم سننلنليةو ن يظننل محنن للتعويل ن تكون لمدعي المسلنلية مصلحة اي إقامة الدعوى. ناليرر– كما و معلوم- الركا الثاني اي المسلنلية المدنية ا يكتف لتحققانا أن يقنع الخطا ما المتسب بل يي أن يسنب الخطنا نررا محققنا نبيننا و نالمينرنر نو النقق يكلم بإًباو اليرر ال قق نقع عليه بطر اإلًباو المعلومة قانونا أ ونه و القق يدعيه. الخطا ما النوااي اللغويةو نالقانونيةو نالشرعية: :الخطأ لغة الخطا نالخطاء: ند الصنواتو نأْ طْناْ الطَّرٌ ينقْ: عْندْف عننه. نأْ طْناْ الرَّامٌني ْ الغْرْ ضْ : لم يعصٌ ب هو نأْ طْاْ نْو عه إٌذا طْلْن :ااجتْنهو النم يْنن يْا و نلنم يعصٌ ن دنيئا . نالخٌ ط ناْ ع أْرض يعخ طٌئاا المطر نيعصٌ ني ع أع نرى قعر بْانا. نأْ طْناْ يعخ طٌنئع إٌذا سْنلْاْ سْنبيلْ الخْطْناْ عْم ندا ي ي ناليرر– كما و معلوم- الركا الثاني اي المسلنلية المدنية ا يكتف لتحققانا أن يقنع الخطا ما المتسب بل يي أن يسنب الخطنا نررا محققنا نبيننا و نالمينرنر نو النقق يكلم بإًباو اليرر ال قق نقع عليه بطر اإلًباو المعلومة قانونا أ ونه و القق يدعيه. الخطا ما النوااي اللغويةو نالقانونيةو نالشرعية: َّ ي :الخطأ لغة الخطا نالخطاء: ند الصنواتو نأْ طْناْ الطَّرٌ ينقْ: عْندْف عننه. Abstract: د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م ب تتمثل مشكلة البحث الحالي اي الوقنو علن النصنوص القانونينة المتعلقنة بمسنلنلية اوطفاف وير المميصيا التقصيرية اي قانون اإلجراءاو المدنية العماني مقارننة بالشنريعة اإلس مية و نمدى الحاجة إل إعا النظر اي تلا الموا ما عدمااأ ما ايث التعنديل أن التطوير ناقا للتطور الحينارق النقق تشناده سنلطنة عمنانأ نمنا دنمله ذلنا التطنور منا تغيراو سلوكيةو نإنسانيةو ناقتصا يةو نًقاايةو ناكريةو ن ينية. :أسجلة البحث إآ :رحإل البحا الحإلى اإلجإب ليو الف ا ال برث اآلتى طددإ جدد التبددإر بددر الشدد رع اإلفدديطر لىددإنل الطعددإطيت الط نردد بفدديمن لطددإ فددى طجإ الطف للر امميإ ر الططر ر التقصر ر فى ىإنل اإلج اءات الط نر العطإنى طقإ ن بإلش رع اإلفيطر ؛ حرا انب قت طنج ام: فبي اآلتر 1 - بطإةا رع َّ الض و لال مأو لالتع ي ط النلاحى اليغلر و لالش لر لالقإنلنر ؟ 2 - ه تلج ف لا جله ر بر الطفبللرتر الط نر لالجنإبر ؟ 3 - طإ ال ل القإنلنى الةي تقل بج الطحلط العيرإ فى فيمن لطإ ؟ أ:هداف البحث تتمثل أ دا ا لبحث الحالي اي: 0 - المقارنة بيا النصوص القانونية المتعلقة بمسلنلية اوطفناف وينر الممينصيا التقصنيرية اي قانون اإلجراءاو المدنية العماني ب الشريعة اإلس ميةأ ما أجل تو يا رجة التبنايا بيا الشريعة اإلس مية نقانون المعام و المدنينة اني ميناف المسنلنلية التقصنيرية لعنديم التمييص بسلطنة عمان اي ركنني الفعنل الينار (الخطنا،و نالينرر– اني اناف نجو نا– ما عدمه. Abstract: نأْ طْناْ الرَّامٌني ْ الغْرْ ضْ : لم يعصٌ ب هو نأْ طْاْ نْو عه إٌذا طْلْن :ااجتْنهو النم يْنن يْا و نلنم يعصٌ ن دنيئا . نالخٌ ط ناْ ع أْرض يعخ طٌئاا المطر نيعصٌ ني ع أع نرى قعر بْانا. نأْ طْناْ يعخ طٌنئع إٌذا سْنلْاْ سْنبيلْ الخْطْناْ عْم ندا نسْا وا أ نيقاف: ْطٌئْ بمعن أْ طْاْو نقيل: ْطٌئْ إٌذا تْعْمَّدْو نأْ طْاْ إٌذا لم يتعمد. نيقاف ل ما ْأْرا ديئا افعل ويره أْن اعل وير الصوات: أْ طْا. نالخاطٌئع: ما تعمَّد لما ينبغيو نتقوف: وْن تعخ طٌنئ اني العلنم أْيسْنرع منا أْن تعخ طٌنئ اني الدٌّيا. نيقاف: قد ْطٌئ تع إٌذا أًٌْم تو ااْنا أْ طْاع نأْنا اطٌئا (ابا منظورو0224 : 15 - 11 .، نبناء عل ما ت :قدم اإن الخطا ما النااية اللغوينة يناتي بعند معنانا منانا النو مو نالظناو نالكننقتو نمقابلننه الصننوات نالحقيقننة. نمننا ًننمو ااننو يعننني العنندنف نالخننرنج عننا جننا .الصواتو نعدم إصابة الاد المقصو و نعدم تحقيق النيات المطلوت كما يدف معن الخطا ما النااية اللغوية عل أاعاف مشينة نمعيبنة نسنيئة مثنل: اإلًنمو نالقن و نارتكنات المعاصني نالسنيئاو نالكبنا ر. نينقسنم الخطنا النديني إلن طنا متعمند نمقصو يستلصم العقات نالتوبيخ نالتاني و ن طنا وينر متعمند أساسنه السناوو نالنسنيانو .ناإل مافو نالغلطو نالتقصير اي تقدير اودياء نموازنة اومور أما ما النااية القانونية اكلمة الخطا نر و بعد تعريفاوأ نظرا لعدم نجنو تعرينم جامع دامل متفق عليه بيا اقااء القانون لتعريم الخطاو نعل الروم منا تلنا الصنعوبة اننإن رجنناف القننانون اننانلوا جا ننديا اسننتنباط تعريننم جننامع نمننانع للخطنناو اتنن كثننرو التعريفنناو نا تلفننت بننا ت النصعننا و الفر يننة لننديامو نا ننعيا نصنن أعينننام منندى التطور ا جتماعي نا قتصا ق للميتمع القق يعيشون ايهو امنام ما عمل علن تينييق ا رته ليحد ما قيام المسلنليةو نمنام ما ذ عك ذلا اعمل عل توسنيعاا لمسناعد الميرنر للوصوف إل التعنويل باسنال الطنر و نيمكنا تقسنيم أراء اقاناء ا لقنانون اني :تعريم الخطا إل أربعة آراء أ :الرررأي األول يعننر الخطننا بانننه عمننل ننار ويننر مشننرنعأ أق العمننل اليننار المخننالم للقننانونو نيل ننق علنن ننقا الننرأق أن القننانون ينننص علنن اواعنناف ويننر المشننرنعة أن اوعماف التي تتناس معه عل سبيل الحصر ليستدف مناا عل اوعماف المخالفة له. Abstract: :الررأي الاراني ن نو رأق الفقينه ب نينوف(Planiol) أ اينث يعنر الخطنا باننه " إ ن ف بالتصام سابق " نيحصر ب نيوف ا لتصاماو التي يشكل اإل ف باا طا ما المسلنف اي أربع ميموعاو ي ا متناع عا العنمو نالكم نا متناع عا الغشو نعدم اإلقدام عل عمننل لننم تايننا لننه اوسننب ات مننا القننو نالماننار ناليقظننة انني تا يننة ناجنن الرقابننة علنن اودخاص أن عل اودياء( السناورقو0252 : 20 - 22 .، نيعتبر تعريم ب نيوف و اوسا القق انطلقنت مننه معظنم القنوانيا المعاصنر اني مختلم الدنف عند محانلتاا التوسع اي اكر الخطاو نيعتقد ب نينوف أن الواجن القنانوني السننابق المقصننو بننه لنني اإل نن ف بننالتصام قننا م بننيا طننراياأ إنمننا اإل نن ف بنناق مننا .ا لتصاماو العامة التي تقع عل عاتق كل دخص ناق المبا ئ العامة للقانون كما أن قا التعريم يعندن كوننه محانلنة لتصننيم الخطناو نتقسنيمه إلن عند أننواعأ ون ب نيوف لم يقدم المعيار الدقيق نالوا ا لتحديد طبيعنة الفعنل الخناطئ نأنواعنهأ إنمنا و مجلة العلوم التربوية والدارسات اإلنسانية 312 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م نيعتبر تعريم ب نيوف و اوسا القق انطلقنت مننه معظنم القنوانيا المعاصنر اني مختلم الدنف عند محانلتاا التوسع اي اكر الخطاو نيعتقد ب نينوف أن الواجن القنانوني السننابق المقصننو بننه لنني اإل نن ف بننالتصام قننا م بننيا طننراياأ إنمننا اإل نن ف بنناق مننا .ا لتصاماو العامة التي تقع عل عاتق كل دخص ناق المبا ئ العامة للقانون كما أن قا التعريم يعندن كوننه محانلنة لتصننيم الخطناو نتقسنيمه إلن عند أننواعأ ون ب نيوف لم يقدم المعيار الدقيق نالوا ا لتحديد طبيعنة الفعنل الخناطئ نأنواعنهأ إنمنا اكتف بو ع قا مة بالواجباو العامة ننما نجو المعيار المقنا لتلا او طاء. Abstract: :الرأي الاالث ن و رأق ليفني اينث يعنر الخطنا باننه إ ن ف بالثقنة المشنرنعة ًنم يبنيا معيار قه الثقة ايقوف أن تحديد الخطا يقتيي التوايق بنيا مقندار معقنوف منا الثقنة يولينه النا للشخصو نبالتالي لانم الحنق أن يحينم عنا اوعمناف التني تينر بانمو نبنيا مقندار نيعتبر تعريم ب نيوف و اوسا القق انطلقنت مننه معظنم القنوانيا المعاصنر اني مختلم الدنف عند محانلتاا التوسع اي اكر الخطاو نيعتقد ب نينوف أن الواجن القنانوني السننابق المقصننو بننه لنني اإل نن ف بننالتصام قننا م بننيا طننراياأ إنمننا اإل نن ف بنناق مننا .ا لتصاماو العامة التي تقع عل عاتق كل دخص ناق المبا ئ العامة للقانون ا أ إل تق م ال طا ا لة لت ك م قا الت ك ا أ معقوف ما الثقة يوليه قا الشخص لنفسه ايتولد له اق عل النا أن يقوم عل أق عمنل ننما أن توقع او رار بالغيرأ بحينث تنتم مسناءلة الشنخص عن ا اعلنه إ إذا تصنر بشكل يتفق مع الثقة المشرنعة للنا ايهو ن يكون النا مسلنليا ما قبنل الغينر إذا كانت تصرااتام تخرج عا قه الثقة المشرنعة . نقنند نجاننت لاننقا الننرأق عنند انتقننا او لعننل مننا أ ماننا: أن ننقا التعريننم يتيننما ننابطا يبننيا السننلوك الننقق يعصننم الشننخص مننا الخطننا إذا سننلكه. كمننا أن المعيننار الننقق ن عه ليفي يعدن محانلة لو ع الخطا اي قال السنفي أكثنر منا كوننه معينارا محند ا للخطننا ذاتننه ( السننناورقو0252 ،و ن(سننلطانو2227 ،و ن(الفننارو2221 ،و ن(اليننندقو 2205 .، ، الرأي الرابع: يرى أنصار قا الرأق نجوت تحليل ركا الخطنا إلن عن صنرياو العنصنر اونف و ا عتداء عل اق مع إ راك المعتدق عتدا هو نالعنصر الثاني ن و اإل راك. نما أنصار قا ا تياه ساااتيه القق يعر الخطنا باننه إ ن ف بواجن قنانوني منع علنم .المخل بإ له أن كان باستطاعته أن يتبيا قا الواج نأن يلتصمه نيعتقد ساااتيه أن الواج القانوني يكون مصندره القنانون أن العقند أن يكنون ناجبنا أ بينا محنند ا يننامر بفعننل أن ينانن عننا اعننل أن عبننار عننا ناجنن عننام يقينني بعنندم اإل ننرار .بالغير نقد نجات لاقا الرأق عد انتقا او مناا أنه يقرر نجو التصام عنام علن كنل دنخص بعنندم اإل ننرار بننالغيرو نالقننوف بننا لتصام العننام يحنند معننن للخطننا بننل ننو بحاجننة إلنن .تحديد .ي نما أنصار قا ا تياه أييا الفقي ه جوسران القق يعر الخطا بانه” انتااك لحرمة اننق يسننتطيع مننا انتاننا ارمتننه أن يعار ننه بحننق أقننوى أن بحننق مماًننل” . Abstract: نبننقاو الطريقة يعر الف قيه يموج الخطا ايث يقوف بانه”اعتدا ء عل انق يندرك المعتندق اينه جان ا عتداء “ ( السناورقو0252،و ن(ز ر و2204 .، نالفر بيا تعريم جوسران ن يمنوج أن يمنوج يينيق منا اكنر الخطنا إذ يشنترط المسا بحق معنيا للغينر أمنا جوسنران ايوسنع منا اكنر اإلاسنا بحنق الغينر لييعلانا تشمل أعم الحقو ن أقلاا تحديدا . نلكا ك ل مناما لم ييع تعريم للخطا نلم يقندم معينارا قيقا للتعنر عل ينهو كمنا أن”ا عتنداء علن انق و ناإل ن ف بالوا جن نالحنق اوقنوى أن الحق المماًل و كل قه اولفاظ تحند معنن الخطنا بنل ني ذلتانا اني ااجنة إلن تحديند “ (امدانقو2202 .، إ نما أنصار قا ا تياه أييا الفقي ه جوسران القق يعر الخطا بانه” انتااك لحرمة اننق يسننتطيع مننا انتاننا ارمتننه أن يعار ننه بحننق أقننوى أن بحننق مماًننل” . نبننقاو الطريقة يعر الف قيه يموج الخطا ايث يقوف بانه”اعتدا ء عل انق يندرك المعتندق اينه جان ا عتداء “ ( السناورقو0252،و ن(ز ر و2204 .، نالفر بيا تعريم جوسران ن يمنوج أن يمنوج يينيق منا اكنر الخطنا إذ يشنترط المسا بحق معنيا للغينر أمنا جوسنران ايوسنع منا اكنر اإلاسنا بحنق الغينر لييعلانا تشمل أعم الحقو ن أقلاا تحديدا . نلكا ك ل مناما لم ييع تعريم للخطا نلم يقندم معينارا قيقا للتعنر عل ينهو كمنا أن”ا عتنداء علن انق و ناإل ن ف بالوا جن نالحنق اوقنوى أن الحق المماًل و كل قه اولفاظ تحند معنن الخطنا بنل ني ذلتانا اني ااجنة إلن تحديند “ (امدانقو2202 .، إ ق (، ( نيعننر الشننرقانق0290 :، الخطننا بانننه" اإل نن ف بواجنن قننانوني سننواء أكننان ننقا الواجنن ناجبننا اصننا أق التصامننا و أم ناجبننا عامننا مننا الواجبنناو التنني تفننرض علنن كننل دخص يعيش اي جماعة يحكماا القانون بان يحترم اقو الغينر ناريناتام ن أ يرتكن مساسا باقه الحقو نالحريا. " ( نيعتقد عبد الدا م2222 ، أن الخطنا منا نو إ "انحنرا ." الشخص عا السلوك المعتا مع إ راكه لاقا ا نحرا مجلة العلوم التربوية والدارسات اإلنسانية 330 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م ( نيعننر الشننرقانق0290 :، الخطننا بانننه" اإل نن ف بواجنن قننانوني سننواء أكننان ننقا الواجنن ناجبننا اصننا أق التصامننا و أم ناجبننا عامننا مننا الواجبنناو التنني تفننرض علنن كننل دخص يعيش اي جماعة يحكماا القانون بان يحترم اقو الغينر ناريناتام ن أ يرتكن مساسا باقه الحقو نالحريا. Abstract: " ( نيعتقد عبد الدا م2222 ، أن الخطنا منا نو إ "انحنرا ." الشخص عا السلوك المعتا مع إ راكه لاقا ا نحرا ( نيعراه مرق0210، بانه: "إ ف بواج قانوني مقترن بإ راك المخل إياه ." ( نيعر دن0212 ، الخطا ما النااية القانونية باننه: "انحنرا عنا سنلوك الشنخص المعتا الموجو اي نف الظرن الخارجية لمرتك اليرر مع "إ راك ذلا الواجنن ناجبننا اصننا أق التصامننا و أم ناجبننا عامننا مننا الواجبنناو التنني تفننرض علنن كننل دخص يعيش اي جماعة يحكماا القانون بان يحترم اقو الغينر ناريناتام ن أ يرتكن مساسا باقه الحقو نالحريا. " ( نيعتقد عبد الدا م2222 ، أن الخطنا منا نو إ "انحنرا ." الشخص عا السلوك المعتا مع إ راكه لاقا ا نحرا ( نيعراه مرق0210، بانه: "إ ف بواج قانوني مقترن بإ راك المخل إياه ." ( نيعر دن0212 ، الخطا ما النااية القانونية باننه: "انحنرا عنا سنلوك الشنخص المعتا الموجو اي نف الظرن الخارجية لمرتك اليرر مع "إ راك ذلا ع ( نيعراه مرق0210، بانه: "إ ف بواج قانوني مقترن بإ راك المخل إياه ." ( نيعر دن0212 ، الخطا ما النااية القانونية باننه: "انحنرا عنا سنلوك الشنخص المعتا الموجو اي نف الظرن الخارجية لمرتك اليرر مع "إ راك ذلا (المجلد5 ،) ( العدد10 ) يونيو3030م ( نيعنننر سنننلطان2225 ، الخطنننا باننننه: "انحنننرا سنننلوك الشنننخص منننع إ راكنننه لانننقا ا نحرا ." ( نيعنننر سنننلطان2225 ، الخطنننا باننننه: "انحنننرا سنننلوك الشنننخص منننع إ راكنننه لانننقا ا نحرا ." نمننا نن ف اسننتعراض التعريفنناو السننابقة يمكننا القننوف أن بعينناا قنند اسننتبعد اكننر ( الخطا ونه ييعل او نرار بحنق الغينر طنا كمنا نر اني تعرينم الشنرقانق0290 ،و أمنننا تعرينننم منننرق( 0210 ، اينقصنننه اصنننر الواجبننناو القانونينننة الملقنننا علننن عننناتق الشخص ن قا أمر مستحيل كما سبق اإلدار إليه . Abstract: إ ( أما تعريفاو كل ما عبد الدا م2222 ( ،و ن دن0212 ( ،و نسلطان2225 ، اإنانا تعتقنند أن ا نحننرا عننا سننلوك معننيا يععنند طننا إ أن التعريفنناو آنفننة الننقكر تيننع ابطا محد ا اوف ما ية السلوك المعيا الواج اإلتباع .اي مياف الخطا ( أما تعريم الخطا ما النااية الشرعية اقد عراه ابا را2220 ، بقوله: "الخطا: نو أن يقصد بفعله ديئا ايصا اعله وير ما قصدهو مثنل أن يقصند قتنل كناار اصنا قتلنه مسلما و أن يظا أن الحق اي جاتهو ايصا .وير ذلا نقاف اليرجاني: الخطا و ما لي لإلنسان ايه قصد ن و عقر صنالا لسنقوط انق هللاا تعننال إذا اصننل عننا اجتاننا نيصننير دننباة انني العقوبننة اتنن يننلًم الخنناط ء ن يلا ق بحد ن قصاص نلم ييعل عقرا اي اق العبا ات نجن علينه نمان العندنانو ننجبننت بننه الديننة كمننا إذا رمنن دخصننا ظنننه صننيدا أن اربيننا اننإذا ننو مسننلمأ أن ور ننا ااصات آ ميا و نما جرى ميراه كنا م ًم انقل عل رجل اقتله (اليرجانيو0295 ،. التعدق ما النوااي اللغويةو نالقا نونيةو نالشرعية: م ي ق :التعدي لغة تاتي بعد معانا مناا ا عتنداء بمعنن الععند نْ انو نالععند نْ ان لغنة بمعنن : الظملنم الصمراتو نقد عدا عْند ن ا نعْندْاء نععندعنو ا نععند نْ ان ا نعٌند نْ ان ا نععند نْ ى نتْعْندَّى ناع تْندْىو كعلمنه َّظْلْمهو نتيانز الحد (ابا منظورو0224 : 20 / 05 .، َ (، كما يعر التعدق منا النااينة اللغوينة باننه: مينانز الحند نالقندر نالحنقو يقناف: عْدَّي تعنه اتعدى أق تيانزو نتعْدَّيت الحنق ناع تْديتنه نعْدْن تعنه أق جانزتنهو نيقنوف ابنا انار عنا ما– ع ن- : إن العيا نالداف نالحنر المعتنل أصنلا نااند صنحيا ترجنع إلينه جمينع ارنع قا الباتو ن و أصلا يدف عل تيانزا اي الشيء نتقدما لما ينبغني أن يقتصنر علينه (الننرازقو0272 : 242 / 4 .، أمننا تعريننم التعنندق مننا الناايننة القانونيننةو اقنند أنر اقانناء :القانون عد تعريفاو مناا مجلة العلوم التربوية والدارسات اإلنسانية 331 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... Abstract: د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م (ق، أمننا تعريننم التعنندق مننا الناايننة الشننرعية اقنند نر اسننتعماله كثيننرا انني أبننوات الفقننه المختلفةو نيختلم المعن المرا مننه بنا ت المو نوع النقق اسنت عمل اينهو إ أن نقه ا ستعما و– مع ا ت ااا– ينتظماا معن نااد و: اعل اإلنسان ما لي له اعلنهو ن نو معننن يطننابق المعننن اللغننوق للتعنندقأ إذ أن مننا اعننل مننا لنني لننه اعلننه اقنند تيننانز الحنند نالقدر نالحق النقق ينبغني لنه ا قتصنار علينهو نايمنا يلني نمناذج منا اسنتعما و الفقاناء :لكلمة التعدق ناقا لآلراء الفقاية المختلفة يعننر الت عنندق عننند اوانننا : (الثنناني: أن القننبل المنناذنن ايننه يكننون تعننديا و ونننه يفننوو ينند المالننا ن ننمان إ علنن المتعنندق ( الكاسننانيو0291 : 207 / 1 ،. االتعنندق أطلق نا– كما و مفاوم العبار– عل القبل وير الماذنن ايه. أما المالكية ايعراون التعدق اي بات الشركة باننه: ن يكنون متعنديا با نقه القنراض إ إذا أ قه بغير إذن دريكه نكان العمل اينه يشنغله عنا العمنل اني مناف الشنركة ًنم إننه اني ااف تعديه يكنون ذلنا التعندق مانعنا منا اسنتبدا ه بنالربا نالخسنر (الدسنوقيو0291 : 205 / 2 .، نالقدر نالحق النقق ينبغني لنه ا قتصنار علينهو نايمنا يلني نمناذج منا اسنتعما و الفقاناء :لكلمة التعدق ناقا لآلراء الفقاية المختلفة أما المالكية ايعراون التعدق اي بات الشركة باننه: ن يكنون متعنديا با نقه القنراض إ إذا أ قه بغير إذن دريكه نكان العمل اينه يشنغله عنا العمنل اني مناف الشنركة ًنم إننه اني ااف تعديه يكنون ذلنا التعندق مانعنا منا اسنتبدا ه بنالربا نالخسنر (الدسنوقيو0291 : 205 / 2 .، نالقننراض انني التعريننم السننابق يقصنند بننه المينناربة ن ننو: أن يننداع دننخص آل ننر مننا .ليتير به نيكون الربا بيناما عل ما يتفقنان علينه بعند إ نراج رأ المناف االتعندق جناء اي قا المو ع بمعن أ ق الشريا ما اوجنبي ما ليعمنل بنه مقار نةأ إذا كنان العمنل ايه يشغله عا العمل اي ماف الشركةو نكان بغير إذن دريكه. نيعنر الشنااعية التعندق اني بننات الينايناو بقنولام: نمننا تعندى بشنرت ناء مصيننل العقننل: ننل يينن عليننه القصنناص؟ قيننل: كمعتننوهو نالمننق القطننع بوجننوت القصنناص لتعديننه بفعننل مننا يحننرم عليننه (الدسننوقيو .مو252 / 2،و ن(الشننااعيو0224 و012 / 2 .، االتعدق نا بمعن ارتكات اعل محرم ن و درت الدناء المصيل للعقل. Abstract: د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م :القانون عد تعريفاو مناا يقصد بالتعدق الظلم نالعدنان نميانز الحدو ايث يعد التعندق عمن نارا بندنن انق أن جننواز دننرعي (عبنندهللااو2225:20 .، ن ننناك مننا يعتبننر التعنندق التصامننا قانونيننا بعنندم ا عتننداء علنن الغيننر ناإل نن رار باننمو ن ننقا ا لتننصام ننو التننصام ببننقف عنايننة يوجنن علنن الشننخص أن يراعنني انني سننلوكه اليقظننة نا نتبنناهو ن التبصننر اتنن ييننر بننالغيرو اننإذا انحننر عننا ننقا السننلوك الواجنن أ اقنند ارتكنن تعننديا يمثننل العنصننر المننا ق للخطنناأ اننإذا تواار بيان قا التعدق القدر علن التميينص كنان نقا ا نحنرا طنا يوجن مسنلنليته التقصيرية( السناورقو0252 : 0292 .، أمننا تعريننم التعنندق مننا الناايننة الشننرعية اقنند نر اسننتعماله كثيننرا انني أبننوات الفقننه المختلفةو نيختلم المعن المرا مننه بنا ت المو نوع النقق اسنت عمل اينهو إ أن نقه ا ستعما و– مع ا ت ااا– ينتظماا معن نااد و: اعل اإلنسان ما لي له اعلنهو ن نو معننن يطننابق المعننن اللغننوق للتعنندقأ إذ أن مننا اعننل مننا لنني لننه اعلننه اقنند تيننانز الحنند يقصد بالتعدق الظلم نالعدنان نميانز الحدو ايث يعد التعندق عمن نارا بندنن انق أن جننواز دننرعي (عبنندهللااو2225:20 .، ن ننناك مننا يعتبننر التعنندق التصامننا قانونيننا بعنندم ا عتننداء علنن الغيننر ناإل نن رار باننمو ن ننقا ا لتننصام ننو التننصام ببننقف عنايننة يوجنن علنن الشننخص أن يراعنني انني سننلوكه اليقظننة نا نتبنناهو ن التبصننر اتنن ييننر بننالغيرو اننإذا انحننر عننا ننقا السننلوك الواجنن أ اقنند ارتكنن تعننديا يمثننل العنصننر المننا ق للخطنناأ اننإذا تواار بيان قا التعدق القدر علن التميينص كنان نقا ا نحنرا طنا يوجن مسنلنليته التقصيرية( السناورقو0252 : 0292 .، مجلة العلوم التربوية والدارسات اإلنسانية 331 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... Abstract: م ق ن جاء تعريم التعدق عند الحنابلة اي بات الوكالة: ن تبطل الوكالة بالتعندق ايمنا نع كٌّنل ايهو مثل أن يلب الثوت نيرك الدابة ( ابا قدامة المقدسيو0219 و221 / 7 .، ن جاء تعريم التعدق عند الحنابلة اي بات الوكالة: ن تبطل الوكالة بالتعندق ايمنا نع كٌّنل ايهو مثل أن يلب الثوت نيرك الدابة ( ابا قدامة المقدسيو0219 و221 / 7 .، االتعدق قصد به اي نقا المو نع مخالفنة الوكينل مقتين الوكالنة كنان يلنب الثنوت أن يركنن الدابننة اللننقيا نع كننل انني بيعامنناو نإ ننااة إلنن مننا سننبق اقنند اسننتعمل الفقانناء لفننظ .(التعدق، بمعن ا عتداء عل اومواف ناوبدان اي بابي الغص ناليناياو (ي، االتعدق قصد به اي نقا المو نع مخالفنة الوكينل مقتين الوكالنة كنان يلنب الثنوت أن ركنن الدابننة اللننقيا نع كننل انني بيعامنناو نإ ننااة إلنن مننا سننبق اقنند اسننتعمل الفقانناء لفننظ .التعدق، بمعن ا عتداء عل اومواف ناوبدان اي بابي الغص ناليناياو ي ي ق، ( نإذا كان الفقااء قد استعملوا لفظ (التعدق، عل النحنو ال منقكورو انإن لبعينام– منا نجها آ ر– اصط اا اصا اي قا اللفظ يختلم ما ايث السعة نالييقأ إذ ما نل ء . ما جعل (التعدق، مصطلحا عل جميع مدلو ته تقريبا ع ق، ( ( نيقوف ابنا جنصق0425 : 209 ، النقق عقند للتعندق بابنا صْندَّره بقولنه: (ن نو أعنم منا .الغص و ون التعدق يكون اي اومواف نالفرنج نالنفو ناوبدان نإجمننا يمكننا القننوف- بننناء علنن مننا تقنندم- أن التعنندق الننقق ننو دننرط انني السننب الموج لليمان و ما كان بنالمعن المواانق لمعنن (التعندق، منا النااينة اللغوينةو انإذا صدر السب عا المتسب عل نجه لي للمتسب الحق اي اعله ما ذلنا الوجنه اتصنم السب بالتعدقو نما أمثلة ذلا: داا الصنرو ناقتناء الحيواناو اليار و ناإلكر اه بغينر اقو نالحفر اي الطريق العامو نإيقا السيار اي مكان تمنع اونظمة المرنرية الوقو .ايه للمصلحة العامةو نوير ذلا ما أمثلة تطبيقية لمعن التعدق ق إن ادتراط التعدق اي السب محل اتفا بيا الفقااء تدف علينه نصوصنام نتعلني تام للقوف بالينمان اني مسنا ل التسنب و نلكنا النقق يقنع اينه الخن بنيا الفقاناء نو تحقنق التعدق اي السنب و نمنا نصنوص الفقاناء اني ادنتراط التعندق: منا نر عنند الحنفينة اني قننولام: نالتسننب إذا لننم يكننا تعننديا يكننون سننببا لوجننوت اليننمان (الكاسننانيو0291 : 272 / 7 .، ، ( نيقوف ابا عراة2229:091 / 1 :، ن نو منا اقاناء المالكينة السنب العنرٌ قّ عنا قصند التلننم إن كننان عننداء. Abstract: انند ل انني ذلننا مننا نننانف صننبيا سنن اا يقنندر علنن مسننكه امنناو الصبيو نإن كان عداء إذا لم يكا السب ايه عداءو كما قاف اني المدنننة: كمنا افنر بئنرا اي اره أن اي طريق اي جان اا طه إذا لم يكا ايه رر ا مان اني ذلناو نكنقلا كل ا.عل ماذنن نإذا كان التعدق دنرطا للينمان بالسنب انإن اسنتمرار اتصنا السنب بالتعندق إلن اصننوف اليننرر بننه دننرط لتحقننق التعنندق انني السننب و الننو زاف التعنندق عننا السننب قبننل اصوف اليرر به برئ المتسب ما مان ما يحصنل بنه منا نرر بعند ذلناو كمنا لنو افر دخص اي أرض مملوكة للغينر أن أنقند ايانا ننارا ًنم ادنترى اورض قبنل أن يتلنم ديءا بالحفر أن النارو نكما لو أنقنم دنخص سنيارته اني مو نع يمننع الوقنو اينه نلنم يحصل رر بسب إيقاااا ات أعلغي المنعو نالتعدق ي مكا اصره– ما ف اسنتقراء صوره– اي ً ث جااو: إأ ي ::األولر أن يتصنر اإلنسنان بمنا انق لنه اينه أصن و كمنا اني إيقنا الننار اني الطريننق العامو نكترك رجل اإلسعا نقل المصات مع القدر. إ عإ م :الاانية أن يتصر اإلنسان بما و اق لهو نلكنه يتيانز اد اقه اي مثل قا التصنر ما جان الصيا و نيسم (اإلاراط، كان يوقد الشخص اي ملكه نارا كبير او المعتا. :الاالاة أن يتصر اإلنسان بما و انق لنهو نلكننه يعقْصَّنر بتنرك ا اتيناط المطلنوت اني مثل ذلا التصر نيسم (التفريط، ن(التقصير،: كان تسير ناقلة زيتو نقد ترك الصينت يتسرت مناا إل الطريق اتنصلق به السياراو. إ نبننناء علنن مننا تننم استعرا ننه اننوف الخطننا ناإل ننرار نالتعنندق ي اننظ أن المفننر او الننث ث يمكننا اسننتخداماا انني مينناف المسننلنلية التقصننيرية نذلننا لعنندم نجننو اننوار جو رية ايما بيناناأ نعلينه يمكنا اسنتخدام أينا منانا لتنل ق ذاو المعنن أ بنل أن اسنتعماف لفظة "اإل رار" ي اوكثر ديوعا ما الناايتيا الفقاية نالقانونية اي الوقنت المعاصنرو ن ي ذاو اللفظة المستعملة .اي قانون المعام و المدنية اي سلطنة عمان :لفرق بين المسؤولية المدنية والجنائية ة :الفرق بين المسؤولية المدنية والجنائية ة :الفرق بين المسؤولية المدنية والجنائية :تعريف المسؤولية مجلة العلوم التربوية والدارسات اإلنسانية 332 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م :تعريف المسؤولية قبل الخوض اي تو يا الفر بيا المسنلنلية المدنينة نالمسنلنلية الينا ينة بند منا تو يا معن المسلنلية ما النوااي اللغوية نا صط ايةأ ذلنا أن كلمنة المسنلنلية لنم تكننا معراننة لنندى اقانناء القننانون إ انني العصننر الحنناليأ ايننث تعتبننر مننا المصننطلحاو الحديثنة اني ميناف القنانونأ ذلنا أن إلنسنان ي منا اينث المبندأ ي انر اني جمينع تصنرااته أقننوا كانننت أم أاعننا أ نلكننا ننقه الحريننة قنند تصننطدم بحنناجص آ ننر ن ننو عنندم اإل ننرار بالغيرو نما نا منشا نظرية التع سم اي استعماف الحق. نبناء عل ذلا اإن الحرية اي التصر تتقيد بقيو نادن و ن نقه القينو نالحندن قند تفر اا او و ناوعرا و نالقنيم نالمعثنل العلينا التني تسنو ميتمنع مناأ نقند يفر ناا القانون. نبمير تيانز قه الحدن سواء أكانت أ قية أم قانونينة انإن الشنخص يتحمنل قبل الخوض اي تو يا الفر بيا المسنلنلية المدنينة نالمسنلنلية الينا ينة بند منا تو يا معن المسلنلية ما النوااي اللغوية نا صط ايةأ ذلنا أن كلمنة المسنلنلية لنم تكننا معراننة لنندى اقانناء القننانون إ انني العصننر الحنناليأ ايننث تعتبننر مننا المصننطلحاو الحديثنة اني ميناف القنانونأ ذلنا أن إلنسنان ي منا اينث المبندأ ي انر اني جمينع تصنرااته أقننوا كانننت أم أاعننا أ نلكننا ننقه الحريننة قنند تصننطدم بحنناجص آ ننر ن ننو عنندم اإل ننرار بالغيرو نما نا منشا نظرية التع سم اي استعماف الحق. تبعنة ذلناو ن ننقه ني ال مسننلنلية نيتبننيا منا ذلننا أن المسنلنلية إمننا أن تكنون أ بيننةو أق أ قيةو نإما قانونية. :تعريف المسؤولية نبصور عامة االمسلنلية ما النااية اللغوية ترجع إل السيا نالامص نال مو كلمنة :ناانند و يقنناف سننافو يسننافو سننلا نمسننالةو ناسننم الفاعننل منننه: السننا لو ناسننم المفعننوف المسلنفو نالمصدر الصناعي: المسلنلية (ابا اار و0272 : 0244 .، نيقاف: ساله بكقا نعا كنقا: اسنتخبره عننه نطلن مننه معراتنهو نسناله عنا كنقا: ااسنبه :عليه نآ نده بنهو نسناله الشنيء: طلبنه مننهو نسناله الوعند: طلن ناناءه نإنينازهو نسناله طلنن معرناننه نإاسننانه و نسنناله بننا أن يفعننل كننقا: أقسننم عليننه أن يفعنن ل كننقا (الصبينندقو 0221 : 215 - 217 / 7 .، أما تعريم المسلنلية ما النااينة الشنرعية ااني تعنني بصنور عامة أن يتحمل اإلنسان نتا اواعاف المحرمنة التني ياتيانا مختنارا ن نو مندرك لمعانيانا .ننتا ياننا ( نيعرااننا الحليبنني0224 : 70 ، باناننا أ ليننة الشننخص أن يكننون مطالبننا دننرعا بامتثاف ا.لماموراوو ناجتنات المناياوو نمحاس علياا :كما يعرااا عو ( .ن222 / 0 ، بانانا: ا سنتعدا الفطنرق النقق جبنل هللاا تعنال علينه اإلنسان ليصلا للقيام برعاية ما كلفه به ما أمنور تتعلنق بديننه ن نيناهو انإن نان منا علينه ما الرعاية اصل له الثواتو نإن ارط ايانا اصنل لنه الع.قنات أمنا منا النااينة القانونينة اقد عرااا اقااء القانون بعد تعريفاو مناا: أ لية الشخص ون ينس اعله إليه نيحاسن عليه (أمامو0220 : 202 .، ( نيعرااا التايه0222 : 27 ، باناا: التبعة التي تترت نتيينة .قوف أن اعل صا ريا ما المسئوف نينبني علياا آًار نيوية نأ رنية الفرق بين المسؤولية المدنية والمسؤولية الجنائية اناليصاء اني المسلنلية المدنية و التعويل (أق اليمان،و نصاا الحق اينه نو المينرنرو ن نو القق يملا راع الدعوى بهو كما يملا التنازف عنه أن التصنالا علينهو نإذا مناو المسنلنف .جاز مطالبة نرًته بالتعويل أما اليصاء اي المسنلنلية الينا ينة اانو النر ع عنا طرينق توقيع عقوبة ما العقوباو منصوص علياا قانونا و نتكون العقوبة دخصنيةو نالنقق يملنا المطالبة بتوقيع العقوبة و النيابة العامة أن ا عاء العنام كمنا نو معمنوف بنه اني سنلطنة عمننان باعتبار مننا ممثلننيا للميتمننعو ن تملننا النيابننة العامننة أن ا عنناء العننام الصننلا أن .التنازف اي المسلنلية الينا ية وناا اق متعلق بالميتمع نكمبدأ قنانوني عنام انإن المسنلنلية المدنينة م زمنة للمسنلنلية الينا ينة ون الينرا م الينا ية تكون عا جرا م مدنيةو كما و ا لحاف اي جرا م ا عتداء علن الننف أن المنافأ إ أن قه القاعد ليست مطلقة ون منا اواعناف منا يرتن مسنلنلية الينا ينة للفاعنل نن المسلنلية المدنية إما نتفاء اليرر أن لصعوبة تقديره كينرا م التشنر و نامنل السن تو نمخالفاو المرنرو نا تفا الينا يو نبعل جرا م الشرن.ع نبالمقابننل اقنند تقننوم المسننلنلية المدنيننة نن المسننلنلية الينا يننة كمننا ننو الشننان انني اوا ث العملو نالمنااسنة وينر المشنرنعةو نالعلنة اني ذلنا أن المسنلنلية المدنينة انسنع َّنطاقننا مننا المسننلنلية الينا يننةو ون القاعنند القانونيننة تقننوف: "إن كننل اعننل نناطئ سننب ررا للغير يلنصم منا ارتكبنه بنالتعويل". نالقاعند اني الفقنه ا سن مي أنسنع منا ذلنا وناا تكتفي باليرر سنتحقا الينمان علن انيا أن القاعند اني المسنلنلية الينا ينة جريمة بدنن ننص. نعلن نقا اوسنا قند يرتن الفعنل الينار مسنلنلية الفاعنل المدنينة . نن المسلنلية الينا ية نكمبنندأ عننام اننإ،ن المسننئولية المدنيننة (اليننمان– انني الفكننر القننانوني– تختلننم عننا المسئولية الينا ية (العقوبة، ما عد نواتاو نترتبطان ببعياما– أايانا– ارتباطنا ينلًر اي أاكام كل منامنا. امنا اينث أنجنه ا نت بنيا المسنلنليتيا ي انظ أن نناك عند ارنقنناو بننيا المسننلنليتيا علنن النحننو اآلتنني (ز ننر و2204 ،و ن(السننناورقو0252 ،و ن(سلطانو2227 ،و ن(الفارو2221 ،و ن(اليندقو2205 :، 1 :: أوجه االفتراق مجلة العلوم التربوية والدارسات اإلنسانية 335 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... الفرق بين المسؤولية المدنية والمسؤولية الجنائية مجلة العلوم التربوية والدارسات اإلنسانية 332 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م الفرق بين المسؤولية المدنية والمسؤولية الجنائية أما الفر بيا المسلنلية المدنية نالمسنلنلية الينا ينة اني اظ أن المسنلنليتيا تشنتركان انني السننب الموجنن لامننا ن ننو مخالفننة أاكننام الشننارع الملصمننةو نإتيننان المحظننوراو الشرعية التي نا هللاا سبحانه نتعال عناما (سراجو7 2202:1 .، كما يشتركان-ما نااية أ رى- اي اوسا الشرعي النقق يقنوم علينه كنل منامناو ن نو ملا ق كل نف بما ارتبكت نمسئوليتاا عنا إ نرار ا بنالغير. ن نو اوسنا النقق أكنده : القرآن الكريم اي أكثر ما مو عأ كقوله تعنال﴿ كعنلم نْف ن ا بٌمْنا كْسْنبْت رْ ٌينْنةا ﴾ .(القنرآن المدًر74 : 29 :،و نقوله﴿ لْاْا مْا كْسْبْت نْ عْلْي اْا مْا اك تْسْبْت﴾ (القرآن. البقر0 : 291 .، إن المتتبننننع لا بينننناو القانونيننننة اننننوف المسننننلنليتيا المدنيننننة نالينا يننننة ي اننننظ أن المسننلنليتيا تيتمعننان علنن الفعننل اليننارأ إذ ياننتم القننانون المنندني بالفعننل اليننار الننقق يصي الشخص نيرت ذ لا قيام مسلنلية الفاعل المدنيةو كما ياتم القانون اليصا ي علن .بالفعل اليار القق يصي الميتمعو نيرت عل ذلا مسلنلية الفاعل الينا ية نبالمقابل انإن المسنلنلية المدنينة تختلنم عنا المسنلنلية الينا ينة منا اينث اوسنا نما ايث اليصاءأ ااسنا المسنلنلية المدنينة اعتنداء علن انق الغينر يتمثنل ايمنا يسنم بالخطا المدنيأ ن قا الخطا يمكا اصره اي صنور محند بعيناناو نذلنا ون اواعناف الصا ر ما مرتكبيانا نيصند عليانا نصنم الخطنا المفيني إلن الينرر أكثنر منا أن .تحصر إن اوسننا الننقق تقننوم عليننه المسننلنلية الينا يننة ااننو ا عتننداء علنن اننق مننا اقننو الميتمعو أق أن أساساا القيام بيريمة ما اليرا مو ناليرا م- كما و معلوم- محند اني ق أما الفر بيا المسلنلية المدنية نالمسنلنلية الينا ينة اني اظ أن المسنلنليتيا تشنتركان انني السننب الموجنن لامننا ن ننو مخالفننة أاكننام الشننارع الملصمننةو نإتيننان المحظننوراو الشرعية التي نا هللاا سبحانه نتعال عناما (سراجو7 2202:1 .، القانون عل سبيل الحصنرأ اسنتنا ا إلن القاعند« ا جريمنة ن عقوبنة إ بننص،و نلنقا تعتبر المسلنلية المدن.ية أنسع نطاقا ما المسلنلية الينا ية القانون عل سبيل الحصنرأ اسنتنا ا إلن القاعند« ا جريمنة ن عقوبنة إ بننص،و نلنقا تعتبر المسلنلية المدن.ية أنسع نطاقا ما المسلنلية الينا ية ع إن اليصاء يختلم اي كلتا المسنلنليتيا تبعنا نت أسنا كنل منامنا. الفرق بين المسؤولية المدنية والمسؤولية الجنائية د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م :ًأوال تختلم المسئولية المدنية عا المسئولية الينا ية ما ايث الاد االمسئولية الينا ية تاد إل اماية الميتمع با قتصاص مما أ ل بامنه ناستقرارهو نزجر ا لياني عما يرتكبه ما جرا م أن محظوراو درعيةو بينما تاد المسئولية المدنية إل جبر اليرر التي أصات الميرنر اي جسمهو أن مالهو أن دراه نسمعتهأ بسب اعل نقع إ بالتصام عقدق أن قانوني. نيترت عل ذلا أن المسئولية المدنية ترتبط نجو ا نعدما بوجو رر. اإذا كان الاد مناا و جبر اليررو اإن مبرر نجو ا ينتفي ما أساسه إذا انتف اليرر. كما أن التعويل يكون بمقدار قا اليرر بصر النظر عا مدى جسامة التعدق. كل ذلا عل عك المسئولية الينا ية ايث توقع العقوبة عل المتام بصر النظر عا نقوع اليرر نمداه. االعقوبة تطبق لر ع المتام نلي لتعويل الميني عليهو مالم يكا القانون يشترط لتكامل عناصر جريمة ما أن يكون .اليرر أاد قه العناصر :ًثانيا تختلم المسئولية المدنية عا المسئولية الينا ية ما ايث أن توقيع العقوبة عل المتام اي المسئولية الينا ية اق للميتمع يطال بتوقيعاا النيابة العامة باعتبار ا ممثلة للميتمعو أما اي المسئولية المدنية اإن ما يطال التعويل و الميرنر نفسه بحسبانه و صاا الحق نيترت عل ذلا أن الميرنر يستطيع– بحسبانه صاا الحق الوايد– التنازف عا اقه بإبراء المسئوف ما التعويل المستحق له. ن و يملا ذلا سواء قبل المط البة بالتعويل أن بعد المطالبة به نتقديره نقبل تحصيله ما ام أن الفعل اليار قد نقع بالفعلأ نبالمقابل اإن المسلنلية الينا ية تييص الصلا أن التنازف عا العقوبة ما قبل الميني عليه باعتباره أن إنصاف اليصاء عل التام و اق للميتمع بوجه عام نلي اقا للميني عليه .ناده :ًثالاا نظرا و مية المسئولية الينا ية نارتباطاا باما الميتمع ناستقرا ره بصور عامةو نلما يترت علياا– عا– ما توقيع عقوباو تم جسم اإلنسان ناريتهو اإناا تقوم عل مبدأ " جريمة ن عقوبة إ بنص"أ ايث تكون اليرا م منصوصا علياا اصرا ناقا للنصوص . القانونية التي تيرم نوعية الفعل المرتك أ إذ تكون العقوباو مقدر سلفا نما ًمو ييوز للقا ي أن يييم جريمة إل ما عدّ ه المشرع أن يليا إل القيا بغرض قه اإل ااة. قا عل عك المسئولية المدنية التي تقوم عل قاعد عامة مقتيا ا عدم جواز اإل رار بالغير نن اصر لحا و اإل رارو ن نن تحديد لمقدار التعويل اسابيا . الفرق بين المسؤولية المدنية والمسؤولية الجنائية إذ يكفي أن تتواار درنط القاعد العامة ات يلصم القا ي المسئوف بتعويل الميرنر بالقدر الكااي نال زم ليبر قا اليرر. اا لقاعد العامة التي قرر ا المشرع– اي نطا المسئولية المدنية– تتسع لتشمل صنواا عديد ما التطبيقاو يصع . اصر ا مقدما أ نظرا لكثرتاا نتنوعاا اي ناقع النا المتفاعل ايوية ناركة ننشاطا :ًرابعا إن نجو النية رنرق لقيام المسئولية الينا ية عل اوقل اي الغالبية العظم ما اليرا م. نما قه النااية تقترت المسئولية الينا ية ما المسئولية المدنيةو نلكا النية ناد ا تكفي لقيام المسئولية الينا ية بحسباناا أمرا ا ليا بطبيعتااو نإنما يي أن تدف علياا قرا ا نمظا ر ارجية تقطع بوجو ا. أما اي المسئولية المدنيةو ا يشترط . بقياماا نجو النيةو نأكثر ما يكون التعدق تقصيرا نإ ما عمدا 3: أوجه االر:تباط مجلة العلوم التربوية والدارسات اإلنسانية 332 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م إذا كانت المسئولية المدنية تختلم– كما تم تو يحه سلفا– عا المسئولية الينا ية ما عد نجوهو إ أن ناك– أييا– أنجه "ترابط"و أن ارتباط بيناما. نيتحقق ذلا إذا كان الفعل اليار يشكل– ذاو الوقت– " جريمة جنا يةو اتنشا عا قا الفعل" الوااد عويان: إادا ما عو عامة ن ي الدعوى الينا يةو ناو رى اصةو ن ي الدعوى المدنية. نتاد اونل إل إنصاف العقوبة عل اليانيو بينما تاد الثانية إل جبر اليرر النادئ عا اليريمة. مثاف ذلا إاداث عا ة مستديمة بالميني عليه أن الس نالقق نا وتصاتو نالسرقةو نالقتلو نوير ذلا ما اليرا م التي تحدث ررا– ما يا نأ بيا– بالميني عليه. ناي مثل قه الحالة ييوز للميني عليه (الميرنر،راع أمام الق ياء المدني نتخيع الدعوى– اي الحالتيا– لقواعد القانون المدني سواء ما ايث القواعد المو وعية أن ما ايث قواعد اإلًبا و. (إتحا ية عليا باإلماراوو02 أكتوبر0222 و ميموعة اواكامو السنة05 و العد الثالثو رقم012و ص02022 .، :نتتيل مظا ر قا "ا رتباط" بيا المسلنليتيا المدنية نالينا ية ما عد نوات 1 - :مُضي المدة المانع من سماع الدعوى 1 - :مُضي المدة المانع من سماع الدعوى عوى التعويل أما اي قانون المعام و المدنية الععمانيو اتنص الما095 :عل أنه أما اي قانون المعام و المدنية الععمانيو اتنص الما095 :عل أنه 0 – تسمع عو تعويل النادئة عا الفعل اليار بعد انقياء م سنواو ما اليوم .القق علم ايه الميرنر بحدنث اليرر نبالمسئوف عنه 2 – إذا كانت عوى التعويل نادئ ة عا جريمة نكانت الدعوى اليصا ية ماتصاف قا مة .بعد انقياء الميعا المقكور اي الفقر السابقة اإن عوى التعويل يمتنع سماعاا 2 – تسمع عوى التعويل اي جميع اواواف بانقياء مسة عشر سنة ما يوم نقوع ."الفعل اليار 0 – تسمع عو تعويل النادئة عا الفعل اليار بعد انقياء م سنواو ما اليوم .القق علم ايه الميرنر بحدنث اليرر نبالمسئوف عنه 2 – إذا كانت عوى التعويل نادئ ة عا جريمة نكانت الدعوى اليصا ية ماتصاف قا مة .بعد انقياء الميعا المقكور اي الفقر السابقة اإن عوى التعويل يمتنع سماعاا 2 – إذا كانت عوى التعويل نادئ ة عا جريمة نكانت الدعوى اليصا ية ماتصاف قا مة .بعد انقياء الميعا المقكور اي الفقر السابقة اإن عوى التعويل يمتنع سماعاا 2 – تسمع عوى التعويل اي جميع اواواف بانقياء مسة عشر سنة ما يوم نقوع ."الفعل اليار .بعد انقياء الميعا المقكور اي الفقر السابقة اإن عوى التعويل يمتنع سماعاا 2 – تسمع عوى التعويل اي جميع اواواف بانقياء مسة عشر سنة ما يوم نقوع ."الفعل اليار مجلة العلوم التربوية والدارسات اإلنسانية 332 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م ع م ع ي ع ."الفعل اليار صعوبة اي اومر– اي وء قا النص– إذا كا نت الدعوى المدنية قد قامت عل طا مدني يشكل جريمة. ااي تخيع– عند إذن– وقصر المدتيا: م سنواو أن مسة عشر سنة اس اواواف. أما إذا كانت الدعوى المدنية نادئة عا جريمةو اإناا تخيع اي سقوطاا كما يمكا إ ااة قيد أ ر– ناقا للنص القانوني السابق- للمدتيا ا لسابقتيا ن و أن الدعوى المدنية تسقط بانقياء المواعيد المقكور طالما أن الدعوى الينا ية لم تسقط مجلة العلوم التربوية والدارسات اإلنسانية 332 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م صعوبة اي اومر– اي وء قا النص– إذا كا نت الدعوى المدنية قد قامت عل طا مدني يشكل جريمة. ااي تخيع– عند إذن– وقصر المدتيا: م سنواو أن مسة عشر سنة اس اواواف. 1 - :مُضي المدة المانع من سماع الدعوى تخيع الدعوى المدنية النادئة عا جريمة جنا ية ايما يتعلق بميي المد لمبدأ ناد ميي المد بالنسبة لكل ما الدعوى المدنية نالدعوى الينا يةو نيعني ذلا أن الدعوى المدنية تخيع بالنسبة لمد عدم سماع الدعوىو لقاو المد التي تخيع لاا الدعوى الينا ية. لكا قا المبدأ يعطلق عل إط قة سواء اي القانون الفرنسي أن القانون الععماني. ااوصل اي القانون الفرنسي اس نص الما02 ما قانون اإلجراءاو الينا ية– نالمعدلة بقانون22 يسمبر0292 – أن تخيع الدعوى المدنية لميي المد المقرر اي القانون المدني إذا راعت أمام القياء المدني. أق أن الدعوى المدنية تخيع اي تقا ماا للم ا المقرر اي القانون المدني طالما راعت أمام القياء سواء كانت نادئة عا اليريمة أم . أما إذا راعت الدعوى المدنية أمام القياء الينا يو اإناا تسمع إذا كانت الدعوى الينا ية ذاتاا قد انقيت بميي المد . أق أن الدعوى تسقط بقاو المد التي تتقا م باا الدعوى ال ينا ية. نبقلا يتيا أن المشرع الفرنسي قد عدّف عا قاعد ا رتباط المطلق بيا الدعوييا المدنية نالينا ية نقصره– اقط– عل االة راع الدعوى المدنية أمام القياء الينا ي. لكا الدعوى المدنية تستقل عا الدعوى الينا ية– بالنسبة لميي المد– إذا ما ا تار المدعي المدن ي الطريق المدني لراع . 1 - :مُضي المدة المانع من سماع الدعوى أما إذا كانت الدعوى المدنية نادئة عا جريمةو اإناا تخيع اي سقوطاا كما يمكا إ ااة قيد أ ر– ناقا للنص القانوني السابق- للمدتيا ا لسابقتيا ن و أن الدعوى المدنية تسقط بانقياء المواعيد المقكور طالما أن الدعوى الينا ية لم تسقط صعوبة اي اومر– اي وء قا النص– إذا كا نت الدعوى المدنية قد قامت عل طا مدني يشكل جريمة. ااي تخيع– عند إذن– وقصر المدتيا: م سنواو أن مسة عشر سنة اس اواواف. أما إذا كانت الدعوى المدنية نادئة عا جريمةو اإناا تخيع اي سقوطاا بميي المد اتبق الدعوى المدنية– اي قه الحالة– قا مة روم انقياء مد عدم سماعااأ ون الدعوى الينا ية مازالت قا مة لم تسقط. أما إذا كانت مد سماع الدعوى قد انق يت بالنسبة للدعوى الينا يةو اقد تبق الدعوى المدنية قا مة إذا كانت مد عدم سماعاا لم تنقيي بعد. يستوق اي كل ذلا أن تكون الدعوى المدنية النادئة عا جريمة قد راعت أمام القياء المدني أن أمام القياء الينا ي. لكا سقوط الدعوى الينا ية بميي المد يمنع– ايما نرى– ما ا عاء أمام القياء المدني بالمسئولية النادئة عا اعل الشيء (ز ر و2204 .، 3 – :الجنائي يقيد المدني قد يفيل الميرنر المطالبة بالتعويل المستحق له أمام القياء المدنيو اي الوقت القق ايه الدعوى الينا ية النادئة عا ذاو الخطاأ منظور أمام القياء الينا ي اي ق ه :الحالة يبدن ا رتباط نا حا بيا الدعوييا ما ناجيتيا ::األول نقم السير اي الدعو المدنية إل ايا الفصل اي الدعوى الينا يةأ اللمدعي بالحق المدني الحق اي المطالبة بالتعويل أمام القياء المدنيو نتكون عواه– عند ق– مقبولة. لكا الحكم اياا يي أن يتا ر إل ايا .صدنر اكم ناا ي اي الدعوى المدنية ني اظ أن الفقه يق إل نجوت نقم السير اي الدعوى المدنيةو اومر القق يعني– اتما– نقم التحقيق اياا كلية إل ايا الفصل اي الدعوى الينا ية. ن نيمكا القوف أنه رنر لوقم السير اي إجراءاو الدعوى المدنية تماما و إنما يكفي أن يتا ر إصدار الحكم اي الدعوى المدنية إل ايا صدنر اكم ناا ي اي الدعوى الينا ية. نما ًمو الي ناك ما يمنع القا ي المدني ما التحقيق اياا نسماع الشاو مث . اي أناا نتيية اتمية لمبدأ تقيل القا ي المدني بالحكم الينا ي ما ناايةو نأناا تحوف– ما نااية أ رى– .إل عدم نقوع تيارت بيا أاكام القياء ن ي قاعد تتعلق بالنظام العامأ نما ًم ييوز وق ما الطرايا التمسا به اي أية مرالة تكون علياا الدعوى نلو ونف مر أمام محكمة النقل. كما يي عل القا ي– إذا لم يتمسا باا أاد الخصوم– أن يحكم باا ما تلقاء نفسه ن ييوز ل لخصوم العدنف عا الطل بعد إبدا ه أن راع الدعوى المدنية– بعد ذلا– .أمام القياء الينا ي نوالبا ما تطبق قه القاعد اي نطا المسئولية المدنية التي تقوم عل أسا الخطا إذ أن القا ي الينا ي يفصل– اتما– اي مسالة طا الماتم نما إذا كان ًابتا اي اقه . أم لكا ذلا يمنع ما تطبيق القاعد أييا اي مياف المسئولية بدنن طا. نيرجع ذلا– اي الواقع– إل أن ااتماف التناقل بيا القا ي المدني نالقا ي الينا ي مازاف قا ما أييا اي قا الفرض. االقا ي الينا ي يفصل اقط اي مدى ًبوو الخطاأ بل يفصل كقلا اي ًبوو اليرر نع قة السببية ن ي مسا ل يعرض لاا القا ي المدني نيخش أن ياتي اكمه متعار اا اي قا الخصوص مع القياء الينا ي. (ز ر و 2204 .، مجلة العلوم التربوية والدارسات اإلنسانية 332 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... 3 – :الجنائي يقيد المدني د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م ن يشترط– كما و الحاف بالنسبة لقاعد ايية الحكم الينا ي أمام القياء المدني – ناد المو وع نالسب اي الدعوييا المدنية نالينا يةأ بل ي كفي بتطبيق نقم الفصل مجلة العلوم التربوية والدارسات اإلنسانية 332 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م ن يشترط– كما و الحاف بالنسبة لقاعد ايية الحكم الينا ي أمام القياء المدني – ناد المو وع نالسب اي الدعوييا المدنية نالينا يةأ بل ي كفي بتطبيق نقم الفصل اي الدعوى المدنيةو أن يوجد طر التناقل بيا الحكم الينا ي نالحكم المدني. نيتحقق ذلا– عم– إذا كانت المسالة أن المسا ل المطرناة أمام القا ي الينا ي تتطابق– نلو جص يا– مع المسا ل التي يي أن يفصل اياا القا ي المدني. إذ يكون الحكم ا لصا ر ما القا ي الينا ي ملًرا– اي قه الحالة– عل القا ي المدني. نيترت عل ذلاو أن القا ي المدني يي أن يرال طل نقم السير اي الدعوى المدنية كلما كان اكمة مستنا عل عناصر ننقا ع تختلم عا تلا المطرناة أمام القا ي الينا ي كما يشترط– لتطبيق قه القاع د– ناد الخصوم اي الدعوييا المدنية نالينا ية اااتماف التيارت بيا القا ي المدني نالقا ي الينا ي يظل قا ما ات نلو ا تلم الخصوم اي الدعوييا. نيحدث ذلا– عم– إذا اركت النيابة العامة الدعوى الينا ية د المتامو نراع الميرنر الدعوى المدنية د المتبوع مث. :الاانية قو اومر المقيي: يترت عل نقم السير اي الدعوى المدنية أن يصدر الحكم الينا ي قبل صدنر الحكم المدني. اإذا أصبا الحكم الينا ي ناا يا ااز قو اومر المقيي ننج عل القا ي المدني– ما ًم– التقيد به. ن ييوز له– بناء عل ذلا – أن يخالم ما اصل ا يه القا ي الينا ي. ن ي قاعد قررتاا الما51 ما قانون اإلًباو الععماني رقم19 لسنة2229 بقولاا: " يرتبط القا ي المدني بالحكم اليصا ي إ اي الوقا ع التي اصل قا الحكمو نكان اصله اياا رنريا نمع ذلا اإنه يرتبط بالحكم الصا ر بالبراء إ إذا قام عل نف."ي نسبة الواقعة إل المتام ن كقا يفقد القا ي المدني اريته جص يا اي التصدق للوقا ع المطرناة أمامه ايث يي عليه أن يقبل كحقيقة تقبل النقاش ما انتا إليه القا ي الينا ي بإ انة المتام نًبوو الخطا اي اقه. 3 – :الجنائي يقيد المدني كل ذلا عل عك القا ي الينا ي القق يظل محتفظا بكامل سلطته اي التقديرو نالتصدق بحرية للوقا ع المطرناة أمامه ات لو كان القا ي المدني قد اصل اي المو وع– بالوقا ع التي اصل اياا القا ي الينا ي نكان اصله رنريا كما تقوف المحكمة ا تحا ية العليا بدنلة اإلماراو اي عباراو يعوز ا الو وتأ "... نايث أن قا النص اي وير محله ذلا أن المناط اي قاعد ارتباط القياء المدني بالحكم الينا ي و أن يكون الحكم اليصا ي قد اصل اص زما اي نقوع الفعل المكون لاسا بيا الدعوييا اليصا ية نالمدنيةو ناي الوصم القانوني لاقا الفعل ننسبته إل ااعلهو اإن اصلت المحكمة اليصا ية ا ي قه اومور اإنه يمتنع عل المحاكم المدنية أن تعيد بحثاا نيتعيا علياا أن تلتصماا اي بحث الدعوى المتصلة باا كي يكون اكماا مخالفا للحكم اليصا ي السابق له". نقد بررو المحكمة ا تحا ية العليا قه القاعد بقولاا: " ما المقرر أن الحكم الصا ر اي الدعوى اليصا ية له اييته المطلقة تياه الكااةو نأن لاقه الحيية قوتاا أمام المحكمة المدنية... نالعلة اي قو قه الحيية ليست مستقا ما اتحا الخصوم نالمو وع نالسب اي الدعوييا نإنما مستمد ما تواار اليماناو المختلفة المقرر اي اإلجراءاو اليصا ية ابتغاء الوصوف إل الحقيقة لما اياا ما ارتباط بالحرياو ناورنات نبمصلحة اليماعةأ مما أنج أن تكون اواكام اليصا ية محل ًقة مطلقة وير معر ة إلعا النظر اي المو وع القق اصلت ايه ات يل ق إل ت"خطئتاا ما جان جاة قيا ية أ رى (ز ر و2204 .، (المجلد5 ،) ( العدد10 ) يونيو3030م نيترت عل ذلا أن القياء المدني يتقيد بما قرره القا ي الينا ي ما ًبوو الخطا الينا ي أن نفيه اي اق المتامو نلي له– ما ًم– أن يحكم بالتعويل ب عد تبر ة المتام ما ارتكات الخطا– لكا اكم البراء الصا ر ما القا ي القق يكون له أًر أمام القا ي المدني إذا كان طل التعويل أمام قا او ير قد أنبن عل ال مسئولية المفتر ة تتطل– للحكم بالتعويل– ًبوو الخطاو ما لم يكا الحكم بالبراء قد ارتكص عل انتفاء رابطة السببية. كما أنه لي ناك ما يمنع القا ي المدني ما النظر اي نقوع طا ما المدع عليه إذا كان الحكم الصا ر بالبراء قد استند عل أن الفعل وير ميرم جنا ي ا و أن لبط ن التفتيش مث . 3 – :الجنائي يقيد المدني نقد طبقت المحكمة ا تحا ية العليا قه اواكام تطبيقا صحيحا بقولاا:" نايث أن النفي اي محلهو ذلا ون ايية الحكم الينا ي أمام المحكمة المدنية تقتصر عل المسا ل التي كان الفصل اياا رنريا لقيامه. ن ي طا المتام نرابطة السببية بي ا الخطا ناليرر ن يلصمه اي ذلا بحث مدى مسا مة الغير اي إاداث اليرر إ اي مياف تقدير العقوبة بيا ادياا او ن ناوعل و ن ي ما اومور الثانوية بالنسبة للحكم باإل انةو نما ًم اإن الحكم الينا ي نإن كانت له ايية ما ايث قيام ركا الخطا الموج إل انة المتام جنا يا نبالتالي مساءلته مدنيا و إ أن قه الحيية تمتد إل الداع بمسا مة الميني عليه اي الخطا ايا تقدير التعويل المستحق لا ير أمام المحكمة المدنية التي لاا ن ي اي سبيل ذلا أن تقرر مسئولية المتام ناده عما لحق الميني عليه ما رر أن مسا مة او ير معه اي إادا ث قا اليرر...". كما قررو محكمة تمييص بي"... أن الحكم الصا ر اي الموا اليصا ية تكون له ايية اي الدعوى المدنية أمام المحاكم المدنية إ إذا كان قد اصل اص زما اي نقوع الفعل المكون لاسا المشترك للدعوييا اليصا ية نالمدنية ناي الوصم القانوني لاقا ا لفعل ننسبته إل ااعلهو أما إذا كان الحكم اليصا ي الصا ر بالبراء مبنيا عل أن الفعل يعاق عليه القانون الينا ي سواء كان ذلا نتفاء القصد الينا ي أن لسب آ ر اإنه تكون له ايية الشي المحكوم ايه أمام المحكمة المدنيةو نبالتالي اإنه يمنع قه المحكمة ما البحث اياا إذا كان قا الفعل مع تير ه ما صفة اليريمة يصلا أساسا للمطالبة بالحقو اي الدعو المدنية. ن قا الدااع يتعلق بالنظام العامو نعل المحكمة– ما ًم– أن تقيي به ما تلقاء نفسااو نييوز إًارته ونف مر أمام محكمة النقل (ز ر و 2204 .، مجلة العلوم التربوية والدارسات اإلنسانية 320 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... 3 – :الجنائي يقيد المدني د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م الفرق بين المس ؤولية العقدية والتقصيرية: :المسؤولية العقدية هى الج اء ليو اإل ي بدإلت ا لقد يو ففدى طفد للر طصد هإ العق ؛ لةلا رجل اإلليإء طنفإ ل تع ر حلإطفإ بطقتضو العقد و لالتعدلرف فرفدإ رلدل ل الض الطتلى منج ر فى حفإب الطتعإى ر :المسؤولية التقصيرية تع الطف للر التقصر ر ليو نفإ: " الج اء ليدو اإل دي بإلت ا القإنل العإ بع اإلض ا بدإلغر و لهدةه الطفد للر تنشدأ لد القدإنل ؛ لدةلا ال رجددل االتيددإا ليددو تعدد ر حلإطفددإ ل اإلليددإء طنفددإو لالقددإنل هددل الددةي رحدد الضدد الةي رشطيج التعلرف ا الطف للر العق ر تعنى اإل ي بإلت ا لإ فإفدج لطصد ه العقد و طدإ الطفد للر تقصر ر فتعنى اإل ي بلاجب ىإنلنى ى ه القإنل لرت تب ليردج ضد ليغرد ؛ لليردج : فرطل التطرر بر الطف للرتر ط ي القلال القإنلنر اآلتر ي ا الطف للر العق ر تعنى اإل ي بإلت ا لإ فإفدج لطصد ه العقد و طدإ الطفد للر تقصر ر فتعنى اإل ي بلاجب ىإنلنى ى ه القإنل لرت تب ليردج ضد ليغرد ؛ لليردج : فرطل التطرر بر الطف للرتر ط ي القلال القإنلنر اآلتر (المجلد5 ،) ( العدد10 ) يونيو3030م 1 - :األهلية يشترط اي المسلنلية عا الفعل اليار بالغير بلو غ الفاعل سا الرددأ بل يشترط ايه التمييص اقط اي ااف المسلنلية التقصيريةو أما المسلنلية العقدية ايعتبر .بلوغ سا الردد أن ما يسم باو لية ما درنط المسلنلية العقدية 3 - :الخطأ تقتصر المسلنلية العقدية عل تغطية الخطا اليسير القق يعلحق ررا بالمتعاقد اآل رأ ا ي ايا أن المسلنلية التقصيرية تشمل الخطا اليسير ناليسيم المتعمد .أن القصدق 2 - :اإلثبات ايما يتعلق بع ء اإلًباو ايي النظر إل محل التصام المديا القق يل ق اإل ف به إل تقرير مسلنليتهأ اإذا كان قا المحل القيام بعملأ اع ء إًباته يقع عل المدياو نإذا كان ت اركا اي االع ء يقع عل عاتق الدا ا أن القاعد العامة اي اإلًباو ي أن البيئة عل ما ا ع أ اإذا كان محل التصام المديا قيام العملو ااو القق ا ع قيام المديا بعمل كان يي عليه عدم القيام بهأ اعليه يقع ع ء إًباتهو ا ع قة لع ء اإلًباو بنوع المسلنليةو بل محل التصام المديا. 3 – :الجنائي يقيد المدني د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م الفرق بين المس ؤولية العقدية والتقصيرية: :المسؤولية العقدية هى الج اء ليو اإل ي بدإلت ا لقد يو ففدى طفد للر طصد هإ العق ؛ لةلا رجل اإلليإء طنفإ ل تع ر حلإطفإ بطقتضو العقد و لالتعدلرف فرفدإ رلدل ل الض الطتلى منج ر فى حفإب الطتعإى ر :المسؤولية التقصيرية تع الطف للر التقصر ر ليو نفإ: " الج اء ليدو اإل دي بإلت ا القإنل العإ بع اإلض ا بدإلغر و لهدةه الطفد للر تنشدأ لد القدإنل ؛ لدةلا ال رجددل االتيددإا ليددو تعدد ر حلإطفددإ ل اإلليددإء طنفددإو لالقددإنل هددل الددةي رحدد الضدد الةي رشطيج التعلرف ا الطف للر العق ر تعنى اإل ي بإلت ا لإ فإفدج لطصد ه العقد و طدإ الطفد للر تقصر ر فتعنى اإل ي بلاجب ىإنلنى ى ه القإنل لرت تب ليردج ضد ليغرد ؛ لليردج : فرطل التطرر بر الطف للرتر ط ي القلال القإنلنر اآلتر مجلة العلوم التربوية والدارسات اإلنسانية 320 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... 3 – :الجنائي يقيد المدني نلما كان المديا اي المسلنلية التقصيرية عدم ا رار بالغيرو اإن ع ء اوًباو يقع عل عا ق المديا كقلاو نلي .اومر كقلا اي المسلنلية العقدية 2 – :مدى التعويض السب اي أن التعويل اي المسلنلية التعاقدية قاصر عل اليرر المبادر المتوقع و أن المت عاقديا لم يتوقعا اليرر المبادر وير المتوقعو نما اسبا .اسابه الي ما العدالة المسلنلية عنه 5 – :التضامن لما كان الخطا و السب المبادر لليرر اي المسلنلية التقصيريةو ( االقانون نا يلصمام باليمان اي تعويل اليررو كما نر اي نص الما15 2 ، ما القانون المدني" اور ني إذا تعد المسلنلياو عا اعل ارو كان كل منام مسلن بنسبة نصيبه ايه. نللمحكمة أن تقيي بالتسانق أن بالتي اما أن التكاال ايما يتعلق بينام "نأيياْ اي القانو( ن المدني الكويتي اي الما229 ، :" أ - إذ تعد اودخاص القيا ادث اليرر بخطئام التصم كل منام اي م واجاة الميرنر .بتعويل كل رر 5 – :التضامن لما كان الخطا و السب المبادر لليرر اي المسلنلية التقصيريةو ( االقانون نا يلصمام باليمان اي تعويل اليررو كما نر اي نص الما15 2 ، ما القانون المدني" اور ني إذا تعد المسلنلياو عا اعل ارو كان كل منام مسلن بنسبة نصيبه ايه. نللمحكمة أن تقيي بالتسانق أن بالتي اما أن التكاال ايما يتعلق بينام "نأيياْ اي القانو( ن المدني الكويتي اي الما229 ، :" أ - إذ تعد اودخاص القيا ادث اليرر بخطئام التصم كل منام اي م واجاة الميرنر .بتعويل كل رر ت – نيتوزع ورم المسلنلية ايما بيا المسلنليا المتعد يا بقدر نر الخطا كل منام ." اي إاداث اليررأ اإن تعقر تحديد قا الدنرو نزع عليام ورم المسئولية بالتسانق أما المسلنلية العقدية اإنه يفترض اياا التياماأ إ إذا اتيات اإلرا المشتركة .للمتعاقديا ناتفقا عل ذلا صرااة 2 – :من حيث إعفاء المسؤولية ييوز ا تفا عل اإلعفاء ما المسلنلية العقديةأ .نبالمقابل ا ييوز اإلعفاء ما المسلنلية اي المسلنلية التقصيرية ونه نظام عام مجلة العلوم التربوية والدارسات اإلنسانية 321 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... 3 – :الجنائي يقيد المدني د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م :الضرر كأساس المسؤولية التقصيرية لعديم التمييز الضرر:كأساس للمسؤولية التقصيرية لعديم التمييز في الشريعة اإلسالمية تتيددا الشدد رع اإلفدديطر طدد طددإ ةهددب الرددج فقفددإء القددإنل فددى تع ردد الضدد الطلجددب ليطفددد للر التقصدددر ر لنللردددج لهطدددإ: الضددد الطدددإ ي لالضددد الطعندددلي– فدددرأتى ش حفطإ الحقإ-؛ اال الش رع اإلفيطر ضإفت نلل إ إل دإ ال لهدل : الضد الط تد لهددل نددل ل فددج جددإ اليقددجو لهددل ريحددا الضدد فددى العددإ ل بإلشدد الطصددإب ليددو طصددإلحج الطإ ردد ل الطعنلردد بدد هددةا الضدد ال رقتصدد حرإنددإ ليددو الطضدد ل لح هو ب ىد ر تد ل يننعك علن أدنخاص آ نريا يصنيبام دخصنيا بوقوعنه أ نرارا أ رىو نيسم قا باليرر المرتد كاليرر القق يصي اوسر التي يمنوو عنا لام اني .،اا ًة (ما ق نمعنوق ا لضرررر سأسررام للمسررؤولية التقصرريرية لعررديم التمييررز فرري قررانون اإلجررراءات المدنيررة :بسلطنة عمان يكفي لقيام المسلنلية التقصيرية أن يقع طا نإنما يي أن يترتن عنا نررأ نيمكنا تعريم اليرر بصفة عامة باننه: اوذى النقق يصني الشنخص نتيينة المسنا بمصنلحة .مشرنعة له أن اق ما اقوقه ر نيقسم اقااء القانون اليرر إل نوعيا أساسييا ما الينرر المنا ق نالينرر المعننوقو نيييم إليه الفقه نالقياء اليرر المرتد– نالقق سبق بيانه- نيمكا تو يا نوعي اليرر عل النحو:اآلتي نيقسم اقااء القانون اليرر إل نوعيا أساسييا ما الينرر المنا ق نالينرر المعننوقو نيييم إليه الفقه نالقياء اليرر المرتد– نالقق سبق بيانه- نيمكا تو يا نوعي اليرر عل النحو :اآلتي 1 :) الضرر المادي و ما يصي الشخص اي جسمه أن اي مالهو ايتمثل اليرر اينئقا ( اي الخسار المالية التي تترت عل المسا بحق أن مصلحة، سواء كان الحق ماليا (كالحقو العينية أن الشخصية أن الملكية الفكرية أن الصناعية ،أ نيكون ررا ما يا إذا نيم عا قا الم سا انتقاص للمصايا المالية التي يخولاا نااد ما تلا الحقو أن وير مالي كالمسا بحق ما الحقو المتصلة بشخص ا نسان كالحرية الشخصية نارية العمل نارية الرأق كحب دخص نن اق أن منعه ما السفر للعمل يترت عليه . 3 – :الجنائي يقيد المدني رر ما ق أييا أ دريطة أن تكون المصلحة مشرنعة 3) الض:رر المعنوي أو األدبي و اليرر القق يلحق الشخص اي اقوقه المالية أن اي مصلحة وير ماليةأ ااو ما يصي الشخص اي كرامته أن اي دعوره أن اي دراه أن اي معتقداته الدينية أن اي عاطفته ن و أييا ما يصي العواطم ما آ م نتيية لفقدان دخص عصيصو نقد توسع القياء اي مفاوم المصلحة او بية ااعتبر ررا أ بيا كل ما يصي الشخص ما جراء الس أن القق ما إيقاء للسمعة أن عا آ م النف إل نطا .ما المحااظة عل اسم الشخص نارمة عا لته ندرااا أقر المشرع العماني اي قانون المعام و المدنية صرااة انتقاف التعويل عا اليرر المورنث إل الور ًة نن تفرقة بيا نوعي اليرر الما ق نالمعنوقأ االيرر بنوعيه ينتقل إل الورًة باعتباره أاد موجو او تركة الميرنر قبل نااتهو ن و ينشا بمير نقوع الفعل اليارو نينتقل إل نرًة الميرنر اور اإلع ن عا نااته سواء كان التعويل قد تحد قداره أم لم يتحد و نسواء طال به الميرنر قبل نااته با تفا .أن بالقياء أن لم يطال به نبناء عل ما تقدم يمكا القوف أن التعويل النات عا اليررأ يعد جصءا ما .التركةو نيي توزيعه عل الورًة ناقا لقواعد الميراث الشرعية :شروط الضرر الموجب التعويض يشترط لتحقيق اليرر الموج التعويل:الشرنط اآلتية 0 - اإل ف بحق مالي (مصلحة مالية،: يي لوقوع اليرر أن يكون ناك إ ف بحق الميرنر أن بمصلحة مالية له كاإل ف بحق الميرنر إذا أار دخص منصفا آل ر أن أتلم زرعهأ ايي مساءلة المعتدق ونه م باعتدا ه اقا يحميه القانون نيستوق اي قا أن يكون الح ق ماليا و ناي قا يشترط أن تكون المصلحة مشرنعة لوجوت تعويل .او رار :شروط الضرر الموجب التعويض يشترط لتحقيق اليرر الموج التعويل:الشرنط اآلتية 0 - اإل ف بحق مالي (مصلحة مالية،: يي لوقوع اليرر أن يكون ناك إ ف بحق الميرنر أن بمصلحة مالية له كاإل ف بحق الميرنر إذا أار دخص منصفا آل ر أن أتلم زرعهأ ايي مساءلة المعتدق ونه م باعتدا ه اقا يحميه القانون نيستوق اي قا أن يكون الح ق ماليا و ناي قا يشترط أن تكون المصلحة مشرنعة لوجوت تعويل .او رار 0 - اإل ف بحق مالي (مصلحة مالية،: يي لوقوع اليرر أن يكون ناك إ ف بحق الميرنر أن بمصلحة مالية له كاإل ف بحق الميرنر إذا أار دخص منصفا آل ر أن أتلم زرعهأ ايي مساءلة المعتدق ونه م باعتدا ه اقا يحميه القانون نيستوق اي قا أن يكون الح ق ماليا و ناي قا يشترط أن تكون المصلحة مشرنعة لوجوت تعويل .او رار 2 - أن يكون اليرر محققا : كي يتوار اليرر بد أن يكون قد نقع اع أن أنه ملكد :الوقوع اي المستقبل ناي قا يي التمييص بيا ً ًة أقسام لليرر المستوج التعويل أ- اليرر الواقع: قا الواقع اع ن مشكلة تثار اوف نقوعه كإصابة الشخص نتيية . 3 – :الجنائي يقيد المدني رر ما ق أييا أ دريطة أن تكون المصلحة مشرنعة 1 :) الضرر المادي و ما يصي الشخص اي جسمه أن اي مالهو ايتمثل اليرر اينئقا ( اي الخسار المالية التي تترت عل المسا بحق أن مصلحة، سواء كان الحق ماليا (كالحقو العينية أن الشخصية أن الملكية الفكرية أن الصناعية ،أ نيكون ررا ما يا إذا نيم عا قا الم سا انتقاص للمصايا المالية التي يخولاا نااد ما تلا الحقو أن وير مالي كالمسا بحق ما الحقو المتصلة بشخص ا نسان كالحرية الشخصية نارية العمل نارية الرأق كحب دخص نن اق أن منعه ما السفر للعمل يترت عليه . :إعبء إثبات الضرر يقع ع ء اإلًباو عل ما يدعيهو نذلا ناقا لما تقيي به القاعد العامة ما أن المندعي نو المكلم بإًباو" ما يدعينه " البيننة علن منا ا عن نإًبناو الينرر أن نفينه منا اومنور الواقعينة التي تقدر ا محكمة المو وع ن رقابة اياا للمحكمة العلياو أما تحديد اليررو نبيان عناصنره نموجباته نتكييفهأ كلاا تخيع لرقابة المحكمة العليا وناا كلاا منا مسنا ل القنانون التني يخينع اياا قا ي المو وع للرقابة. ن يكتفي ما المدعي بإًباو اليرر القق أصابه ن طا المندعي عليه بل عليه أن يثبت اليرر القق يدعيه إنما و ن ادئ عنا طنا المندعي علينه مبادنر أق أن .يثبت الع قة المبادر بيا اليرر نالخطا المسب لليررو نتلا ي الع قة السببية الخطأ سأسام للمسؤولية التقصيرية لعديم التمييز: م إن المسلنلية عا العمنل الشخصني ني تلنا التني تترتن علن عمنل يصندر منا المسنلنف نفسننه نأن المسننلنل ية التقصننيرية كالمسننلنلية العقديننة أركاناننا ً ًننة ن نني الخطنناو ناليننررو نع قة السببية بيناماو كما يتيا بان أسا قه المسلنلية نو الخطنا الواجن ا ًبناوو نعلن الميرنر إًباتهو اإذا ًبت الخطا نترت عليه رر للغير اإن مرتكبه يلتصم بتعويل الغير عا قا اليررو نللقا ي او سا اق تقدير قيام الخطاو كما له اق تقدير انتفا هو وينر أننه يخينع لرقابنة المحكمنة العليننا اني عمليننة تكييفنه القننانونيو نيمكنا تننانف ركننا الخطنا لعننديم التميينص مننا نننننناايتيا إاننننندا ما دنننننرعية ناو نننننرى قانونينننننة كننننناآلتي (الينننننندقو2205أز نننننر و2204 أ عرااوو2204 :، ، الخطأ سأسام للمسؤولية:التقصيرية لعديم التمييز في الشريعة اإلسالمية :تعريف الخطأ يتوقم تحديد اركان الخطا عل التعرينم النقق يعطن لنه نلنو أن إعطناء تعرينم للخطا لي باومر الاياو إذ لم يختلم الفقااء اي أمر كا ت اام اي قا المو وعو نلنقا تبايننت تعريفنناو الخطننا تباينننا كبيننرا . ويننر أن الفقنن ه يميننل إلنن او ننق بننالتعريم التقلينندق للخطنناو ن ننو انحنرا اني سنلوك الشنخص منع ا راكنه لانقا ا نحنرا . 3 – :الجنائي يقيد المدني اا ث السيار ت- رر ملكد الوقوع: و رر لم يقع بعد نلكا نقوعه ملكد اسب اليرر قد تحقق نلكا آًاره كلاا أن بعياا ترا ت اي المستقبلأ كإصابة عامل بعا ة مستديمة تيعله عاجصا عا الكس مستقب أ ايعوض عا اليرر القق نقع عليه اع جراء عيصه عا العمل اي الحافو نعا اليرر القق سيقع اتما نتيية عيصه عا العمل اي المستقبل االتعويل دمل اليرر الحاليو ناليرر المستقبل المحقق الوقوعو أن تادم منصف يكون اتمي ن بد ما نقوعه نتيية لعمل آ و مصنع ميانر أ و إل او رار باوسا و اإن ال.يرر اي قه الحالة يكون ملكد الوقوع ج- اليرر ا اتمالي: و اليرر القق لم يقع بعدأ نلكا نقوعه مستقب وير محقق الوقوعو ااو يختلم عا اليرر المستقبلي ن تقوم عليه المسلنلية المدنيةأ بل ينتظر ات يصبا ا اتماف يقينا ا تعويل عنه إ إذا تحقق اع أ كان يعحدث دخصا بخطئه ل اي منصف جاره ااو رر محقق يلصم المسلنف بإص اه أما ما قد يل ق إليه الخلل ما انادام المنصف اي المستقبل ااو ما قبيل اليرر المحتمل ن تعويل عنه إ إذا .انادم اع نتيية قا الخلل نينبغي عدم الخلط بيا اليرر المحتملو ناليرر المتم ثل اي تفويت ارصة ن ي ارمان الشخص ارصة كان يحتمل أن تعو عليه بالكس أ االفرصة أمر محتمل نلكا تفويتاا أمر محققو كان يصدم دخص كان اي طريقه إل أ اء امتحان اي مسابقةو اقد اوتت عليه الفرصة أن الفوزو ن قا القدر كا لتحقق اليرر القق يقع اع ااو .مستوج التعويل 2 - أن يكون اليرر دخصيا: ن قا الشرط ينصر القصد ايه إل أنه إذا كان طال التعويل و الميرنر أص ايي عليه أن يثبت ما أصابه دخصيا ما رر نإذا .كان طل التعويل بصفة أ رى ااإلًباو يكون لليرر الشخصي لما تلق الحق عنه 4 - أن يكون قد سبق تعوييه: إذ أنه يي وز أن يحصل الميرنر عل أكثر ما تعويل إلص ت رر بعينهو اإذا قام معحدث اليرر بما يي عليه ما تعوييه .ا تيارا أ اقد أنا بالتصامهو ن محل بعد ذلا لمطالبته بالتعويل وير أنه إذا كان الميرنر ملمنا عل نفسه د ما قد يصيبه ما اوا ثأ اإنه يمكنه بعد الحصوف عل تعويل دركة التاميا أن يطال بعد ذلا محدث اليرر بالتعويل .بما لم يشمله مبلغ التاميا كما يمكا القوف-نناقا لما تم عر ه- أن اليرر او بي كاليرر الما قأ يي أن يكون محققا ندخصيا نلم يسبق التعويل عنه ات يمكا للقا ي التعويل عنه ناومر اياا يخيع تقديره لم .حكمة المو وع كما يمكا القوف-نناقا لما تم عر ه- أن اليرر او بي كاليرر الما قأ يي أن يكون محققا ندخصيا نلم يسبق التعويل عنه ات يمكا للقا ي التعويل عنه ناومر اياا يخيع تقديره لم .حكمة المو وع :إعبء إثبات الضرر :إعبء إثبات الضرر ن نقا التعرينم كمنا نو ظنا رأ يبنني الخطننا علنن ركنننيا أساسننييا: أانند ما مننا ق ن ننو ا نحننرا نالتعنندقو ناآل ننر معنننوق ن ننو ا راكأ إ أن الشريعة اإلس مية تعتبر الفعنل الينار نو الن قق يطلنق علينه التعندق اني قنانون المعام و المدنية العماني (ز ر و2204 .، إ نجدير بالقكر أن الفقه اإلس مي له قص السبق اي قا الميمار (عبدالمييد الحكيمو522 ، اقد تقدم كل التشريعاو بص اء أربعة عشر قرنا اي التسليم بالمسئولية الما يةو ناي إقرار مبدأ الغرم بننالغنم ن لننم يابننه بعنصننر الخطننا انني التصننرااو الفعليننةأ اقنند جعننل مرتكنن الفعننل اليننار ننامنا عواق اعله بصر النظر عا التعمندو نالتعندق أق عنا ا راك نالتميينصو االمبادنر ن نو منا .أادث اليرر اما نأن لم يتعمد أن يتعد (المجلد5 ،) ( العدد10 ) يونيو3030م االمسئولية المالية لإلنسان تبدأ منق مولده نبغل النظر عا مراال اياتنه نتفنانو ا راكنهو نمع ذلا اقد أقنر الفقنه ا سن مي اكنر الخطنا اني نطنا نيق نسنماه تقصنيرا أن عندم تحنرزو . نتطلبه اي اا و الخطا المفرنض وير قابل إلًباو العك اي القوانيا المدنية المعاصر الخطا كاسا للمسلنلية التقصيرية لعديم التمييص اي قانون ا لمعام و ا:لمدنية بسلطنة عمان ( تنننص المننا071 ، مننا قننانون المعننام و المدنيننة العمنناني علنن أن " كننل إ ننرار بننالغير يلننصم ااعلننه نلننو كننان ويننر مميننص بننالتعويل"و نتشننير المننا سننالفة الننقكر إلنن أنننه يشننترط انني قيننام المسلنلية تواار ً ًة أركان تتمثل اي الفعل الينارو نالينررو نالع قنة السنببي ة منا بنيا الفعنل ،ناليرر. االمشرع العماني يقيم المسلنلية التقصيرية عل أسا نجنو الفعنل الينار (الخطنا نصور ذلا أن ياتي الشخص بفعل وير مشرنع "اإل رار" نيفام ما ذلا أن كل اعل أن عدم الفعننل يلحننق اليننرر بننالغير يسننتوج تعوييننهأ نبالتننالي كننل إ ننرار بننالغير يعتبننر عمنن ويننر مشرنع أن مخالم للقانون. باعتبار أن المشرع العمناني لنم يا نق بالخطنا كنركا لقينام المسنلنلية التقصيرية مكتفيا لناوض مسلنلية مرتك الفعل أن يكنون قند ارتكن اعن وينر مشنرنع ألحنق ررا بالغير نلانقا يشنترط لقينام المسنلنلية التقصنيرية عنا الفعنل الينار أن يكنون مرتكن الفع. :إعبء إثبات الضرر ل اليار مميصا إإ االمسلنلية التقصيرية تنشا نتيية اإل ف بالتصام قانوني عام ن و (ا لتصام بعندم اإل نرار بننالغير، نأن ننقا ا لتننصام بطبيعتننه التننصام ببننقف عنايننة بمننا يوجنن علنن الشننخص أن يتبننع سننلوك المتيننرر انني أمننوره محتننرز انني تعاملننه مراعيننا القننوانيا ناونظمننةأ بحيننث يننل ق اعلننه إلنن .إ رار الغير ( ناإل رار بالغير بد لة الفقر2 ( ، ما الما071 ، ما قانون المعام و المدنية العمنانيو قد يكون بالمبادر أن التسنب "إذا كنان اإل نرار بالمبادنر لنصم التعنويل نإن لنم يتعندىو نإذا كان بالتسب ايشترط التعندق". نكمنا مثناف للننوع او نف: قينام دنخص بكسنر إاندى نواانق مننصف جارهو ايث اعل اليرر مبادر نبالتالي يشترط لقيام المسنلنلية أن يكنون نناك تعند. أمنا إذا كان اليرر بالتسب كما قطع ابل قننديل معلنق اسنقط القننديل علن اورض نأنكسنر يكنون قند أتلم الحبل بالمبادر نكسر القنديل بالتسب و نبالتالي ادت رط المشرع العماني اي قه الحاف أن يكون ناك تعد أق تعمدو نبمعن آ ر ات يلصم الشخص أن يكون الفعنل النقق أتناه مفينيا إلن .اليرر معيار التعدق (الفعل اليار،: يعتبنر النركا المنا ق اني الخطنا نو التعندق أن ا نحنرا اني السلوكو نأن التعدق يقابل الفعل اليار أق الفع ل القق يل ق إل الينرر اني ذاتنهو ن نقا الفعنل .ناده يستوج اليمان اي الفقه اإلس ميو نقانون المعام و المدنية العماني نلكا كيم يقا التعدق أن بعبار أ رى ما نو المعينار النقق يرجنع إلينه اني تحديند ا نحنرا ؟ يفننر اقانناء الشننريعة اإلسنن مية ناقانناء القننانون انني ننقا المعيننار بننيا الفعننل المتعمنند نويننر المتيعييميدو ااذا كان الفعل متعمدا أق قصد به صاابه اإل رار بالغير اإن المعينار يكنون عند نق معيارا ذاتيا أق دخصيا بمعن أن القا ي يرجع إل المسلنف نفسه ليبحنث اني مكننون نميره .ن فايا صدره إ مجلة العلوم التربوية والدارسات اإلنسانية 325 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... :إعبء إثبات الضرر د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م ر ي ن أما اذا كان العمل أن الفعل اليار وير متعمد أق نقع نتيية اإل مافأ ا يل ق اني قينا التعندق بالمعيار القاتي ون قا المعينار مبنناه رجنة اليقظنة لندى الشنخصأ ينل ق إلن محاسنبة الرجنل الشديد اليقظة عل أقل فو تصدر منهأ نبالمقابل يل ق إل إا و معتا اإل ماف ما مسنلنليته عنا الخطنا اليسننيرو نقا باإل نااة إلنن منا يقتينيه ننقا المعينار منا بحننث اني عنا او المسننلنف لمعراة رجة يقظتنهو ًنم يقنا منا نقنع مننه الن المنالو منا سنلوكه لمعرانة منا إذا كنان يعتبنر انحرااا أم و ن يخف ما اني نقا البحنث منا نصن نمشنقة نلانقا اسنتقر الفقنه نالقيناء ع لن أما اذا كان العمل أن الفعل اليار وير متعمد أق نقع نتيية اإل مافأ ا يل ق اني قينا التعندق بالمعيار القاتي ون قا المعينار مبنناه رجنة اليقظنة لندى الشنخصأ ينل ق إلن محاسنبة الرجنل الشديد اليقظة عل أقل فو تصدر منهأ نبالمقابل يل ق إل إا و معتا اإل ماف ما مسنلنليته عنا الخطنا اليسننيرو نقا باإل نااة إلنن منا يقتينيه ننقا المعينار منا بحننث اني عنا او المسننلنف لمعراة رجة يقظتنهو ًنم يقنا منا نقنع مننه الن المنالو منا سنلوكه لمعرانة منا إذا كنان يعتبنر انحرااا أم و ن يخف ما اني نقا البحنث منا نصن نمشنقة نلانقا اسنتقر الفقنه نالقيناء ع لن او ننق انني ننقه الحالننة بمعيننار ميننر و ايقننا سننلوك المسننلنف بسننلوك الشننخص العننا قو ن ننو .دخص يمثل أناسط النا و ا و دديد اليقظة ن و معتا ا ماف نا عتدا بسلوك الشخص العا ق لقينا ا نحنرا أن التعند قو إذا كنان يتطلن عندم او نق بالظرن الدا لية الخاصة بال مسلنفو ن ي الظرن التي ترجنع إلن طبيعنة الشنخص ننفسنيته نعا اته ناالته الصحية كظرن المرض أن تادم اوعصات أن عم ا بصارو أن التي ترجع إل سنه بان كان صبيا أن دابا أن ديخا و أن إل نوعه ا جتماعي بان كان رج أن امرأ و إ أن قا القيا يفترض عدم إسقاط الظر ن الخارجيةأ كظر المكان أن الصمانو بمعنن أننه يين ا عتنندا انني قيننا مسننلا المسننلنف بمننا يكننون عليننه مسننلا الشننخص العننا ق لننو نجنند انني مثننل ظرناننه الخارجيننةو اسننا ق السننيار الننقق يقننع منننه ا نحننرا مننث و يعتنند انني مسننلكه عننند قياسننه بمسلا الشخص العا ق بالمكان القق كان يسير ايهو ب ان كنان مديننة أم قرينةو طريقنا مص امنا أم طريقا وير مطرن و كثير المنحنياو أم مستقيما و كمنا يعتند بظنر الصمنان كاللينل أن النانارو أن باق ظر ارجي آ ر كحالة اليو بان كان صحوا أن ممطرا (اليندقو2205أز نر و2204 أ عرااوو2204 ،. :إعبء إثبات الضرر :التعدي أو االضرار بالفعل أو بالترك اار ايمنا يتعلنق بالتعندق أق اإل نرار أن يتخنق انحنرا الشنخص مظانرا إييابينا أن مظانرا سلبيا و ذلنا أن ا متنناع أن التنرك يعتبنر عمن وينر مشنرنع إذا كنان نناك ناجن قنانوني ينان . عنهو كوجوت إ اء مصابيا السيار لي ناك ما اقااء القانون ما يفر بنيا مينر التنركو ن بيا ا متناع المصحوت بنشاط سنابق منا المسنلنف امينر التنرك اينث يوجند التنصام سنابق بعمل يترت أية مسلنلية مدنية عل الشخصو كما لو امتننع عنا انقناذ ورينقو ون اني القنوف بغير ذلا اعتداء عل الحرية الفر ية نانتقاص لااو ون مد يد المساعد إل الغير ناج أ قي نلي بواج قانوني. أما ا متناع المصحوت بنشاط سابق ما الفر اموج لمسلنليتهو كما لنو امتنع سا ق السيار عا إ اء مصابيحاا لي أن عا استعماف آلة التنبيه عند اللصنم. أ أن والبية اقااء القنانون يميلنون إلن نقه التفرقنة نيقنررنن مسنلنلية الممتننع منا ام قند انح ر اني سنلوكه عنا سنلوك الشنخص العنا قو سنواء اني ذلنا كنان نقا ا نحنرا مصنحوبا بنشنناط سننابق مننا جانبننه أم كننان ميننر تننرك (السننناورقو الوسننيط بننند542 ،و (وننانمو0219 و 421 .، :التعدي أو االضرار بالفعل أو بالترك اار ايمنا يتعلنق بالتعندق أق اإل نرار أن يتخنق انحنرا الشنخص مظانرا إييابينا أن مظانرا سلبيا و ذلنا أن ا متنناع أن التنرك يعتبنر عمن وينر مشنرنع إذا كنان نناك ناجن قنانوني ينان . عنهو كوجوت إ اء مصابيا السيار لي ناك ما اقااء القانون ما يفر بنيا مينر التنركو ن بيا ا متناع المصحوت بنشاط سنابق منا المسنلنف امينر التنرك اينث يوجند التنصام سنابق بعمل يترت أية مسلنلية مدنية عل الشخصو كما لو امتننع عنا انقناذ ورينقو ون اني القنوف بغير ذلا اعتداء عل الحرية الفر ية نانتقاص لااو ون مد يد المساعد إل الغير ناج أ قي نلي بواج قانوني. أما ا متناع المصحوت بنشاط سابق ما الفر اموج لمسلنليتهو كما لنو امتنع سا ق السيار عا إ اء مصابيحاا لي أن عا استعماف آلة التنبيه عند اللصنم. ع م أ أن والبية اقااء القنانون يميلنون إلن نقه التفرقنة نيقنررنن مسنلنلية الممتننع منا ام قند انح ر اني سنلوكه عنا سنلوك الشنخص العنا قو سنواء اني ذلنا كنان نقا ا نحنرا مصنحوبا بنشنناط سننابق مننا جانبننه أم كننان ميننر تننرك (السننناورقو الوسننيط بننند542 ،و (وننانمو0219 و 421 .، مجلة العلوم التربوية والدارسات اإلنسانية 322 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... :إعبء إثبات الضرر د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م ، :سلطة محكمة الموضوع في تقديرها لتعويض المضرور عن الضرر الذي لحق به بالنظر إل الفتانى القيا ية المتعل قة بالمسنلنلية التقصنيريةو نالصنا ر عنا المحكمنة العلينا اني سلطنة عمان ي اظ أنانا قند أصندرو اتنانى قينا ية تتعلنق بتعنويل المينرنر جبنرا للينرر القق لحقهأ اقند قينت المحكمنة العلينا بنان " تقندير التعنويل الينابر للينرر منا ا تصاصناو محكمننة المو ننوعو ن رقابننة علنن تقنندير ا مننا ام أ ن القننانون لننم يوجنن اتبنناع معننايير معينننة للتقديرو نيكفي إًباتاا لعناصر اليررو نتقدير التعويل إجما ". (الطعنون مندني أرقنام214 و ن219و ن252 / 2221 جلسة20 / 4 / 2227 أ ميموعة أاكام المحكمة العليا لسننة7 ص229 ،و نالطعننا التيننارق رقننم040 / 2202 جلسننة07 / 4 / 2202 .ميموعننة اواكننام السنننة02و ن04 و ص595 .، كما أصدرو ذاو المحكمنة اكمنا قينا يا يقيني "بوجنوت تعينيا منا يندااع عنا القاصنر تحقيقنا للعدالة" (طعا درعي رقنم02 / 2202 جلسنة22 / 02 / 2202 .ميموعنة أاكنام المحكمنة العلينا السنة02/ن04و ص2 .، :الدراسات السابقة :سو يتم استعراض الدراساو السابقة ناقا لتسلسلاا الصمني عل النحو اآلتي :سلطة محكمة الموضوع في تقديرها لتعويض المضرور عن الضرر الذي لحق به بالنظر إل الفتانى القيا ية المتعل قة بالمسنلنلية التقصنيريةو نالصنا ر عنا المحكمنة العلينا اني سلطنة عمان ي اظ أنانا قند أصندرو اتنانى قينا ية تتعلنق بتعنويل المينرنر جبنرا للينرر القق لحقهأ اقند قينت المحكمنة العلينا بنان " تقندير التعنويل الينابر للينرر منا ا تصاصناو محكمننة المو ننوعو ن رقابننة علنن تقنندير ا مننا ام أ ن القننانون لننم يوجنن اتبنناع معننايير معينننة للتقديرو نيكفي إًباتاا لعناصر اليررو نتقدير التعويل إجما ". :إعبء إثبات الضرر (الطعنون مندني أرقنام214 و ن219و ن252 / 2221 جلسة20 / 4 / 2227 أ ميموعة أاكام المحكمة العليا لسننة7 ص229 ،و نالطعننا التيننارق رقننم040 / 2202 جلسننة07 / 4 / 2202 .ميموعننة اواكننام السنننة02و ن04 و ص595 .، ، كما أصدرو ذاو المحكمنة اكمنا قينا يا يقيني "بوجنوت تعينيا منا يندااع عنا القاصنر تحقيقنا للعدالة" (طعا درعي رقنم02 / 2202 جلسنة22 / 02 / 2202 .ميموعنة أاكنام المحكمنة العلينا السنة02/ن04و ص2 .، ( قامننا اسننام النندياو نإيمننان نننابوش2201 ، بدراسننة تنانلننت المسننلنلية التقصننيرية لعديم التمييصو ن دات إل التعر عل قوانيا بعل الدنف العربية اي ميناف المسنلنلية التقصي ريةو نأنجه ا تفا نا اترا ايما بيناا اي قا الميافأ إلن جانن تحديند أسنبات التبنايا بنيا قنوانيا تلنا الندنف انني ميناف المسنلنلية التقصنيرية لعنديم التميينصأ إذ قننررو بعننل تلننا النندنف نبشننكل أصننلي نجننو ننقه المسننلنلية اسننتنا ا لمننا ذ نن اليننه الفقننه اإلسنن مي نمناننا مننا تنناًر بالقننان ون الفرنسنني انني عنندم تقريننر ننقه المسننلنليةو نآ ننرنن مصجوا بيا ك الناييا. نتوصن إلن أن والبينة الندنف صناوت قوانينانا نقنم منا قنرره الفقننه اإلسنن مي انني تقريننر المسننلنلية التقصننيرية الكاملننة نالشخصننية ناوصننلية لعننديم التمييننص بننالروم مننا نجننو ا ت انناو دننكلية انني الصننياوة القانونينن ة نا ننت انني بعننل .التفاصيل بيا تلا القوانيا - ( أدار دمحم صبرق اليندق2205 ، اي راسته بعنوان المسلنلية التقصيرية "المسلنلية عا الفعل اليار" راسة اي الفقه الغربي نالفقه اإلس مي نالقانون المدني اور ني إل المسلنلية عا الفعل الشخصي. ن دات الدراسة إل التعر ع ل المسلنلية عا الفعل الشخصي نأنواعاا نالتمييص بيناا ما اليان اليصا ي نالمسلنلية العقدية نالمسلنلية التقصيرية. كما دات إل البحث عا أس المسلنلية ناإلرا نأ ميتاا ما ف تتبع بعل النظرياو كنظرية تحمل التبعةو ننظرية الغرم بالغنمو ننظرية مخاطر السلطة ننظ رية اليمانو إل جان البحث اي مفاوم التعدق نإبراز صوره نمبرراته. نتوصل البااث إل أن نقطة الخ بيا اقااء القانون تتحد اي اليرر كاسا المسلنلية عا الفعل الشخصي. كما أن القوانيا المختلفة اي مياف المسلنلية التقصيرية أ قو برأق الفقه اإلس مي اي قا الشان. حمدإ ي ي لإ ق - ( ناقام دمحم المرسي ز ر2204 ، بدراسة بعنوان المصا ر وير اإلرا ية ل لتصامو اي القانون العماني الفعل اليار نالفعل النااع والبحث اي أاكام الفعل اليار نأاكام الفعل النااع اي مياف المسلنلية المدنية. نقد اتبع البااث أسلوت عدم مساير المشرع جص يا تينبا للتكرا ر ن راسة اواكام العامة نالمسلنلية عا اوعماف الشخصية معا نذلا داا لو ع نظرية عامة واكام المسلنلية الشخصية. :إعبء إثبات الضرر نقد أدار البااث اي راسته إل متغير الفعل اليار ما عد أبعا ن ي: المسلنلية عا اوعماف الشخصيةو نالمسلنلية عا اعل الغيرو المسلنلية عا اعل الشيء (اودياء الحية/ اودياء وير الحية،و نأًر المسلنلية ناسر اياا التعويل نمقداره نكيفيته نسلطة القا ي اي تقدير التعويل نوير ا. كما تنانف البااث المتغير الثاني ن و الفعل النااعأ ايث بيا البااث موقم الفقه اإلس مي ما اإلًراء. كما ذكر بعل القواعد العامة لإلًراء ك الكس ب سب و نتطبيقاتاا. نتوصل البااث إل أن للقا ي المدني السلطة التقديرية تحديد مقدار التعويل ن اصة اي الدية ناقا للظرن ا قتصا ية للدنلةو نأن الشريعة اإلس مية تمنع اي جو ر ا او ق بمبدأ اإلًراءو نالقق يعد مصدرا عاما ل لتصام. مجلة العلوم التربوية والدارسات اإلنسانية 322 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م نتوصل البااث إل أن للقا ي المدني السلطة التقديرية تحديد مقدار التعويل ن اصة اي الدية ناقا للظرن ا قتصا ية للدنلةو نأن الشريعة اإلس مية تمنع اي جو ر ا او ق بمبدأ اإلًراءو نالقق يعد مصدرا عاما ل لتصام. - ( نقامت نا يا عرااو2204 ( ، بدراسة بعنوان المسلنلية التقصيرية لعديمي التمييص بيا النظرية التقليدية نالنظرية الحديثة، راسة مقارنةأ بان المسلنلية التقصيرية ما منطلق الما022 الفقر الثالثة ما قانون الموجباو نالعقو اور ني القق ينص عل المسلنلية ا ستثنا ي ة لعديم التمييص نإلصامه بالتعويل. ن دات البااثة إل تفسير اوسا القانوني القق يستند عل القانون نا جتاا الفرنسي لاقه الفقر التي لم تيد تفصي نا حا ما قبل الفقه نا جتاا المقارن. ايث تنانلت البااثة مفاوم اليرر نصورهو نالتعدقو نمفاومه نالحا و التي يس تبعد اياا التعدق نأًر استبعا ه عل نشوء اليمانو نرابطة السببية اي الفقه اإلس ميو ناي القانون اور ني نالقياء .اور ني نالمقارن ي أظارو نتا البحث إمكانية إعا تنظيم اواكام القانونية المختصة بمساءلة عديمي التمييص اي القانون المقارنو ناقترات كيفية تام يا الحماية لام نليحايا مو نذلا بإقرار قانون مان مسلنلية عديمي التمييص. - ( قام أامد بوكرزاز2204 ، بدراسة بعنوان المسلنلية المدنية للقاصرو راسة مقارنة(رسالة كتوراه علوم اي القانون الخاص،. تنانف اياا اإلدكا و القانونية اوف المسلنلية التقصيرية نالمسلنلية ا لعقدية. :إعبء إثبات الضرر ايث دات الدراسة إل تمييص ائة ااقدق او لية بخصوصياو نأاكام اصةو نالتعر عل ا ت مركص م القانوني اي القوانيا المقارنة نالقانون اليصا رقو نالتعر عل نجه الحقيقة ما المسلنلية المدنية .العقدية ن التقصيرية لاقه الفئة اتبع البااث المنا التحليلي المقارن لتحليل نصوص القانون نالمقارنة بيا ميموعة ما القوانيا المدنية نالتشريع اإلس ميو كما استخدم المنا التاريخي نذلا للتعر عل تاريخ بعل النصوص القانونية التي يعو أصلاا للقانون الرنماني نالفرنسي نالمصرق ناليصا رق. نأظارو نتا الدراسة أن المسلنلية الت قصيرية .مشكلة معقد نمتدا لة بيا اق الميرنر نأ مية اماية عديم التمييص اي نف الوقت كما أظارو أن مسلنلية عديم التمييص عا او رار التي سبباا اعله يعد إ بالنظم ،ا جتماعية نبعدا عا السلوك القويمو ن ي ما المنطلق الشرعي ( رر ن رار ايث أن المسلنلية تقوم عل تبعة الفعلو نأظارو الدراسة تناقيا نعدم انسيام اي القانون اليصا رق اي تنانف مو وع المسلنلية التقصيرية ايث يعتمد المشرع اليصا رق عل الفقه ( اإلس مي اي تشريع قانون اوسر ، نالقانون المصرق نالفرنسي اي تشريع القانون المدني ايث أقر المشرع المسلنلية التقصيرية للقاصر المميص عا جميع او رار كما أبقت نف الما القانونية عل مسلنلية المكلم برقابة القاصر ن و ما نصت عليه التعدي و القانونية او ير للقانون اليصا رق المدني. ايث ذكرو التعدي و بان القاصر وير المميص وير مسلنف عا او رار التي يسبباا للغير نذلا نعدام تمييصه مما ينت عنه انعدام طاه التقصيرق نبالتالي يبق التسا ف ما و المسلنف عا مصير اق الميرنر عند انعدام المكلم بالرقابة. مجلة العلوم التربوية والدارسات اإلنسانية 322 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م م - ( قام إيا جا الحق2202 ، بدراسة بعنوان مدى لصنم (الخطا، كركا ما أركان المسلنلية التقصيرية اي مشرنع القانون المدني الفلسطين ي. نقد تنانف البحث تو يا مفاوم الخطا نعناصره للوصوف إل مدى لصنمه كركا ما أركان المسلنلية التقصيرية اي مشرنع القانون المدني الفلسطينيو باإل ااة إل مدى اعتبار ا متناع عا القيام بعمل تعديا يستوج مساءلة محدث اليرر تقصيريا و نقد توصل البااث إل أن المشرنع أ ق بالنظرية الشخصية للمسلنلية التقصيرية نإل لصنم ركا الخطا كركا ما أركان المسلنلية التقصيرية اي مشرنع القانون المدني الفلسطينيو باإل ااة إل اعتبار ا متناع القيام بعملأ تعديا يستوج مساءلة الممتنع تقصيريا. :إعبء إثبات الضرر - ( ناي راسة كيلي ريتشا2200، بعنوان ما القق يي عل ما جنا اواداث مختلفيا عا الميرميا الكبار التي دات إل التعر عل صا ص جرا م عديمي التمييص نأنجه ا ت مع جرا م البالغيا نذلا ما ف تحليل التقارير الوار ما الملسساو القانونية نأييا ا ستفا ما نتا الدراساو السابقة اوف ذاو المو وع. ايث أدار إل أن سي و الشرطة أاصت أن عديمي التمييص ناواداث القيا تترانت أعمار م بيا02 ن07 سنة م اوقلية بيا اليرا م المسيلة لديام. بينما كانت والبية القيايا المسيلة اي مرالة المرا قة بيا09 - 02 سنة. نأدارو درطة ايكتوريا بان إاصا ياو2229 / 2222 أظارو أن نسبة جرا م عديمي التمييص مثلت 20 اي ايا ذكرو سي و كوينص ند أن نسبة اليرا م المسيلة لااداث بيا% 02 - 07 سنة مثلت09 ما جرا م المقاطعة. نذكر بان معظم اليرا م المسيلة ي% الكتابة عل اليدران نالتخري نالتارت ما اوجر و بينما نا را ما يتم تسييل جرا م طير كالقتل ناليرا م الينسية. نناقش البااث أ م أسبات قيام اواداث باليرا م ايث ذكر بان صا ص اواداث مختلفة عا صا ص الكبارو اقد أدار إل أن البااثيا لصوا إل العشر سنواو او ير ما سا المرا قة تحدث اياا تغيراو كثير نسريعة اصة اي المناطق المرتبطة.بالدماغ نتعتبر اإلعاقة الق نية ناومراض العقلية ناإلعاقاو الفكرية اوكثر ديوعا بيا اواداث المسيليا اي نظام العدالة الينا ية. نتوصل البااث إل أن اواداث المخالفيا للقانون لديام ااتياجاو معقد نوالبا ما يكون الينا اواداث أكثر تعقيدا اي ا اتياجاو ما الميرميا الكبار. نعل الروم ما ،أن العديد ما قه المشاكل (تعاطي المخدراوو المرض العقلي ن/ أن اإلعاقة المعراية نالتي قد يعاني مناا الكبارأ اإن الينا اواداث يحتاجون إل جان أعل ما الرعايةو امث بسب ن عام كقاصريا قانونييا اإنه يي عل الدنلة أن توار مراقبة نالدية اي سيا اواداث ن الحرص عل ما م اي العمر المدرسي للتعلم. كما أنص البااث عل أ مية زيا نسبة الموظفيا نذلا ما ف تكثيم اإلدرا نرعاية اواداث لاقه اوسباتو ايث أن اإلدرا عل سيون اواداث يمكا أن يكون كبيرا نيحتاج إل موار مالية عالية. مجلة العلوم التربوية والدارسات اإلنسانية 322 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م - ( نتنانف برنالد ن يمورا2227 ، اي راسة بعنوان المرا قيا اي صراع مع قانون المرا قيا. نذكر البااثان بان المرا قيا القق يتصراون بعنم قد يكونون م اي اوول حايا للعنم. :إعبء إثبات الضرر نقاما بيمع بياناو الدراسة ما سي و السيناء اي سيا سان وسيه ريو بريتو . نا تار البااثان متغيراو: مكان السكاو العمرو مستو ى التعليمو نوع اليريمةو تعاطي المخدراوو ل اوسر و مستوى تعليم الوالدياو نالوظيفة. نأظارو نتا التحليل الديمغرااي للبياناو أن ائة المرا قيا ما عمر07 سنة قد نصلت لمستوى تعليم ابتدا ي اقط. نكانت أكثر اليرا م ديوعا بينام السطو ًم السرقة المنظمةو نالقتلو نت يار المخدراوو نالسطو المفيي إل الموو. كما أظارو الدراسة بان معظم العينة يد نون التبغ نالمارجواناو نتنانف المشرنباو الكحولية. كما أدارو النتا بان العينة التي تم راستاا تربت اي بيئة مليئة بالمخاطر نسط ظرن أسرية صعبة ن ل منخفل ننظا م بسيطة باإل ااة إل تعاطي أاد الوالديا أن كلياما للمشرنباو الكحولية. كل قه اوسبات أ و إل .تحويل ل ء المرا قيا إل حايا الميتمع كما أظارو الدراسة إل ا طرار والبية اومااو للعمل لإلنفا عل اوسر عند وف الصنج للسيا نتكون كوسيط بيا صراع المرا ق مع القانون نالمحاكم نا لميتمع. ننظرا رتفاع تكاليم قيايا العنم بيا اودخاصأ اقترت البااثان أ مية ن ع استراتييية للتعامل مع مثل قه اون اع لاطفاف نالمرا قيا اي سان وسيه ريو بريتو. الكحولية. كما أدارو النتا بان العينة التي تم راستاا تربت اي بيئة مليئة بالمخاطر نسط ظرن أسرية صعبة ن ل منخفل ننظا م بسيطة باإل ااة إل تعاطي أاد الوالديا أن كلياما للمشرنباو الكحولية. كل قه اوسبات أ و إل .تحويل ل ء المرا قيا إل حايا الميتمع إ كما أظارو الدراسة إل ا طرار والبية اومااو للعمل لإلنفا عل اوسر عند وف الصنج للسيا نتكون كوسيط بيا صراع المرا ق مع القانون نالمحاكم نا لميتمع. ننظرا رتفاع تكاليم قيايا العنم بيا اودخاصأ اقترت البااثان أ مية ن ع استراتييية للتعامل مع مثل قه اون اع لاطفاف نالمرا قيا اي سان وسيه ريو بريتو. :إعبء إثبات الضرر - ( نذكرو أسماء موس أسعد أبو سرنر2221 ، اي راستاا بعنوان ركا الخطا اي المسلنلية التقصيريةو راس ة مقارنة بيا القانون المدني المصرق نالقانون المدني اور ني ( رسالة ماجستير، بان المسلنلية التقصيرية تقوم إ عل الخطا ايث .طرات البااثة عد تسا و قامت بالر علياا ما ف اإلطار النظرق للدراسة ايث دات الدراسة إل البحث اي ركا الخطا ن رنرته ناقا لطب يعته نأركانهو نالتحقق ما تمام التوااق بيا المسلنلية كمنا يسع لتحقيق جبر لا رار نإعا .التوازن للقمم المالية ي م و زن استخدمت البااثة المنا التحليلي للبحث عا المنا المقارت اي مو وع المسلنلية التقصيرية ن و القق يتم اتباعه اي الفقه اإلس مي ن و اليمان. ن لصت نت ا البحث إل أن التمييص يعد عنصراْ أساسيا إلناطة المسلنليةو نإن رنر التكليم ابتداء ترت نجو مناط التكليم بالمخاط باقا التكليم- نيقصد به العقل أق التمييص- و التخلي عا التمييص كركا اي الخطا نإييا ما عر بالخطا المو وعي نذلا للتمكا ما جبر او رار الواق عة ما عديم التمييص. نأنصت البااثة با مية التوسع اي مفاوم التمييص نالتشد اي إًباو نجو ه نأ مية ابتكار مفاوم الخطا المفترض الغير قابل إلًباو عكسه بحيث تنتفي مسلنلية ما أسند إليه إ بالقو القا ر أن السب اوجنبي. التعقيب عل: الدراسات السابقة: ب يب : ر: - أظارو الدراساو السابقة نالمتعلقة بالمسنلنلية التقصنيرية أن أركنان المسنلنلية تتحند اني الخطنا نالينرر نالع قنة السنببيةو نتباينننت القنوانيا المختلفنة التني أجرينت ايانا تلننا الدراساو امناا ما أسنتق أاكامنه منا القنانون الفرنسني كدراسنة (أبنو كنرزاز و2204 أ عرااوو2204 أ ريتشار و2200 أ برنالد ن يمورا و2227 ،. - كما أظارو بعل الدراساو السنابقة أن بعنل قنوانيا الندنف ن اصنة العربينة منانا قند توااقننننننننننت مننننننننننع الفقننننننننننه اإلسنننننننننن مي انننننننننني مينننننننننناف المسننننننننننلنلية التقصننننننننننيرية (اليننندقو2205أز ننر و2204أ عرانناوو2204 ،و نإن ننقه النتييننة تتوااننق مننع قننانون اإلجراءاو المدنية العماني نالقق توااقت موا ه القانونية اي مياف المسنلنلية التقصنيرية مع ما قررته الشريعة اإلس مية. مجلة العلوم التربوية والدارسات اإلنسانية 320 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م - ن ا تلم البحث الحالي مع الدراساو السابقة ما ايث المنا المتبعو ايث ي اظ أن جميع الدراساو السابقة قد ركصو عل المنا الوصفي التحليلي نالمنا التاريخيأ اي ايا أن البحث الحالي اقد استخدم مناييا ما: المنا الوصفي التحليلي نالمنا التيريبي باعتبار ما اونس لتحقيق أ دا البحث الحالي. كما ا تلم البحث الحالي عا الدراساو السابقة اي تنانله لركنيا ما أركان المسلنلية التقصيرية لعديمي التمييص ما: الخطاو ناليرر اي ايا أن الدراساو السابقة قد تنانلت ركنا ناادا ما تلا اوركان المتعلقة بالمسلنلية التقصيرية لعديم التمييص. أوجه االستفادة من الدراسات السابقة: أوجه االستفادة من الدراسات السابقة: ا - استفا البااث ما الدراساو السابقة اي تحديد أسئلة البحث نأ دااه نأ ميتهو كما استفا اي تحديد المنايية العلمية السليمة لتحقيق أ دا البحث بصور تطبيقية للوصوف إل .التصور المقترت المرجو :النتائج والتوصيات ما ا ف استعراض نجااو النظر المختلفة اوف مسلنلية اوطفاف وير المميصيا التقصيرية اي قانون اإلجراءاو المدنية العماني مقارنة بالشريعة اإلس مية و اإن البحث الحالي قد لص إل النتا :اآلتية إ - استفا البااث ما الدراساو السابقة اي تحديد أسئلة البحث نأ دااه نأ ميتهو كما استفا اي تحديد المنايية العلمية السليمة لتحقيق أ دا البحث بصور تطبيقية للوصوف إل .التصور المقترت المرجو :النتائج والتوصيات :النتائج والتوصيات ما ا ف استعراض نجااو النظر المختلفة اوف مسلنلية اوطفاف وير المميصيا التقصيرية اي قانون اإلجراءاو المدنية العماني مقارنة بالشريعة اإلس مية و اإن البحث الحالي قد لص إل النتا :اآلتية إ يآ - عدم نجو تبايا بيا مسلنلية اوطفاف وير المميصيا التقصيرية اي قانون اإلجراء .المدنية العماني مقارنة بالشريعة اإلس مية - عدم نجو تبايا بيا مسلنلية اوطفاف وير المميصيا التقصيرية اي قانون اإلجراءاو .المدنية العماني مقارنة بالشريعة اإلس مية - " المشرع العماني استخدام لفظ "إ رار اي مياف مسلنلية اوطفاف وير المميصيا التقصيرية اي قانون اإلجراءاو المدنيةأ باعتبار أن الفعل الصا ر ما الطفل وير المميص نياه الغير و اي اقيقته رر يستحق الميرنر المطالبة بالتعويل جبرا .لليرر القق لحقه - تعتبر النصوص القانونية المتعلقة بمسلنلية اوطفاف وير المميصيا التقصيرية اي قانون اإلجراءاو المدنية العماني كاايةأ نجابر لليرر ال.قق قد يلحق بالميرنر اإلجراءاو المدنية العماني كاايةأ نجابر لليرر ال.قق قد يلحق بالميرنر - يمكا التوسع اي بحث اق بإ ااة الركا الثالث ما أركان المسلنلي ة التقصيرية " لعديم التمييص ن و."الع قة السببية - يمكا التوسع اي بحث اق بإ ااة الركا الثالث ما أركان المسلنلي ة التقصيرية " لعديم التمييص ن و."الع قة السببية م - أ مية نشر القوانيا المتعلقة بمسلنلية اوطفاف وير المميصيا التقصيرية اي قانون اإلجراءاو المدنية تياه الغيرأ كثقااة توعوية بيا أارا الميتمع افاظا عل الممتلكاو .العامة نالخاصة ما عبث اوطفاف وير المميصيا م - أ مية نشر القوانيا المتعلقة بمسلنلية اوطفاف وير المميصيا التقصيرية اي قانون اإلجراءاو المدنية تياه الغيرأ كثقااة توعوية بيا أارا الميتمع افاظا عل الممتلكاو .العامة نالخاصة ما عبث اوطفاف وير المميصيا مجلة العلوم التربوية والدارسات اإلنسانية 321 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م ا:لمصادر والمراجع .مرق و سليمان0210 .. موجص أصوف ا لتصاماو. . م. أوجه االستفادة من الدراسات السابقة: القا ر : مطبعة لينة البيان العربي .التايننهو أسننامة إبننرا يم علنني0222. مسننلنلية الطبينن الينا يننة انني الشننريعة اإلسنن م ية. الطبعننة . اونل . عمان: ار البيار .الحليبنننيو أامننند بنننا عبننندالعصيص0224 . . المسنننلنلية الخلقينننة نالينننصاء عليانننا. الطبعنننة اونلننن .الرياض: مكتبة الردد .إمننامو دمحم كمنناف الننديا0220 :. المسننلنلية الينا يننة أساسنناا نتطور ننا. الطبعننة الثانيننة. بيننرنو .الملسسة اليامعية الصبينندقو . دمحم مرتينن0221 :. تنناج العننرن مننا جننوا ر القننامو . الطبعننة اونلنن . مشننق .مطبعة الصبات عو و عبدالقا ر. . ن. التشنريع اليننا ي اإلسن مي مقارننا بالقنانون الو نعي. .م. بينرنو: ار .الكتات العربي .ابا اار و أامد با انار بنا زكرينا النرازق0272 . .. معينم مقنايي اللغنة م. بينرنو: ار .الفكر هللا .عبدهللااو اتحي عبد الرايم2225 . راساو اي المسئولية التقصيرية ( نحو مسئولية مو وعية، . . م. اإلسكندرية: منشا المعار دمحدمحدمح إ م ابننا عراننةو .دمحم بننا دمحم بننا دمحم الننورومي التونسنني المننالكي2229 . . المبسننوط. الطبعننة اونلنن .بيرنو: ار الكت العلمية دمحدمحهللا .ابا جصق الكلبيو دمحم با أامند بنا دمحم بنا عبند هللاا 0425 .القنوانيا الفقاينة اني تلخنيص منق المالكية نالتنبيه عل مق الشنااعية نالحنفينة نالحنبلينة. الطبعنة اونلن . بينرنو: ار . النفا ابا قدامة المقدسيو .أبو دمحم موانق النديا عبند هللاا بنا أامند بنا دمحم0219. المغنني . بنا قدامنة . .م. القا ر : مكتبة القا ر دمح م الدسوقيو دمحم با أامند بنا عرانة. .ن. اادنية الدسنوقي علن الشنرت الكبينر. . م. بينرنو: ار .الفكر دمح .الشااعيو أبو بكر با دمحم با عبد المنلما بنا ارينص بنا معلن الحسنيني الحصنني0224 . كفاينة او يار اي ال واية اإل تصار. الطبعة او.نل . مشق: ار الخير .الرازقو امد با اار با زكريا0272 . . معيم مقنايي اللغنة. الينصء الرابنع. الينصء السنا .الطبعة اونل . بيرنو: ار الفكر لإ ي .الرازقو امد با اار با زكريا0272 . معيم مقنايي اللغنة. الينصء الرابنع. الينصء السنا .الطبعة اونل . بيرنو: ار الفكر أ أ .الكاسانيو أبو بكر با مسعو با أامد0291 . بدا ع الصنا ع اي ترتي الشرا ع. الطبعنة .الثانية. بيرنو: ار الكت العلمية ال .منانقو عبد الر ن با تاج العارايا با علي با زينا العابنديا0222 . التوقينم علن . ماماو التعاريم. الطبعة اونل . القا ر : عالم الكت دمح ال .منانقو عبد الر ن با تاج العارايا با علي با زينا العابنديا0222 . التوقينم علن . ماماو التعاريم. الطبعة اونل . القا ر : عالم الكت .الراونن اوصننفاانيو أبننو القاسننم الحسننيا بننا دمحم0402 .. أوجه االستفادة من الدراسات السابقة: المفننر او انني ورينن القننرآن الطبعة اونل . بيرنوو ن مشق: ار القلمو نالدار.الشامية .سنننلطانو أننننور2225 . النظرينننة العامنننة ل لتنننصام– أاكنننام ا لتنننصام- . . الطبعنننة اونلننن اإلسكندرية: ار اليامعة اليديد للنشر م .الراونن اوصننفاانيو أبننو القاسننم الحسننيا بننا دمحم0402. المفننر او انني ورينن القننرآن الطبعة اونل . بيرنوو ن مشق: ار القلمو نالدار.الشامية م .سنننلطانو أننننور2225 . النظرينننة العامنننة ل لتنننصام– أاكنننام ا لتنننصام- . . الطبعنننة اونلننن .اإلسكندرية: ار اليامعة اليديد للنشر إ .سلطانو أنور0292 .. الموجص اي مصا ر ا لتصام. .م. بيرنو: ار الناية العربية اليرجننانيو أبننو بكننر بننا عبنند القننا ر بننا عبنند الننراما0295. كتننا .ت التعريفنناو. .م .بيرنو: ار الكت العلمية دمح . دن و دمحم لبي0212. موجص اي مصا ر ا لتصام . . م. بيرنو: ار الناية العربية. .عبننند الننندا مو أامننند2222 . دنننرت القنننانون المننندني النظرينننة العامنننة ل لتنننصام- مصنننا ر ا لتصام- . . اليصء اونف. الطبعة الثالثة. ال : جامعة ال م .الشننرقانقو جميننل0290 . النظريننة العامننة ل لتننصام-مصننا ر ا لتننصام- . . الطبعننة اونلنن . القا ر : ار الناية العربية ابننا م.نظننورو دمحم بننا مكننرم بننا علنني0224 . لسننان العننرت. الطبعننة الثانيننة. بيننرنو: ار .صا ر .مصننطف و إبننرا يم نآ ننرنن2224 : . المعيننم الوسننيط. الطبعننة الرابعننة. القننا ر كتبننة الشننرن الدنلية. .مصننطف و إبننرا يم نآ ننرنن2224 : . المعيننم الوسننيط. الطبعننة الرابعننة. القننا ر كتبننة الشننرن الدنلية. .السيوطيو ج ف الديا عبدالراما با أبي بكر0292. اودنباه نالنظنا ر اني قواعند نانرنع اقن .الشااعية. الطبعة اونل . بيرنو: ار الكت العلمية .ي ب ن . بيرن : ر ي . .ابننا رجنن و زيننا الننديا عبنندالراما بننا أامنند الحنبلنني2220. جننامع العلننوم نالحكننم انني . مسيا اديثا ما جوامع الكلم. الطبعة السابعة. بيرنو: ملسسة الرسالة .مواايو أامد0227. اليرر اي الفقه اإلس مي: تعريفه و أنواعه و ع قاته و وابطه . .اونل . الخبر: ار ابا عفان .بوكرزاز و أامد2204 .المسلنلية المدن ينة للقاصنر راسنة مقارننة . ( راسنة كتنوراه،. جامعنة .قسطنطينية دمح .جننا الحننقو إيننا دمحم2202 . "منندى لننصنم الخطننا كننركا مننا أركننان المسننلنلية التقصننيرية انني .مشرنع القانون المدني الفلسطيني" راسة تحليلية ميلة اليامعنة اإلسن مية للدراسناو اإلسنن مية . الميلنند العشننرنن. العنند اونف. ص220 -ص221 . .ISSN 1726-6807 http://www.iugaza.edu.ps/ar/periodical . حمد .جننا الحننقو إيننا دمحم2202 . "منندى لننصنم الخطننا كننركا مننا أركننان المسننلنلية التقصننيرية انني .مشرنع القانون المدني الفلسطيني" راسة تحليلية ميلة اليامعنة اإلسن مية للدراسناو اإلسنن مية . الميلنند العشننرنن. العنند اونف. ص220 -ص221 . .ISSN 1726-6807 http://www.iugaza.edu.ps/ar/periodical . p p http://statistics.ahlamontada.com/t44-topic أوجه االستفادة من الدراسات السابقة: p g p p .اليندقو دمحم صبرق2205 . اي المسلنلية التقصيرية- المسنلنلية عنا الفعنل الينار-. الطبعنة .اونل . عمان: ار الثقااة للنشر نالتوزيع .اليننندقو دمحم صننبرق2205 .انني المسننلنلية التقصننيرية عننا الفعننل اليننار . . الطبعننة اونلنن .اور ن: ار الثقااة للنشر نالتوزيع .الحنبلننيو أبنني الفننرج عبنند الننراما بننا رجنن . . و الفقننه اإلسنن مي . . م: ار الفكننر للطباعننة .نالنشر نالتوزيع ع .ايدرو علي2222 .القواعد الكلية ما رر الحكام- درت ميلة اواكام- . طبعنة اصنة. الميلند . اونف. الرياض: ار عالم الكت م .الصايليو ن بة2222 .الفقه ا س مي نأ لته- اليص يا4 ن5 . الطبعة02 .. مشق: ار الفكر .ز ر و دمحم المرسي2204 . .المصا ر وير اإلرا ية ل لتصام الطبعة اونلن . العنيا: ار الكتنات .اليامعي ي .سننراجو دمحم أامنند2202 . ننمان العنندنان انني الفقننه ا سنن مي راسننة اقايننة مقارنننة بااكننام المسنننلنلية التقصنننيرية اننني القنننانون . الطبعنننة التاسنننعة. بينننرنو: الملسسنننة اليامعينننة .للدراساو نالنشر نالتوزيع ع .سلطنة عمان2202 . قانون المعام و المدنية. المرسوم السلطاني رقم22 م عا2202 . لط ة ل ة ل ل ل ة ل ل ل ة ل .سلطنة عمان2202 . قانون المعام و المدنية. المرسوم السلطاني رقم22 م عا2202 . .سلطنة عمان2202 . ميموعة المبا ئو نالقواعد القانونية التي قررتاا المحكمة العليا اي الفتر ما2220 نات2202 م. الدا ر المدنية02 / 0 .(م،. المحكمة العليا: المكت الفني . السناورقو عبد الرزا0252 .الوسيط اي درت القانون المدني اليديد.- نظرية ا لتصام بوجنه عام- مصا ر ا لتصام- .. . م. بيرنو: ار ااياء التراث العربي العثيمننياو دمحم بننا صننالا2224دننرت اوربعننيا النونيننة الطبعننة الثالثننة الرينناض: ار الثريننا .الفنت نقو سنايل اسنيا2202 . مبننا ئ القنانون السنعو ق ( راسنة انني نظريتني القنانون نالحننق مقارنة بقوانيا الدنف العربية.،. الطبعة اونل . عمان: ار نا ل للنشر . قيننمانيو سنناير مصننطف2205 . طننا المتيننرر نأًننره انني المسننلنلية التقصننيرية. الطبعننة .اونل . بيرنو: منشوراو الحلبي الحقوقية ي .اسام الدياو دمحمو نننابوشو إيمنان2201 .". "المسنلنلية التقصنيرية لعنديم التميينص ميلنة العلنوم القانو.نية نالسياسية الميلد02 ( . ISSN 2222-7288 :، 00 . ( ، .مننرق و سننليمان0299 . الننوااي انني دننرت ا لتصامنناو المدنيننة انني الفعننل اليننار نالمسننلنلية المدنينننةو القسنننم اونف- اننني اواكنننام العامنننة . الميلننند اونف. . م. بينننرنو: ار صنننا ر للطباعة نالنشر .منصورو دمحم اسيا2221 .النظرية العام ة اي ا لتنصام- مصنا ر ا لتنصام-. . م: . القنا ر ار .ر ب ر.ص م ر ي ون ي ي ق ر. . أوجه االستفادة من الدراسات السابقة: الطبعة اونل.عمان: ار الثقااة للنشر نالتوزيع .سننلطانو أنننور2227 .مصننا ر ا لتننصام انني القننانون المنندني اور ننني- راسننة مقارنننة بالفقننه اإلس مي- . . الطبعة اونل.عمان: ار الثقااة للنشر نالتوزيع .منصورو دمحم اسيا2221 . النظرية العامة ل لتصام– مصا ر ا لتصام- . . م. اإلسنكندرية: ار .اليامعة الحديثة للنشر دمح القرنزأبننا قو مينند الننديا دمحم بننا يعقننوت . 2227 . القننامو المحننيط. .م. بيننرنو: ار الكتنن .العلمية .قطيطو وسان يوسنم2222 .اوسنبة التقنويم الصنفي. الطبعنة اونلن . عمنان: ار الثقاانة للنشنر .نالتوزيع يأ ع R, Priuli; M de Moraes. 2007. “Adolescents in conflict with the law”. Ciencia & saude coletiva. vol. 12, issue 5 (2007) pp. 1185-1192 أ R, Priuli; M de Moraes. 2007. “Adolescents in conflict with the law”. Ciencia & saude coletiva. vol. 12, issue 5 (2007) pp. 1185-1192 pp Richards, Kelly. 2011. What makes juvenile offenders different from adult offenders? Trends and issues in crime and criminal justice, 409. https://samehar.wordpress.com/2009/09/07/010909 https://samehar.wordpress.com/2009/09/07/01090 http://statistics.ahlamontada.com/t44-topic مجلة العلوم التربوية والدارسات اإلنسانية 322 مسؤولية الأطفال غير المميزين التقصيرية في قانون..... د/عبد الله بن علي بن سالمالشبلي (المجلد5 ،) ( العدد10 ) يونيو3030م
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Somersault of Paramecium in extremely confined environments
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Jana S, Eddins A, Spoon C, Jung S. Somersault of Paramecium in extremely confined environments . Scientific Reports 2015, 5, 1-9 Jana S, Eddins A, Spoon C, Jung S. Somersault of Paramecium in extremely confined environments . Scientific Reports 2015, 5, 1-9 Jana S, Eddins A, Spoon C, Jung S. Somersault of Paramecium in extremely confined environments . Scientific Reports 2015, 5, 1-9 DOI link to article: http://dx.doi.org/10.1038/srep13148 http://dx.doi.org/10.1038/srep13148 Copyright: This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. Somersault of Paramecium in extremely confined environments Saikat Jana, Aja Eddins, Corrie Spoon & Sunghwan Jung received: 19 March 2015 accepted: 20 July 2015 Published: 19 August 2015 received: 19 March 2015 accepted: 20 July 2015 Published: 19 August 2015 We investigate various swimming modes of Paramecium in geometric confinements and a non- swimming self-bending behavior like a somersault, which is quite different from the previously reported behaviors. We observe that Paramecia execute directional sinusoidal trajectories in thick fluid films, whereas Paramecia meander around a localized region and execute frequent turns due to collisions with adjacent walls in thin fluid films. When Paramecia are further constrained in rectangular channels narrower than the length of the cell body, a fraction of meandering Paramecia buckle their body by pushing on the channel walls. The bucking (self-bending) of the cell body allows the Paramecium to reorient its anterior end and explore a completely new direction in extremely confined spaces. Using force deflection method, we quantify the Young’s modulus of the cell and estimate the swimming and bending powers exerted by Paramecium. The analysis shows that Paramecia can utilize a fraction of its swimming power to execute the self-bending maneuver within the confined channel and no extra power may be required for this new kind of self-bending behavior. This investigation sheds light on how micro-organisms can use the flexibility of the body to actively navigate within confined spaces. Paramecia are amongst the most ubiquitous ciliary microorganisms in nature, and their various species are often found to inhabit ponds, lakes and marine water bodies1,2. They have been reported to swim with speeds of a few millimeters per second3 and have also been used as indicators in bioassays to detect bac- teria level in soils4 or the concentration of heavy metals in sludge5. The nature of the ciliary beat around Paramecium and the kinematics of the helical swimming pattern has continued to inspire experimen- talists6–8 and theorists9–12 over the past few decades. The natural habitats of such microorganisms often consist of decayed matter, soil, debris and extremely confined spaces. During Paramecium’s navigation in the complicated natural environment, Paramecium might come into contact with crevices, obstacles or bio-flocs; possibly of shapes and sizes similar to its size13,14. Department of Engineering Science and Mechanics, Virginia Tech, Blacksburg, VA 24061, USA. Correspondence and requests for materials should be addressed to S.Jung. (email: sunnyjsh@vt.edu) Date deposited: Newcastle University ePrints - eprint.ncl.ac.uk www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | 5:13148 | DOI: 10.1038/srep13148 Results iff Different swimming behaviors in quasi-infinite fluid. To understand the swimming character- istics of Paramecium, we undertake an analysis of the recorded tracks. Paramecia swimming in qua- si-infinite fluid media (a large drop on a glass slide) and are observed to execute three different types of trajectories as shown in Fig. 2. The first is the ballistic motion; in which Paramecium swims along a sinusoidal path and shows a large displacement from its starting point (shown as red curves in Fig. 2(a)). In the second case, Paramecia circles around and comes very close to its starting point at different times (shown as blue curves in Fig. 2(a)). The third type is a meandering mode in which Paramecium tends to move around locally without any large displacements from its original position (shown as purple curves in Fig. 2(a)).f g To differentiate the various swimming behaviors, the mean square displacement is used as a primary measure. As a reference, we define an idealized situation in which Paramecium swims with constant velocity along a straight line without any turning motion. Then ideal ballistic motion has the mean square displacement (MSDideal = δ ∆ ( ) x U i t i swim 2 2 2 ~ where Uswim is the swimming speed) and would exhibit a slope of 2 on the log-log plot of mean squared displacement vs. δt. For analysis, all our exper- imentally recorded tracks are truncated to 100 frames, so that the total time T of individual tracks is 3.3 s (the time interval δt is (1/30) s). Truncating the movies to 100 frames allows us to minimize the errors in the Matlab tracking program arising due to intersecting Paramecium tracks and also helps us to ensure that the full trajectories of ballistic swimmers are captured within the field of view. Subsequently, the mean squared displacements ∆xi 2 of recorded tracks are calculated as N i x j i t x j t 1 [ ] j N i 0 2 δ δ ( /( −))∑ →(( + ) ) −→( ) = − where i, j are integers. We then compare the normalized mean squared displacement (MSD) of experimentally recorded trajectories with the case of idealized ballistic swimming which is denoted by the black dotted line as in Fig. 2(a). Somersault of Paramecium in extremely confined environments While chemotaxis15, gravikinesis16, gal- vanotaxis17 and swimming characteristics in the bulk of fluid18 for Paramecium is well characterized, its swimming behavior in confined spaces/geometry remains relatively unexplored19.it gi p g y y p Confined spaces or boundaries often bring about many surprising characteristics in a variety of swimmers, in quite unexpected and different ways. For example, bacteria and spermatozoa exhibit accu- mulation near flat surfaces20,21 or show circular swimming tracks due to hydrodynamic effects22,23. A suspension of spermatozoa, when injected into micro-fluidic geometries show preferential swimming along surfaces24. The length of the cilia/flagella has been found to play an important role in governing the scattering angle of spermatozoa and chalmydomonas after collision with a wall25. A bacteria after running into a wall can reorient and exhibit long residence times on surface26 or reverse its direction by reorienting the flagella, thereby making entry and exit swimming tracks indistinguishable27. A helically swimming Paramecium on collision with a wall exhibits avoidance behavior; during which it slightly moves backwards, gyrates its body and finally resumes its directional swimming6.hl gy yi y g The flexibility of a swimmer coupled with boundaries and/or external cues can trigger an active change in the cell shape or result in a change of the swimming mode. Bacteria in sub-micron constric- tions28 and fabricated micro-structures29 show adaptation to confinements by growing and/or dividing into anomalous shapes. Growing yeast cells when placed in small chambers tend to buckle and exhibit Scientific Reports | 5:13148 | DOI: 10.1038/srep13148 www.nature.com/scientificreports/ Figure 1. Three different kinds of swimming modes seen during locomotion of Paramecium in confined channels : (a) Ballistic helical swimming with a net direction, (b) meandering motion with turns, (c) sudden bending to change the direction of swimming, and (d) zoomed-in image for self-bending. (See the supplement video). Figure 1. Three different kinds of swimming modes seen during locomotion of Paramecium in confined channels : (a) Ballistic helical swimming with a net direction, (b) meandering motion with turns, (c) sudden bending to change the direction of swimming, and (d) zoomed-in image for self-bending. (See the supplement video). Figure 1. Three different kinds of swimming modes seen during locomotion of Paramecium in confined channels : (a) Ballistic helical swimming with a net direction, (b) meandering motion with turns, (c) sudden bending to change the direction of swimming, and (d) zoomed-in image for self-bending. (See the supplement video). Somersault of Paramecium in extremely confined environments bent shapes30 and monotrichous bacteria have been found to utilize buckling of flagellar hook to exe- cute sharp turns31. Larger organisms like fish swim by undulating their body32 and can also execute a C-shaped bending of the body by using muscles in order to abruptly change the swimming direction33. In micro-world, prey change the radii and pitch of their swimming helix to escape the predators34, and Paramecium shoot out trichocysts to exhibit evasive maneuvers in response to a threat35,36.i In this paper, we explore transitions in swimming responses of Paramecium in confined spaces, and report an interesting feature of the cell buckling in response to extreme confinements as seen in Fig. 1. Paramecia that are initially placed in quasi-infinite fluids, and are found to mostly execute directional helices (ballistic swimming) as they explore the free space. When Paramecia are further confined to thin fluid films, many swimmers transition from ballistic swimming to meandering and exhibit a large num- ber of abrupt turns. In rectangular channels, a meandering Paramecium can push on the confining walls and execute self bending of the body to reorient in a completely new direction. We use a force deflection method to measure the elasticity of the cell body and investigate the swimming and bending power of the cell during the different maneuvers. Scientific Reports | 5:13148 | DOI: 10.1038/srep13148 Results iff For the case of sinusoidal path, the plot of (MSDballistic) is almost a straight line which closely follows the black dotted line showing large displacements for short as well as long timescales. In the case of circling motion, the MSD shows Scientific Reports | 5:13148 | DOI: 10.1038/srep13148 2 www.nature.com/scientificreports/ Figure 2. Analysis of swimming tracks and probability distribution of characteristic times for swimmers in semi-infinite fluid medium. (a) Plot of normalized mean square displacements vs. time difference of three different swimming trajectories as they swim in the large drop of fluid: Ballistic, meandering and circling. Only the first 100 frames of the recorded tracks are used for analysis. (Color codes are same in inset and figure). The black dashed line represents the δt2 line corresponding to ideal ballistic motion MSDideal. Black dots represent the times at which significant deviation occurs from the ideal ballistic motion (ε =  20%). (b) Probability distribution of characteristic times δtc in 2D semi-infinite fluid medium for ε =  20%. The red and green bar shows the changes in the characteristic time distribution when ε =  15% or 25% is considered. ysis of swimming tracks and probability distribution of characteristic times for swimmers lf Figure 2. Analysis of swimming tracks and probability distribution of characteristic times for swimmers in semi-infinite fluid medium. (a) Plot of normalized mean square displacements vs. time difference of three different swimming trajectories as they swim in the large drop of fluid: Ballistic, meandering and circling. Only the first 100 frames of the recorded tracks are used for analysis. (Color codes are same in inset and figure). The black dashed line represents the δt2 line corresponding to ideal ballistic motion MSDideal. Black dots represent the times at which significant deviation occurs from the ideal ballistic motion (ε =  20%). (b) Probability distribution of characteristic times δtc in 2D semi-infinite fluid medium for ε =  20%. The red and green bar shows the changes in the characteristic time distribution when ε =  15% or 25% is considered. dips at certain time intervals since the Paramecium comes very close to its original position. It is worth noting that this circling motion is rarely observed, so we will neglect this swimming behavior in the later analysis. Results iff The meandering motion shows ballistic characteristics at very short timescales, however for longer timescales the MSDmeandering drops off abruptly when compared to ballistic swimming, indicating a loss of memory of an initial direction of the swimmer. y To distinguish between the sinusoidal ballistic and the meandering swimmers, we calculate the abso- lute deviation of the mean squared displacement ε = ( ) − ( ) MSD MSD log log log ideal for the trajec- tories, and find the timescale when the MSD deviates 20% from the ideal ballistic case (ε =  1.2). The time when this deviation starts is termed as “Characteristic time” (δtc) and is an indicator that the motion has significantly deviated from the ideal ballistic swimming. By extracting this timescale signature from recorded tracks (a total of 3103 swimming tracks in different configurations), we get a probability distri- bution of the characteristic times by counting trajectories falling into a specific time interval. The prob- ability distribution plots in different film thicknesses allow us to characterize swimming behavior executed by the swimmers in different configurations. For quasi-infinite fluid the probability distribution plot of the characteristic time (δtc) shows a peak at 3.3 s (as shown in Fig. 2(b)), indicating that most of the swimmers have ballistic like characteristics as they swim in semi-infinite fluid. In this study, we classify the swimmers as ballistic (δtc >  3.0 s; 90% of the total swimming duration) or meandering (δtc <  3.0 s) based on the difference in characteristic time. Ballistic to meandering transition in thick and thin fluid films. Paramecia are placed to swim in fluid films of varying thickness, and ballistic and meandering swimmers are distinguished using the method described in the previous section. The probability distribution of characteristic times in thick fluid films (as shown in Fig. 3(a); H =  508 μm) has the highest peak in the bin of 3.0 s <  δtc <  3.3 s; show- ing the trend that most of swimmers display sinusoidal ballistic characteristics. But, what happens if we decrease the thickness of the fluid films in which Paramecia are swimming ? Inset of Fig. 3(b) shows the tracks of Paramecium in the thin film (H =  76 μm); which predominantly shows meandering motion with a large abrupt number of turns. Scientific Reports | 5:13148 | DOI: 10.1038/srep13148 Results iff Probability distribution of characteristic times for Paramecia swimming in fluid films of different thicknesses. (a) Probability distribution of characteristic times in 76 μm thin film, showing high peaks at smaller timescales indicating meandering motions. (b) Probability distribution of characteristic times in 508 μm thin film, showing one single peak at a larger timescale indicating dominating ballistic motions. Insets show the visuals of recorded tracks. (c) Swimming speeds of ballistic and meandering Paramecia in different thickness fluid films. Error bars represent one half standard deviation values. Figure 4. Characteristics of the swimmers in films of varying thickness. (a) Probability of meandering and ballistic swimming depending on the gap thickness. (b) Average number of turns executed by the swimmers and the mean characteristic time in thick and thin fluid films. (c) Ratio of net to gross displacements. The error bars indicate a half standard deviation of data. This ratio measures the tortuosity of the swimming tracks; lower values indicate higher tortuosity. Figure 4. Characteristics of the swimmers in films of varying thickness. (a) Probability of meandering Figure 4. Characteristics of the swimmers in films of varying thickness. (a) Probability of meandering and ballistic swimming depending on the gap thickness. (b) Average number of turns executed by the swimmers and the mean characteristic time in thick and thin fluid films. (c) Ratio of net to gross displacements. The error bars indicate a half standard deviation of data. This ratio measures the tortuosity of the swimming tracks; lower values indicate higher tortuosity. measurements of swimming speeds in thin fluid films as seen in Fig. 3(c) would be an artifact due to the fact that the swimming speeds are measured in the 2D projected plane, which neglects the vertical component and therefore systematically underestimates the swimming speed in larger gap thicknesses. For H =  508 μm, 78% of the swimmers are ballistic whereas in 76 μm-thick films only 27% of Paramecia swim ballistically indicating a transition from ballistic to meandering mode in smaller gaps as seen in Fig. 4(a). The mean characteristic time ( t t T c c δ δ = / ⁎ ) which can be thought of as a measure of directional persistence of the cell also shows an increasing trend in Fig. 4(b); confirming that in small gap thickness the orientational memory is lost at smaller times. Results iff g p In the case of Chlamydomonas, spermatozoa or bacteria the collision with a wall might result in a simple scattering effect or may show long residence times near the boundary. However, in case of Paramecium, the collision with a wall triggers ‘avoidance behavior’, during which the Paramecium backs up and gyrates its body about a mean position. In thin films that have thickness similar to the width of the cell body, the swimming space is severely constrained. This leads to a series of successive collisions with the wall and causes the Paramecium to move back and forth exhibiting meandering behavior with a large number of turns. Since, such a behavior causes very little displacements and frequent reorien- tations, the characteristic time for such swimmers tend to be on the lower side. This is confirmed by the probability distribution of characteristic time for the thin film (H =  76 μm) that shows a new peak for 0.3 s <  δtc <  0.6 s in addition to the ballistic-motion peak (in the bin of 3.0 s <  δtc <   3.3 s); indicating that a large fraction of swimmers switch from ballistic to meandering swimming. The slightly elevated Scientific Reports | 5:13148 | DOI: 10.1038/srep13148 3 www.nature.com/scientificreports/ Figure 3. Probability distribution of characteristic times for Paramecia swimming in fluid films of different thicknesses. (a) Probability distribution of characteristic times in 76 μm thin film, showing high peaks at smaller timescales indicating meandering motions. (b) Probability distribution of characteristic times in 508 μm thin film, showing one single peak at a larger timescale indicating dominating ballistic motions. Insets show the visuals of recorded tracks. (c) Swimming speeds of ballistic and meandering Paramecia in different thickness fluid films. Error bars represent one half standard deviation values. Figure 3. Probability distribution of characteristic times for Paramecia swimming in fluid films of different thicknesses. (a) Probability distribution of characteristic times in 76 μm thin film, showing high peaks at smaller timescales indicating meandering motions. (b) Probability distribution of characteristic times in 508 μm thin film, showing one single peak at a larger timescale indicating dominating ballistic motions. Insets show the visuals of recorded tracks. (c) Swimming speeds of ballistic and meandering Paramecia in different thickness fluid films. Error bars represent one half standard deviation values. Figure 3. Probability distribution of characteristic times for Paramecia swimming in fluid films of fi Figure 3. Results iff We also measure the total number of turns for the tracks; defined by the angle included (greater than 45°) by tangents between the successive recorded positions and find that the number of turns executed in thinner films is about 2.5 times more as compared to the thicker ones (Fig. 4(b)). A measure of the tortuosity/straightness of the path given by the ratio of displacement between the first and last frame to the total distance travelled by organism37 shown in Fig. 4(c); also demonstrates the fact that in smaller gaps the trajectories executed are more jagged. Meandering to bending transition in rectangular channels. We further studied the various beh- viors exhibited by Paramecium in Poly-dimethyl siloxane (PDMS) channels of height (H =  80 μm) and varying widths (W =  120, 140, 150, 160 and 180 μm) as described in the Methods section. Within these quasi-1D channels, ballistic motion as well as meandering motion are observed, however, in certain cases Paramecia are observed to buckle their body and abruptly change their swimming direction. The phenomenon occurs due to the confinement effect which constrains Paramecia to touch and exert forces on the walls. A probability plot in Fig. 5 shows that in the optimal range of 0.4 <  W/L <  1.0 values for Scientific Reports | 5:13148 | DOI: 10.1038/srep13148 4 ntificreports/ Figure 5. Probabilities of different swimming modes within channels. Probability of ballistic swimming, meandering, or bending in PDMS channels with different level of confinements. (L denotes the length of the individual Paramecium and W denotes the width of the channel). www.nature.com/scientificreports/ Figure 5. Probabilities of different swimming modes within channels. Probability of ballistic swimming, meandering, or bending in PDMS channels with different level of confinements. (L denotes the length of the individual Paramecium and W denotes the width of the channel). Figure 6. Force deflection technique to measure the elasticity of Paramemcium. (a) Deflected state of Paramecium and the displacement of the glass fiber from the reference position. (b) Undeflected state of the fiber after being retracted horizontally and the Paramecium has also returned to its original undeflected state. Figure 6. Force deflection technique to measure the elasticity of Paramemcium. (a) Deflected state of Paramecium and the displacement of the glass fiber from the reference position. (b) Undeflected state of the fiber after being retracted horizontally and the Paramecium has also returned to its original undeflected state. Scientific Reports | 5:13148 | DOI: 10.1038/srep13148 Results iff which bending events are more probable. For larger channel widths (W/L >  1), Paramecium cannot touch both walls simultaneously and therefore cannot bend itself. Th lf b d ll h ll h l d l fi d The self-bending allows the cell to change its locomotion direction in extremely confined environ- ments when meandering motion would not suffice. The bending of the cell which has not been reported previously, may be crucial while the cell navigates extremely small spaces within its natural environ- ments. Experimental observations of the self-bending phenomena in Paramecia show that the cell ini- tially touches and slides along both the walls. The posterior end anchors onto the wall and remains fixed; while the anterior end keeps on sliding along the other wall causing the cell body to buckle like a bent bow. This sliding motion is presumably connected to the swimming force that Paramecium uses in wider channels. Once the body reaches the maximum curvature, then the continuous motion of the anterior end relaxes the bent cell back to its original shape. Based on the above description of the self-bending, we hypothesize that the cell expends some of its swimming power by exerting forces on the wall to bend itself. Measurement of Young’s Modulus. To estimate the power spent during self-bending, the stiffness of the cell body needs to be determined. We use force deflection technique38 to quantify the Young’s modulus of the immobilized cell. A glass micro-fiber manufactured by extrusion is calibrated by hanging weights and is used to deflect the free end of the Paramecium. The Paramecium was held by suction using a glass pipette, and the flexible glass micro-fiber of known stiffness (k) was placed against the free end. Then the base of the glass fiber (out of view) was displaced forcing the Paramecium to deflect a distance (δtip) from it’s equilibrium position as shown in Fig. 6(a). Scientific Reports | 5:13148 | DOI: 10.1038/srep13148 5 www.nature.com/scientificreports/ Figure 7. Bent states of Paramecium within channels. (a) Plot of the curvature of a Paramecium while bending at different instances (t). The time at which maximum curvature occurs is denoted by tb. (b) Ratio of bending to swimming power for Paramecia vs t* =  t −  tb. This graph shows that the bending power is always less than the swimming power. Results iff The upper inset shows the normalized bending powers (Pb/max(Pb)) vs t* for the different cases. Figure 7. Bent states of Paramecium within channels. (a) Plot of the curvature of a Paramecium while bending at different instances (t). The time at which maximum curvature occurs is denoted by tb. (b) Ratio of bending to swimming power for Paramecia vs t* =  t −  tb. This graph shows that the bending power is always less than the swimming power. The upper inset shows the normalized bending powers (Pb/max(Pb)) vs t* for the different cases. The glass fiber was the horizontally retracted, and the fiber was straightened which allowed for meas- urement of the fiber’s base displacement (δbase). Force (Ffiber) applied to the Paramecium due to displace- ment was determined by Ffiber =  k(δbase− δtip). By knowing the value of the force exerted (about 20 ~ 60 nN for different trials) we can finally calculate the elastic modulus of the cell as E =  Ffiberc3/3Iδtip where c denotes the length of the cell and I donates the moment of inertia of the cell body. Three different cells and multiple (15 ~ 17 force deflection trials) allowed us to find the elastic modulus to be in range of 4.1 ±  0.4 kPa39; which is significantly lower than the cell wall of prokaryotic bacteria E. Coli40,41 and also the cilium42. Power exerted by Paramecium during bending in quasi 1D channel. As Paramecium maneuvers to bend within the channel; the posterior end gets anchored to one of the walls. The time to anchor varies across individual organisms and is influenced by factors like roughness of the walls and local adhesion between cilia and the wall. During the anchoring period, Paramecium has very little curvature as seen in Fig. 7(a) (t <  0.5 s). The Paramecium then progressively deforms its body and at the point of maximum curvature assumes a bow-like shape during 0.5 s <  t <  2.2 s in Fig. 7(a). The maximum body curvature is followed by a relaxation to the unbent state after which the Paramecium either resumes swimming in the opposite direction, or prepares for another bending event. Results iff pp p p g In the case of swimming Paramecium, the swimming power (Ps) is estimated as Ps =  6πμV2L ~ 14 pW where μ ~ 10−3 Pa·s is the viscosity of water, V ~ 2000 μm/s is the average ballistic swimming speed in 76 μm gap thickness (see Fig. 3c) and L ~ 200 μm is the length of the organism. The bending power (Pb) of the cell can be approximated as Pb =  EILκ∂κ/∂t, where E ~ 4 kPa is the Young’s modulus, I =  πr4/4 ≈ 5 ×  10−12 m4 is the moment of inertia based on the average width (r ≈ 20 μm), κ is the cur- vature of bending Paramecium, and the term ∂κ/∂t denotes the variation of the curvature with time. To measure κ, image sequences of Paramecium during bending events are converted into black and white image. Then, the body centerline is extracted and is best-fitted by a curved line of constant radius (R). The inverse of the fitting radius yields the body curvature (κ =  1/R). As shown in Fig. 7(a), the curvature is monotonically increasing until the maximum is reached. After that, the Paramecium relaxes back to its unbent state. The bending power is measured in cases of only W/L >  0.8. We also observed the bending events when W/L <  0.8, however, the bending becomes highly nonlinear like forming a kink in the mid- dle of the body, and falls outside the scope of our estimates using a linear theory. y p g y Figure  7(b) shows the plot of the ratio of bending to swimming power (Pb/Ps) vs time t*(=  t −  tb; where tb is the time when Paramecium reaches the maximum curvature). The bending power exerted by Paramecia is less than the swimming power since the power ratio never goes beyond 1. In addition, this power ratio increases as the channel width gets smaller, indicating Paramecium uses more power to bend its body in narrower channels. During the bending, the lateral displacement of the cell is quite small, therefore we assume swimming and bending events to be mutually exclusive. This leads us to conclude that a fraction of the power that would be otherwise spent for swimming is utilized to bend the cell as a part of swimming course within the channel, thereby allowing the cell to switch directions. Methods Cell Culture. Cell Culture. Wild type Paramecium Multimicronucleatum (Part No. 131540) initially obtained from ATCC 30842 by Carolina Biological Supply were ordered for our experiments. The cells were centrifuged; washed and are cultured in a medium consisting of boiled spring water and wheat seeds. Washed cells were made to grow in a controlled temperature environment of 22 °C. The cells were isolated during their growth phase and were washed twice in Tris-HCl buffer solution consisting of 9 mM CaCl2, 3 mM KCl and 5 mM Tris-HCl with pH adjusted to 7.2. The cells showed renewed vigor when introduced into this solution and were allowed to equilibrate for 30 minutes prior to starting the experiments. The cells were found to be of a prolate spheroid shape with the major axis of the cell bodies measuring 193 ±  24 μm and the minor axis being 40.3 ±  5.6 μm ( as seen in Fig.8(a)). The propelling organelles known as the cilia project out front the protective covering called pellicle and beat at a frequency of about 30 Hz19 to create metachronal waves. Ballistically swimming Paramecia are found to propel mostly along the ante- rior direction in a left-handed helical trajectory in the quasi-infinite fluid with the velocities measuring 2.397 ±  1.001 mm/s. Experimental Methods—Thin and thick films. To simulate the effect of fluid films of varying thickness we took two clean glass slides (VWR Vistavision) 76.2 ×  25.4 ×  1 mm. Two plastic spacers having a constant thickness (H =  50, 76, 127, 254, 317, 381, or 508 μm) were placed at the edge of the glass slide. A controlled volume of the washed culture media was pipetted onto the slide and the other glass slide was put on top. A fiberoptic light source (Fiber-lite MI 152) was used to provide illumina- tion from the side that allowed us to obtain dark-field images of the swimming organisms as shown in Fig. 8(b). A Sony handycam (Sony HDR-XR100) with a 4 ×  zoom lens was used to record 2D projected swimming tracks of multiple Paramecia at 30 fps. Experimental Methods—Rectangular channels. For quasi 1D confinements, microfluidic chan- nels were used with a fixed height (H =  80 μm) and varying widths (W =  120, 140, 150, 160 and 180 μm). Mould masters ordered from Stanford Micro-fluidics foundry were cleaned with isopropyl alcohol and dried in a stream of compressed air. Discussionh The experimental investigations described here reveals the complicated nature of the swimming tracks executed by Paramecium multimicronucleatum as it interacts with its fluidic environment. We start by Scientific Reports | 5:13148 | DOI: 10.1038/srep13148 6 www.nature.com/scientificreports/ Figure 8. Experimental setups : (a) Schematic showing the dimensions of Paramecium. (b) Fluid films of varying thickness (50 <  H <  508 μm). (c) Rectangular channels of 80 μm height with different widths (120 <  W <  180 μm). Figure 8. Experimental setups : (a) Schematic showing the dimensions of Paramecium. (b) Fluid films of varying thickness (50 <  H <  508 μm). (c) Rectangular channels of 80 μm height with different widths (120 <  W <  180 μm). verifying the conventional notion that Paramecium swims helically in quasi-infinite fluid media, and progressively show different swimming behaviors of Paramecium by introducing various confinements. When Paramecia are placed in thin films, we find that Paramecia meander around due to frequent collision and reorientation events arising from the confining boundaries. In rectangular channels the meandering ciliate anchors itself to the walls and bends its flexible body to change its swimming orien- tation. We measure the Young’s modulus of the cell and show that the organism can use a fraction of its swimming power to bend within the channel. The investigation sheds light on the previously unknown bending maneuver executed by Paramecium in confined geometries and also shows how body deform- ability can be exploited by slender micro-organisms to navigate and/or escape from the complicated geometries in their habitat. verifying the conventional notion that Paramecium swims helically in quasi-infinite fluid media, and progressively show different swimming behaviors of Paramecium by introducing various confinements. When Paramecia are placed in thin films, we find that Paramecia meander around due to frequent collision and reorientation events arising from the confining boundaries. In rectangular channels the meandering ciliate anchors itself to the walls and bends its flexible body to change its swimming orien- tation. We measure the Young’s modulus of the cell and show that the organism can use a fraction of its swimming power to bend within the channel. The investigation sheds light on the previously unknown bending maneuver executed by Paramecium in confined geometries and also shows how body deform- ability can be exploited by slender micro-organisms to navigate and/or escape from the complicated geometries in their habitat. References Kung, C. & Saimi, Y. The physiological basis of taxes in Paramecium. Ann. Rev. Physiol. 44, 519–534 (1982). gh y g y 16. Guevorkian, K. & Valles, J. M. Swimming Paramecium in magnetically simulated enhanced, reduced, and inverted g environments. P. Natl. Acad. Sci. USA 103, 13051–13056 (2006). ( ) 17. Itoh, A. Motion control of protozoa for bio-mems. IEEE-ASME T. Mech. 5, 181–188 (2000).l oh, A. Motion control of protozoa for bio-mems. IEEE-ASME T. Mech. 5, 181–188 (2000).l p 18. Brennen, C. & Winet, H. Fluid mechanics of propulsion by cilia and flagella. Ann. Rev. Fluid. Mech. 9, 339–398 (1977). Winet, H. Fluid mechanics of propulsion by cilia and flagella. An ennen, C. & Winet, H. Fluid mechanics of propulsion by cilia and l 19. Jana, S., Um, S. H. & Jung, S. Paramecium swimming in capillary tube. Phys. Fluids. 24 041901 (2012). 20. Li, G. et al. Accumulation of swimming bacteria near a solid surface. Phys. Rev. E 84, 041932 (2011).f 21. Smith, D., Gaffney, E., Blake, J. & Kirkman-Brown, J. Human sperm accumulation near surfaces: a simulation study. J. Fluid Mech. 621, 289–320 (2009). 22. Lauga, E., DiLuzio, W., Whitesides, G. & Stone, H. Swimming in circles: motion of bacteria near solid boundaries. Biophys. J. 90, 400–412 (2006). 23. Woolley, D. Motility of spermatozoa at surfaces. Reproduction 126, 259–270 (2003). 24. Denissenko, P., Kantsler, V., Smith, D. & Kirkman-Brown, J. Human spermatozoa migration in microchannels reveals boundary- following navigation. P. Natl. Acad. Sci. USA 109, 8007–8010 (2012). 25. Kantsler, V., Dunkel, J., Polin, M. & Goldstein, R. E. Ciliary contact interactions dominate surface scattering of swim eukaryotes. P. Natl. Acad. Sci. USA 110, 1187–1192 (2013). eukaryotes. P. Natl. Acad. Sci. USA 110, 1187–1192 (2013). y 6. Drescher, K., Dunkel, J., Cisneros, L. H., Ganguly, S. & Goldstein, R. E. Fluid dynamics and noise in bacterial cell-cell and cell surface scattering. P. Natl. Acad. Sci. USA 108, 10940–10945 (2011). 27. Cisneros, L., Dombrowski, C., Goldstein, R. & Kessler, J. Reversal of bacterial locomotion at an obstacle. Phys. Rev. E 73, 030901 (2006). 8. Mannik, J., Driessen, R., Galajda, P., Keymer, J. & Dekker, C. Bacterial growth and motility in sub-micron constrictions. P. Natl Acad. Sci. USA 106, 14861–14866 (2009).i 29. Takeuchi, S., DiLuzio, W., Weibel, D. & Whitesides, G. Controlling the shape of filamentous cells of Escherichia coli. Nano Lett. 5, 1819–1823 (2005). 30. Minc, N., Boudaoud, A. Methods Cell Culture. Sylgard 184 polymer and the curing agent were mixed in ratio of 10:1 by weight and poured over the mould master43. The mixture was degassed and allowed to bake at 65 °C. Finally the cured polymer was cut into pieces and bonded to glass slide using a plasma cleaner and Paramecia were introduced into the channels using a pipette. Swimming Paramecia were observed under Olympus CKX-41 microscope at 4 ×  magnification and their swimming tracks are recorded at 100 fps for 4 sec duration using a high-speed camera (Pixel-Link) as shown in Fig. 8(c). Experimental Methods—Elasticity Measurement. Micro-pipettes were used to apply suction to Paramecia and hold them in place for the experiments involving determination of Young’s modulus. Glass pipette tips were secured in the holder of Micro-forge (MF 900, Narishige Inc.) and heat was Scientific Reports | 5:13148 | DOI: 10.1038/srep13148 7 7 www.nature.com/scientificreports/ applied (using a micropipette puller) which caused the hollow section to be extruded under its own weight. Halfway through the length, where the diameter of the extruded section has become sufficiently low (~ 20 μm); heat is applied so that the thin region bends and forms an angle with the vertical. This was done to ensure that the micro-capillary can easily be dipped into the fluid containing Paramecia.l p y y ppl g While the Paramecia were held in place with the micro-pipette, their distal end were deflected using a glass fiber. The fibers were made from borosilicate glass rods with diameters of 0.5 mm. The rods were first extruded and then the tip was heated until a blob of glass formed at the tip of the rod. The heating coil of the Micro-forge was then brought in contact with the blob and quickly withdrawn in a perpen- dicular direction to extrude a thin fiber. The length of the fiber was clipped to match the stiffness of Paramecium and was then used to deflect the Paramecium held in place. References 1. Foissner, W., Chao, A. & Katz, L. Diversity and geographic distribution of ciliates (protista: Ciliophora). In Foissner, W. & Hawksworth, D. (eds.) Protist Diversity and Geographical Distribution, Vol. 8 of Topics in Biodiversity and Conservation, 111–129 (Springer Netherlands, 2009).h p g 2. Wichterman, R. The biology of Paramecium. (Plenum Press, New York, 1986). h h h h k ff p g 2. Wichterman, R. The biology of Paramecium. (Plenum Press, New York, 1986). h h h h k ff d b l l 2. Wichterman, R. The biology of Paramecium. (Plenum Press, N 3. Machemer, H. & Machemer-Rohnisch, S. Is gravikinesis in Paramecium affected by swimming velocity?. Eur. J. Protistol. 32, 90–93 (1996). 4. Habte, M. & Alexander, M. Further evidence for the regulation of bacterial populations in soil by protozoa. Arch. Microbiol. 113, 181–183 (1977). 5. Madoni, P., Davoli, D. & Gorbi, G. Acute toxicity of lead, chromium, and other heavy metals to ciliates from activated sludge plants. B. Environ. Contam. Tox. 53, 420–425 (1994).hh 6. Jennings, H. Behavior of the lower organisms, vol. 10 (The Columbia university press, The Macmillan company, agents, London, 1906). 7. Machemer, H. Ciliary activity and the origin of metachrony in Paramecium: effects of increased viscosity. J. Exp. Biol. 57, 239–259 (1972). ( ) 8. Jung, I., Powers, T. R. & Valles, J. M. Evidence for two extremes of ciliary motor response in a single swimming microorganism Biophys. J. 106, 106–113 (2014). p y 9. Crenshaw, H. C. Kinematics of helical motion of microorganisms capable of motion with four degrees of freedom. Biophys. J 56, 1029–1035 (1989). 10. Crenshaw, H. C. & Edelstein-Keshet, L. Orientation by helical motion II. Changing the direction of the axis of motion. Bull. Math. Biol. 55, 213–230 (1993). 1. Crenshaw, H. C. Orientation by helical motion III. Microorganisms can orient to stimuli by changing the direction of thei rotational velocity. Bull. Math. Biol. 55, 231–255 (1993). 12. Zhu L., Lauga E. & Brandt L. Low-Reynolds-number-swimming in a capillary tube, J. Fluid Mech., 726, 285–311 (2013).l 12. Zhu L., Lauga E. & Brandt L. Low-Reynolds-number-swim 13. Jonsson, P. & Johansson, M. Swimming behaviour, patch exploitation and dispersal capacity of a marine benthic ciliate in f flow. J. Exp. Mar. Biol. Ecol. 215, 135–153 (1997).hi l p 14. Darbyshire, J. The use of soil biofilms for observing protozoan movement and feeding. FEMS Microbiol. Lett. 244, 329 (2005).h ( ) 15. Author Contributions All authors conceived the experiment(s), conducted the experiment(s), analyzed the results, and ontributed to the writing of the manuscript. References 2 B b S A Fl l i idi d l i f ili J E Bi l 56 459 467 (1972) ( ) 41. Tuson, H. et al. Measuring the stiffness of bacterial cells from growth rates in hydrogels of tunable elasticity. Mol. Microbiol. 84, 874–891 (2012). 42. Baba, S. A. Flexural rigidity and elastic constant of cilia. J. Exp. Biol. 56, 459 467 (1972). 43. Xia, Y. & Whitesides, G. Soft lithography. Ann. Rev. Mater. Sci. 28, 153–184 (1998). 43. Xia, Y. & Whitesides, G. Soft lithography. Ann. Rev. Mater. Sci. 28, 153–184 (1998). g . Jung. acknowledges support from the National Science Foundation (PHY-1205642 and CBET-1336038) S. Jung. acknowledges support from the National Science Foundation (PHY-1205642 and CBET-1336038). Scientific Reports | 5:13148 | DOI: 10.1038/srep13148 References & Chang, F. Mechanical forces of fission yeast growth. Curr. Biol. 19, 1096–1101 (2009).l 31. Son, K., Guasto, J. S. & Stocker, R. Bacteria can exploit a flagellar buckling instability to change direction. Nat. Phys. 9, 494 (2013).il 32. Pedley, T. & Hill, S. Large-amplitude undulatory fish swimming: fluid mechanics coupled to internal mechanics. J. Exp. Biol. 202, 3431–3438 (1999).i 3. Tytell, E. & Lauder, G. Hydrodynamics of the escape response in bluegill sunfish, Lepomis macrochirus. J. Exp. Biol. 211 3359–3369 (2008).l 34. Sheng, J. et al. Digital holographic microscopy reveals prey-induced changes in swimming behavior of predatory dinoflagellates. P. Natl. Acad. Sci. USA 104, 17512–17517 (2007). 35. Hara, R. & Asai, H. Electrophysiological responses of Didinium nasutum to Paramecium capture and mechanical stimulation. Nature 283, 869–870 (1980). , ( ) 6. Hamel, A., Fisch, C., Combettes, L., Dupuis-Williams, P. & Baroud, C. Transitions between three swimming gaits in Paramecium escape. P. Natl. Acad. Sci. USA 108, 7290–7295 (2011). Scientific Reports | 5:13148 | DOI: 10.1038/srep13148 8 www.nature.com/scientificreports/ 37. Codling, E. A., Plank, M. J. & Benhamou, S. Random walk models in biology. J. R. Soc. Interface 5, 813–834 (2008).tf ., Plank, M. J. & Benhamou, S. Random walk models in biology. g gy f 38. Spoon, C. & Grant, W. Biomechanics of hair cell kinocilia: experimental measurement of kinocilium shaft stiffness and base rotational stiffness with Euler-bernoulli and Timoshenko beam analysis. J. Exp. Biol. 214, 862–870 (2011). g gy f 38. Spoon, C. & Grant, W. Biomechanics of hair cell kinocilia: experimental measurement of kinocilium shaft stiffness l ff h l b ll d T h k b l l ( ) 38. Spoon, C. & Grant, W. Biomechanics of hair cell kinocilia: experimental measurement of kinocilium shaft stiffness rotational stiffness with Euler-bernoulli and Timoshenko beam analysis. J. Exp. Biol. 214, 862–870 (2011). f 9. Kuznetsova, T., Starodubtseva, M., Yegorenkov, N., Chizhik, S. & Zhdanov, R. Atomic force microscopy probing of cell elasticity Micron 38, 824–833 (2007).f 0. Deng, Y., Sun, M. & Shaevitz, J. Direct measurement of cell wall stress stiffening and turgor pressure in live bacterial cells. Phys Rev. Lett. 107, 158101 (2011).f , ( ) 1. Tuson, H. et al. Measuring the stiffness of bacterial cells from growth rates in hydrogels of tunable elasticity. Mol. Microbiol. 84 874–891 (2012). Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepihi Supplementary information accompanies this paper at http://www.nature.com/srepihi Competing financial interests: The authors declare no competing financial interests.i How to cite this article: Jana, S. et al. Somersault of Paramecium in extremely confined environments. Sci. Rep. 5, 13148; doi: 10.1038/srep13148 (2015). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Com- mons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ 9
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Color polymorphism in Pachycoris torridus (Hemiptera: Scutelleridae) and its taxonomic implications
Revista chilena de historia natural
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ABRIELY K. SOUZA1, TIAGO G. PIKART1, HARLEY N. OLIVEIRA2, JOSÉ E. SERRÃO3 & JOSÉ C. ZANUNCIO1, * SOUZA1, TIAGO G. PIKART1, HARLEY N. OLIVEIRA2, JOSÉ E. SERRÃO3 & JOSÉ C. ZANUNCIO1, * GABRIELY K. SOUZA1, TIAGO G. PIKART1, HARLEY N. OLIVEIRA2, JOSÉ E. SERRÃO3 & JOSÉ C. 1Departamento de Entomologia, Universidade Federal de Viçosa, 36570-000 Viçosa, Minas Gerais, Brazil 2Embrapa Agropecuária Oeste, Caixa Postal 661, 79804-970, Dourados, Mato Grosso do Sul, Brazil 3Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-000 Viçosa, Minas Gerais, Brazil *Corresponding author: zanuncio@ufv.br Pachycoris torridus (Scopoli, 1772) (Hemiptera: Scutelleridae) is widely distributed in Latin America and it is a serious pest to the plantations of Jatropha curcas Linnaeus in Brazil, damaging fruits and seeds (Rodrigues et al. 2011). This species exhibits twenty one different morphs (Monte 1937, Sánchez-Soto et al. 2004, Santos et al. 2004, Pikart et al. 2011), which were misidentified as eight different species (Costa-Lima 1940). Laboratory of Biological Control of Insects of the UFV, where they were killed and mounted for identifi cation. The identity of P. torridus was confi rmed by comparing the individuals collected with material deposited at the Museu Regional de Entomologia (UFVB) of the UFV. The color patterns of adults of P. torridus included six patterns not previously recorded (Monte 1937, Sánchez-Soto et al. 2004, Santos et al. 2004, Pikart et al. 2011). The most frequent color pattern of P. torridus was black body with The high chromatic variation of P. torridus makes necessary to describe new phenotypes of this insect to assure its correct taxonomic identification. However, the source and function of the color variation of P. torridus are unknown. In this study, we describe six new chromatic patterns for P. torridus in Brazil, and provide a brief discussion about the taxonomic and ecological implications of such variation. Fig. 1: Spot numbering system established to des- cribe color patterns of Pachycoris torridus (Scopoli, 1772) (Hemiptera: Scutelleridae). Sistema de numeración de manchas establecido para des- cribir los patrones de color de Pachycoris torridus (Scopoli, 1772) (Hemiptera: Scutelleridae). This study was car ried out at the Universidade Federal de Viçosa (UFV) in Viçosa, Minas Gerais State, Brazil (20º46’ S; 42º52’ W) in November 2009. The Köppen climate classifi cation of this subtropical region is of the Cwb (rainy summers, and cool and dry winters) with average temperatures of 22.4 °C in the summer and 18.3 °C in the winter, and a mean annual rainfall of 1221.4 mm. COLOR POLYMORPHISM IN PACHYCORIS TORRIDUS REVISTA CHILENA DE HISTORIA NATURAL © Sociedad de Biología de Chile Revista Chilena de Historia Natural 85: 357-359, 2012 NATURAL HISTORY NOTE Color polymorphism in Pachycoris torridus (Hemiptera: Scutelleridae) and its taxonomic implications ABRIELY K. SOUZA1, TIAGO G. PIKART1, HARLEY N. OLIVEIRA2, JOSÉ E. SERRÃO3 & JOSÉ C. ZANUNCIO1, * The area of this study includes agricultural crops used for research studies such as corn, cotton, sunfl ower, beans, wheat, and coffee, which are surrounded by a fragment of secondary forest with native species. Fig. 1: Spot numbering system established to des- cribe color patterns of Pachycoris torridus (Scopoli, 1772) (Hemiptera: Scutelleridae). Adults of P. torridus were collected when feeding on Jatropha curcas Linnaeus (Euphorbiaceae) and transferred to the Sistema de numeración de manchas establecido para des- cribir los patrones de color de Pachycoris torridus (Scopoli, 1772) (Hemiptera: Scutelleridae). SOUZA ET AL. 358 and spots 10, 11 and 12 joined forming a continuous strip (Fig. 2C). and spots 10, 11 and 12 joined forming a continuous strip (Fig. 2C). eight spots on the pronotum and fourteen on the scutellum either yellow or red, and with the ventral part of the body metallic green (Monte 1937). Pattern 25: Black body with yellow spots. Spots four and fi ve very close together, but not joined on the pronotum. Spots six and nine joined, respectively, with spots seven and eight on the scutellum (Fig. 2D). The new color patterns were described following the spot numbering system (Fig. 1) suggested by Monte (1937): Pattern 22: Black body with red spots. On the pronotum, spots one and eight joined, respectively, with spots three and seven. On the scutellum, spots seven and eight joined, and spots 10, 11 and 12 joined forming a continuous strip (Fig. 2A). Pattern 26: Black body with yellow, orange or red spots. Spots six and nine joined, respectively, with spots seven and eight, and spots 10, 11 and 12 joined forming a continuous strip on the scutellum (Fig. 2E). Pattern 27: Black body with yellow spots. Spots four and fi ve joined on the pronotum. Spots two, three and four joined forming a continuous strip, and spots six and nine joined, respectively, with spots seven and eight on the scutellum (Fig. 2F). Pattern 23: Black body with yellow spots. On the scutellum, spots 10, 11 and 12 joined forming a continuous strip (Fig. 2B). Pattern 24: Black body with orange spots. Spots seven and eight joined on the scutellum, Fig. 2: Color patterns of Pachycoris torridus (Scopoli) (Hemiptera: Scutelleridae): (A) pattern 22; (B) pattern 23; (C) pattern 24; (D) pattern 25; (E) pattern 26; (F) pattern 27. COLOR POLYMORPHISM IN PACHYCORIS TORRIDUS ACKNOWLEDGEMENTS: To “Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)”, “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)” and “Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)” for fi nancial support. ACKNOWLEDGEMENTS: To “Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)”, “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)” and “Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)” for fi nancial support. ABRIELY K. SOUZA1, TIAGO G. PIKART1, HARLEY N. OLIVEIRA2, JOSÉ E. SERRÃO3 & JOSÉ C. ZANUNCIO1, * Patrones de coloración de Pachycoris torridus (Scopoli) (Hemiptera: Scutelleridae): (A) patrón 22; (B) patrón 23; (C) patrón 24; (D) patrón 25; (E) patrón 26; (F) patrón 27. Fig. 2: Color patterns of Pachycoris torridus (Scopoli) (Hemiptera: Scutelleridae): (A) pattern 22; (B) pattern 23; (C) pattern 24; (D) pattern 25; (E) pattern 26; (F) pattern 27. Fig. 2: Color patterns of Pachycoris torridus (Scopoli) (Hemiptera: Scutelleridae): (A) pattern 22; (B) pattern 23; (C) pattern 24; (D) pattern 25; (E) pattern 26; (F) pattern 27. Patrones de coloración de Pachycoris torridus (Scopoli) (Hemiptera: Scutelleridae): (A) patrón 22; (B) patrón 23; (C) patrón 24; (D) patrón 25; (E) patrón 26; (F) patrón 27. COLOR POLYMORPHISM IN PACHYCORIS TORRIDUS Editorial responsibility: Mario George-Nascimento Received March 7, 2012; accepted May 31, 2012 COLOR POLYMORPHISM IN PACHYCORIS TORRIDUS 359 Some insects are unpalatable to predators and often advertise them using conspicuous warning colors as bright orange, red and yellow (Ruxton et al. 2004). Predators learn to associate the presence of chemical defense with visual signals, and the presence of polymorphisms would play a role as self- reinforcing signals in the avoidance learning by predators (Joron et al. 1999). The new color patterns of P. torridus described in this study are discretely different from the basic pattern, forming a continuum which could facilitate the avoidance learning by predators. Besides, P. torridus is phenotipically similar to two sympatric species: P. klugii Burmeister, 1835 and P. stallii Uhler, 1863. These species present color polymorphism associated with aposematic behavior and toxic compounds sequestration from Euphorbiaceae plants as defense mechanism against predators (Wink et al. 2000, Williams et al. 2001). Due to the similarity in color pattern between them, and because they co-occur in a large area, these species may form a Müllerian mimetic complex. The presence of chemical defenses in P. torridus was not verifi ed but it could sequester toxic compounds during feeding on Euphorbiaceae plants. Further genetic and behavioral studies are required to explain the function of P. torridus polymorphism and the hypothesis of the Müllerian mimetic complex. RODRIGUES SR, HN OLIVEIRA, WT SANTOS & AR ABOT (2011) Aspectos biológicos e danos de Pachycoris torridus em pinhão-manso. Bragantia 70: 356-360. RUXTON GD, TN SHERRATT & MP SPEED (2004) Avoiding attack: The evolution of crypsis, warning signals and mimicry. Oxford University Press, New York. SÁNCHEZ-SOTO S, P MILANO & O NAKANO (2004) Nova planta hospedeira e novos padrões cromáticos de Pachycoris torridus (Scopoli) (Hemiptera: Scutelleridae) no Brasil. Neotropical Entomology 33: 109-111. SANTOS JC, FAO OLIVEIRA, FMV ALMEIDA & GW FERNANDES (2005) Ecology and behavior of Pachycoris torridus (Hemiptera: Scutelleridae): New host plant, color polymorphism, maternal care and parasitism. Lundiana (Brazil) 6: 107-111. WILLIAMS L, PE EVANS & WS BOWERS (2001) Defensive chemistr y of an aposematic bug, Pachycoris stallii Uhler and volatile compounds of its host plant Croton californicus Muell.-Arg. Journal of Chemical Ecology 27: 203-216. WINK M, C GRIMM, C KOSCHMIEDER, F SPORER & O BERGEOT (2000) Sequestration of phorbolesters by the aposematically coloured bug Pachycoris klugii (Heteroptera: Scutelleridae) feeding on Jatropha curcas (Euphorbiaceae). Chemoecology 10: 179-184. LITERATURE CITED COSTA-LIMA AM (1940) Insetos do Brasil: Hemípteros. Escola Nacional de Agronomia, Rio de Janeiro. JORON M, IR WYNNE, G LAMAS & J MALLET (1999) Variable selection and the coexistence of multiple mimetic forms of the butterfl y Heliconius numata. Evolutionary Ecology 13: 721-754. ( ) Al d h MONTE O (1937) Algumas variações nos desenhos e cores de Pachycoris torridus (Scopoli). O Campo (Brazil) 8: 71. Ã PIKART TG, GK SOUZA, TV ZANUNCIO, JE SERRÃO & JC ZANUNCIO (2011) New chromatic pattern and fi rst register of Pachycoris torridus damaging Coffea arabica fruits in Viçosa, Minas Gerais State, Brazil (Hemiptera: Scutelleridae). Entomologia Generalis 33: 207-211.
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Mathematical Modelling For Decision Making of Lockdown during COVID-19
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Mathematical Modelling For Decision Making of Lockdown during COVID-19 Ahona Ghosh   Brainware University https://orcid.org/0000-0003-0498-285X Sandip Roy   (  sandiproy86@gmail.com ) Brainware University https://orcid.org/0000-0002-5447-803X Suparna Biswas   (  mailtosuparna@gmail.com ) Maulana Abul Kalam Azad University of Technology https://orcid.org/0000-0002-6150-9316 Research Article Keywords: Lockdown, COVID-19, Corona virus, Ridge regression, 1st world countries, 2nd world countries, 3rd world countries, SIR model, Finite Impulse Response ¦lter, Python. Posted Date: July 20th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-42770/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Applied Intelligence on May 10th, 2021. See the published version at https://doi.org/10.1007/s10489-021-02463-7. Noname manuscript No. (will be inserted by the editor) Noname manuscript No. (will be inserted by the editor) Noname manuscript No. (will be inserted by the editor) Mathematical Modelling For Decision Making of Lockdown during COVID-19 Ahona Ghosh · Sandip Roy · Suparna Biswas Ahona Ghosh · Sandip Roy · Suparna Biswas the date of receipt and acceptance should be inserted later Suparna Biswas Department of Computer Science and Engineering, Maulana Abul Kalam Azad University of Technology, West Bengal, India E-mail: mailtosuparna@gmail.com Ahona Ghosh Department of Computational Science, Brainware University, Kolkata, India E-mail: ahonaghosh95@gmail.com 1 Introduction COVID-19 (Corona Virus Disease 2019) is the name given by World Health Organization (WHO) to the worldwide spread disease caused by novel corona virus, the most discussed topic nowadays. The virus is totally a new one and previously has not been observed ever, the reason behind calling it ‘novel’, lies here. The present statistics say that in spite of being the most developed country having strong financial background, first world countries like USA, Italy, France, Spain, Germany are suffering more than the other countries in the world due to corona. The origin of this virus was China and the first case was observed in Wuhan of China in December 2019. After that, it began to spread all over the world and affected more than 170 countries till now. The rapid growth in the number of affected and deceased people is very alarming and therefore governments have been forced to take hard decisions like lock- down and sometimes partial lockdown in their respective countries. According to WHO globally 5317625 confirmed cases have been registered, 340214 deaths have been recorded till date [1]. Forecasting methods can help governments to take the right decision and proceed further accordingly, so our approach here is to design a decision- making system for lockdown, which analyses different records of lockdown in different countries and predicts when it will end in the concerned areas. Figure 1 shows that USA is leading in the total number of positive cases of COVID-19 and Spain is after that, where in the second world countries like Kazakhstan, Uzbekistan and Romania and third world countries like Philip- pines, Mexico and Thailand have not been affected much. Figure 2 shows number of positive cases per 1 million population, here comes the concept of population density, because more the population density, more is the chance of spreading the virus. Spain is facing the greatest number of infections per 1 million population, whereas Italy and France are not much behind. The total number of cases till 26th April, 2020 is shown in Figure 3, we have considered five countries from each of the first world, second world and third world, so that the economy of the affected countries can also relate during the analysis. Based on the economy, their correlation with the trend of recovery (if any) has been attempted to detect. Abstract Due to the recent worldwide outbreak of COVID-19, there has been a huge change in our lifestyle and it has a severe impact in different fields like fi- nance, education, business, travel and tourism, economy and several others in all the affected countries.In this scenario, people have to be careful and cau- tious about the symptoms and should act accordingly. Accurate predictions of different factors, like the end date of the pandemic, duration of lockdown and spreading trend can guide us through the pandemic and precautions can be taken accordingly. Multiple attempts have been made to model the virus transmission, but none of them has investigated it at a global level. The nov- elty of our proposed work lies here. In this paper, at first, we have analysed the nature of spreading of the said disease using data collected from various platforms and then, we have presented a predictive mathematical modelling for fifteen different countries from first, second and third world considered by us to have an idea and indicative data for probable future projections of this pandemic. The prediction can be used by planning commission, health care organizations and the government agencies as well for creating suitable arrangements against this pandemic. Recommendations can be created; advi- sories created on the basis of the prediction can be implemented region and country wise according to the situation. Ahona Ghosh Department of Computational Science, Brainware University, Kolkata, India E-mail: ahonaghosh95@gmail.com Sandip Roy Department of Computational Science, Brainware University, Kolkata, India E-mail: sandiproy86@gmail.com 2 Ahona Ghosh et al. Keywords: Lockdown, COVID-19, Corona virus, Ridge regression, 1st world countries, 2nd world countries, 3rd world countries, SIR model, Finite Impulse Response filter, Python. 1 Introduction From 15th February to 26th April, the daily new cases have been recorded and shown country wise in Figure 4.The forthcom- ing situations can be controlled in an efficient manner if systematic forecast is made and guidance from medical persons is provided accordingly. Mathematical Modelling For Decision Making of Lockdown during COVID-19 3 Figure 1. Country wise total cases upto 26.04.2020 Figure 1. Country wise total cases upto 26.04.2020 Figure 2. Country wise total cases per 1 milion population Figure 2. Country wise total cases per 1 milion population Ahona Ghosh et al. 4 Figure 3. Date and country wise COVID-19 total cases Figure 3. Date and country wise COVID-19 total cases Figure 3. Date and country wise COVID-19 total cases Figure 4. Date and country wise daily new cases Figure 4. Date and country wise daily new cases Mathematical Modelling For Decision Making of Lockdown during COVID-19 5 Figure 5. Date and Country wise total active cases Figure 5. Date and Country wise total active cases Figure 6. Date and Country wise total deaths Figure 6. Date and Country wise total deaths Ahona Ghosh et al. 6 Figure 7. Date and country wise daily new deaths Figure 7. Date and country wise daily new deaths Now, in the whole world, the death rate due to COVID-19 per day is 15.82%. The recovery rate from COVID-19 per day is 84.18% and the same for first world, second world and third world countries are shown in Table 1, which is very alarming for all. Date and Country wise total active cases, total deaths and daily new deaths have been shown in Figure 5, Figure 6 and Figure 7 respectively. The date wise worldwide cumulative death rate and recovery rates are shown in Figure 8, where it is clear that death and recovery both are undergoing a normal growth proportional to the currently active cases. 1 Introduction Country Population Population density (/km2) Average recovery rate from COVID 19 per day Average death rate due to COVID 19 per day USA 32.82 crores 36 — 70.56 29.44 France 6.7 crores 117 Spain 4.69 crores 96 Italy 6.04 crores 206 Germany 8.3 crores 227 China 139.27 crores 379 — 95.15 4.85 Russia 14.45 crores 8.4 Uzbekistan 3.3 crores 79 Kazakhstan 1.83 crores 7 Romania 1.94 crores 82 India 135.26 crores 464 — 68.25 17.06 Brazil 20.95 crores 24.66 Thailand 6.94 crores 137 Mexico 12.62 crore 67 Philipines 10.67 crore 368 Table 1 Country wise recovery and death rate mapping with population Country Population Population density (/km2) Average recovery rate from COVID 19 per day Average death rate due to COVID 19 per day USA 32.82 crores 36 — 70.56 29.44 France 6.7 crores 117 Spain 4.69 crores 96 Italy 6.04 crores 206 Germany 8.3 crores 227 China 139.27 crores 379 — 95.15 4.85 Russia 14.45 crores 8.4 Uzbekistan 3.3 crores 79 Kazakhstan 1.83 crores 7 Romania 1.94 crores 82 India 135.26 crores 464 — 68.25 17.06 Brazil 20.95 crores 24.66 Thailand 6.94 crores 137 Mexico 12.62 crore 67 Philipines 10.67 crore 368 Table 1 Country wise recovery and death rate mapping with population Table 1 Country wise recovery and death rate mapping with population Table 1 Country wise recovery and death rate mapping with population Mathematical Modelling For Decision Making of Lockdown during COVID-19 7 Figure 8. Datewise worldwide cumulative death rate and recovery rate in percentage Figure 8. Datewise worldwide cumulative death rate and recovery rate in percentage In the next section, existing literature in our concerned domain have been reviewed. In section 3, the proposed methodology for predicting the lockdown end dates of lockdown in the fifteen countries from first, second and third world has been discussed. The experimental result with comparison and per- formance evaluation have been presented in section 4 and finally the concluding statements and future directions of our proposed work have been discussed in Section 5. 2 Literature survey Several research works are going on with different factors regarding the corona virus outbreak [2] [3] [4]. Somewhere the end dates of COVID-19 in different countries have been predicted [5], the size at the end [6] and somewhere the spread in the concerned countries has been modelled and predicted date wise [7], time series also has played a major role in estimation of transmission rate scaling [8]. Mahalle et al. have [9] discussed some challenges and recommended some strategies to control the outbreak. Different prediction techniques have been adopted using different platforms including mathematical modelling, big data, social media post, data science and machine learning here. Bhola et al. have shown in [10] that the only way to decrease the number of new infective with time is to totally isolate the infective from the susceptible population. Influenza epidemic about a century ago has shown how the situation can gets worse day by day just for the exponential growth due to the large number of susceptible people in India. The same can be repeated if the current situation goes uncontrolled or casually attended. The impact of travel restrictions due to COVID-19 has been discussed in [11] where the modelling results have shown that sustained 90% travel limitations to and from China diffidently affect the epidemic route. The results suggest that isolation, home quarantine and early detection are more effective than the travel restriction, because it has just de- layed the progression of the virus within china by 3-5 days, but could not stop Ahona Ghosh et al. 8 it from propagation. The current situation of China, mainly six provinces having most of the cases has been analysed in [12] [13] [14] whereas the same for Germany [15], Portugal [16], United States [17], Italy [18], Brazil [19] has been discussed in the recent literature. Although the spread of COVID-19 has been exponentially increased all over the world, still the fatality rate is not so alarming which gives residents of every country to re-establish the confidence to overcome and fight this pandemic together. However, the fatality rate for severe cases has been observed as 10% for which, the governments need to adopt proper se- ries of strategies as early as possible. Numerical analysis of COVID-19 has been conducted in [20] where recovery rate and transmission rate character- istics have been tracked and the future trend has been predicted in addition. 2 Literature survey The transmission record of early stage from January Mathematical Modelling For Decision Making of Lockdown during COVID-19 9 Mathematical Modelling For Decision Making of Lockdown during COVID-19 10 to January 24 has been analysed and observed exponential growth in [31]. The experimental results identify the potential of the virus to cause outbreaks in Mainland China. With the increase in the reporting rate from 8-fold to 0- fold, the mean of basic reproduction number has been calculated in [32] which is a little bit higher than the same measure reported by WHO. The measure- ment is typically based on the accuracy of determining Susceptible-Infected, which requires sufficient amount of patient samples over a significant follow up time. In comparison with China, higher death rates and lower recovery rate ob- served due to Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV- 2)has proved that most of the Italian cases are of severe infection because of delayed lockdown and initially lower restrictions [33][34]. Awareness in gen- eral people are mandatory for effective interventions of government, and thus the number of affected people in Iran has been estimated upto 15th April in Zareie et al.’s research work [35]. Twenty countries all over the world have been considered in [36] for providing reliable statistics of the COVID-19 scenario. The transmission model has been characterized using parameters from the existing literature or some reasonable values, which somehow depend on the strength and the speed of drugs to be undertaken. Basic reproduction number, non-affecting period, pre symptom affecting period, probability of severity de- velopment, probability of severity diagnosis, probability of mild case diagnosis as well have been the factors contributing to successful pandemic modelling. Being the second epicentre of COVID-19 spread, Italyhas been the main focus of the study conducted by Russo et al. in [37] where they have used SEIRD model (S: Susceptible, E: Exposed, I: Infected without symptoms, R: Recov- ered, D: Deceased) considering two categories of population, Asymptomatic one not having proper symptoms of the disease and another one. i.e. Symp- tomatic is having significant symptoms. Genetic algorithm has been used to optimize the objective function base on all the combinations of initial predic- tions of pandemic dynamics. Control reproduction number and basic reproduction number have been the two factors of analytical calculation in Nadim et al.’s work [38] for obser- vation of the dynamics of this epidemic in five provinces of China. 2 Literature survey Deterministic Susceptible-Infected-Removed(SIR) model can be enhanced to stochastic model in future to achieve a better precision in the experimental result. The novel feature of this specific one is the ability of it to spread in the latent time, thus the time delay has been considered in differential equa- tions formed by Chen et al. for proposal of a dynamic model to describe the pandemic in China [21]. Transmission route of the virus has been analysed in [22] using decision tree and prior algorithms, but due to no implementation in terms of programming or coding, there is a chance of data to be missed and more algorithms need to be added for checking the overall performance of the proposed approach.Spread of COVID-19 in Hubei of China has been modelled and predicted in [23], where the characteristics of populations like effect of age, people having other health problems, steps taken already for controlling the spread have not been studied, whereas decisions like lockdown and social distancing and its effects have been taken into consideration in our model, the novelty of our proposed model lies here. Yafia has presented a modelling and dynamics with social distancing and isolation effect applied to Moroccan people [24]. Level of abstraction for the available data has been a major concern in [25]where modelling of biological systems has taken place to understand the spreading pattern of covid-19. Li et al. have presented a time series and kinetic analysis model where they have proved that emergency interventions regarding the restriction in people going out by Chinese government have a great impact on the spread of the pandemic [26]. Experimental results find out that bodies of deceased people do not result in additional infection and recovered people get an advantage of the prepared antibodies in their body which prevent reinfections later.Delay in identifying the symptoms and Chinese medicine preparation have been considered as the factors contributing to modified viral shading dynamics patterns of COVID- 19 as mentioned by He et al. in [27].The first 425 cases have been considered by Guan et al. for determination of epidemic characteristics in terms of mean incubation time [28] [29], mean serial interval, confidence interval, basic repro- duction number and concluded that transmission took place from the middle of December 2019 in [30]. 2.1 Motivation In the current situation due to the pandemic, where the worldwide infection and decease rate are really very alarming, the world needs quick recovery and for that, a proper prediction regarding the transmission range, transmission trend and decision making of lockdown measures are needed. From the existing literature, it is clear that there have been many attempts to predict the disease- spreading trend but all of them have considered only either their own country [2] [3] or maximum two or three countries from all over the world. However, as the isolation and social distancing guidelines need to follow the steps taken in the already recovered countries, it is required to analyse the decisions made by all of them [1] and the factors like population density due to which, the decision should vary country wise, also should be taken into account for better understanding and analysis of the situation. In this regard, the objective of our proposed work has come into picture and the approaches like the well known SIR model and regression, which have history of accurate prediction of earlier diseases [15], have been applied in our work to get a better accuracy and efficiency in the prediction. 2 Literature survey Research outcomes show that for reduction of the burden, management of quarantined people is more effective than the management of isolated people. The effect of border control on the transmission of COVID-19 has been studied by investi- gating the outbreak trend in mostly visited ten cities of China in [39] where risk assessment has been done. The platform can be used in multiple affected sources to multiple target destinations by keeping the computational complex- ity constant using different control measurements. Volpert et al. have analysed disease transmission data and found three possible patterns, i.e. growth-decay- growth dynamic, growth-decay dynamics and growth dynamics discussed in [40]. Imposed quarantine should be stricter to be effective, because the current one is not sufficient.The evolution of COVID-19 has been performed and fu- 10 Ahona Ghosh et al. ture possibilities have been predicted using the extended version of composite Monte-Carlo model integrated with Deep learning and fuzzy rule in [41] where the main focus was decision making where the other existing works have con- centrated on disease modelling only. The duration and severity of lockdown should be scheduled against their impact on economy, as whenever these mea- sures will be relaxed a bit, the disease transmission trend will go back to its original exponential growth [42]. Impact of COVID-19 on economy has been evaluated in[43] by solving a stylized production-based asset pricing model with the use of transmission rate estimated by SIR model of China, Japan and Korea. Four mostly infected regions of China and five mostly infected regions of Italy have been the focus of research in [44] where the assumption of COVID-19’s omission during summer has been investigated by considering three environmental factors, i.e. highest wind speed, maximum temperature and maximum relative humidity. Research findings show that the first and the third one is negligible, whereas the impact of the second factor to the virus transmission can be ranged between negligible to moderate. However, prelim- inary data has been used here, it can hold stronger conclusion whenever more data becomes available. 2.2 Contribution This paper contributes in the concerned field in terms of prediction for fif- teen countries from first, second and third world and in decision making for lockdown there. None of the existing works have considered worldwide disease transmission trend to predict the nature of the virus infection, so the novelty Mathematical Modelling For Decision Making of Lockdown during COVID-19 11 of our proposed work lies here. The main contributions can be summarized as follows – The time dependent discrete SIR model has been able to predict and anal- yse the disease transmission trend. – The time dependent discrete SIR model has been able to predict and anal- yse the disease transmission trend. – Change in the basic reproduction number, which is the indicator of the transmission trend, not the infection rate has shown that after lockdown it has decreased which eventually proves that the decision of lockdown was essential globally to control the massive impact of COVID-19 on the common people, especially to the population of the third world countries where the economic condition to handle the situation is really very poor. – However, more strictness in the steps taken by the government authorities to maintain the social distancing and lockdown decisions could make the lockdown a success. The comparative summary of the existing works and the motivation behind taking up our proposed work from their limitations have been presented in Table 2. Ref. no. Objective Data source used Area considered Method and Simulation platform Simulation parameter Limitation found (if any) [3] Virus transmission rate identification https://www. who.int States of India SIR model and Mathematica Total case, recovered case & death No consideration of lockdown and social distancing parameters [6] Estimation of final affected size 1.https://our worldind ata.org/cor onavirus source-data 2.https://www. worldometer s.infocorona virus Not defined MATLAB Total case, recovered case & death No consideration of country population density [7] Prediction of pandemic end https://ourwo rldindata. org/corona virussource -data Singapore and Italy MATLAB New cases per day No consideration of country population density & social distancing [10] Prediction of undetectable infected population http://www. nhc.gov. cn/xcs/yq tb/listgzbd .shtml China MSIER & Python Total case, recovered case &daily new case No comparison with the other countries transmission trend Prop osed TDD SIR Decision making of lockdown https://www. 2.2 Contribution worldomete rs.info/cor onavirus Countries all over the world Time dependent discrete SIR and Scikit learn of Python Total case ,recovered case, daily new case, new death Stochastic model & more regression methods can be applied to test the result Table 2 Comparative study of proposed work with existing works Table 2 Comparative study of proposed work with existing works 3 Proposed methodology Hethcote has analysed different mathematical models in [45] and applied them in specific diseases according to the trend of spreading. Contact number, ba- sic reproduction number, replacement number of SIR (S: Susceptible cases, I: 12 Ahona Ghosh et al. Infected cases, R: Removed cases), MSEIR (M: People having passive immu- nity, S: Susceptible population, E: People exposed to the disease, but not yet affected, I: Infected and spreading people, R: Recovered people having perma- nent immunity) [46] and SEIR models [47] have been reviewed and discussed which is used to estimate various disease patterns later. The basic reproduction number denoted as R0 can be defined as the average number of infected people due to entrance of an infected one into a susceptible population. Age structure, spatial structure and heterogeneity have been considered as the main factors contributing to the trend of disease spread here. 3.1 SIR model for COVID-19 SIR model for detecting any disease as epidemic says that if is the density of susceptible birth, then N = S + I + R = 0 (1) (1) Where when λ=0, N=total population S=total susceptible people I=total infected people R=total removed people (dead/recovered) and the rate of change in suscepti- ble population w.r.t. time is Where when λ=0, N=total population S=total susceptible people I=total infected people R=total removed people (dead/recovered) and the rate of change in suscepti- ble population w.r.t. time is S=total susceptible people S total susceptible people I=total infected people R=total removed people (dead/recovered) and the rate of change in suscepti- ble population w.r.t. time is R=total removed people (dead/recovered) and the rate of change in suscepti- ble population w.r.t. time is ds dt = λ −r(t)(1 −α(t))(1 −µ(t))S(t)I(t) −α(t)S(t) (2) f change in infected population w.r.t. time is ds dt = λ −r(t)(1 −α(t))(1 −µ(t))S(t)I(t) −α(t)S(t) (2) (2) The rate of change in infected population w.r.t. time is dI dt = r(t)(1 −α(t))(1 −µ(t))S(t)I(t) −a(t)I(t) −µ(t)I(t) (3) (3) The rate of change in removed population w.r.t.time is dR dt = a(t)I(t) + µ(t)I(t) + α(t)S(t) (4) (4) Where r=rate of getting infected, Where r=rate of getting infected, a=average rate of recovery death, a=average rate of recovery death, a=average rate of recovery death, α=lockdown rate of susceptible, α=lockdown rate of susceptible, α=lockdown rate of susceptible, µ=isolation rate of infectious. µ=isolation rate of infectious. µ Therefore, the total change rate Therefore, the total change rate dS dt + dI dt + dR dt = 0 (5) dS dt + dI dt + dR dt = 0 (5) The susceptible fraction of the population s(t) = S(t) N (6) s(t) = S(t) N (6) Mathematical Modelling For Decision Making of Lockdown during COVID-19 13 The infected fraction of the population The infected fraction of the population The infected fraction of the population i(t) = I(t) N (7) i(t) = I(t) N (7) (7) The removed fraction of the population The removed fraction of the population r(t) = R(t) N (8) r(t) = R(t) N (8) and (8) and and s(t) + i(t) + r(t) = 1 (9) (9) It has been assumed that 0≤l≤1 and 0≤i≤1 where l is the lockdown of suscep- tible rate and i is the isolation of infectious rate. 3.1 SIR model for COVID-19 The fraction of susceptible protected people is (1-l)S and the fraction of un-isolated infectious people is (1-i)S.The basic reproduction number denoted by R0 has been expressed in different forms during the first, second and third phase of outbreak in different countries. The duration of first, second and third stages of outbreak in all the fifteen countries considered by us have been represented in Table 3 where the 1st phase denotes the appearance stage, the second stage deals with the local transmission and the thirds stage is concerned about community transmission. The initial stage is where the disease is first introduced and active cases start to emerge for the very beginning, in this stage, almost everyone is protected, because very few people have come across the persons with travel history. The local transmission is the spread of the disease from a person who has a travel history to his local people (family, neighbours and friends) or the spread from a directly contacted infectious person. The community transmission is the most dangerous stage where the source of transmission is very difficult to trace. The newly affected people have no history of being in contact with foreign sourced people or the people belonging to hotspot areas. Phase 4 or the widespread outbreak has been faced by China only until the number of cases and deaths begin to grow exponentially; Italy and USA are apparently in this stage now. 3.2 Time Dependent Discrete SIR (TDDSIR) model Using time dependent discrete SIR model, equations (2), (3), (4) have been modified Using time dependent discrete SIR model, equations (2), (3), (4) have been modified S(t + 1) −S(t) = −r(t)S(t)I(t) N (10) I(t + 1) −I(t) = r(t)S(t)I(t) N −a(t)I(t) (11) R(t + 1) −R(t) = a(t)I(t) (12) (10) (11) (12) (11) R(t + 1) −R(t) = a(t)I(t) (12) (12) At the very first stage, the number of confirmed cases is very low and it can be assumed that most of the total population is in susceptible stage (N≈S(t)). At the very first stage, the number of confirmed cases is very low and it can be assumed that most of the total population is in susceptible stage (N≈S(t)). 14 Ahona Ghosh et al. So,by modifying equation (11), we get So,by modifying equation (11), we get I(t + 1) −i(t) = r(t)I(t) −a(t)I(t) (13) (13) Daily recovery rate and transmission rate can be denoted by Daily recovery rate and transmission rate can be denoted by a(t) = R(t+1)−R(t) I(t) (14) a(t) = R(t+1)−R(t) I(t) (14) (14) and r(t) = R(t+1)−R(t)+I(t+1)−I(t) I(t) (15) and and r(t) = R(t+1)−R(t)+I(t+1)−I(t) I(t) (15) (15) 3.2.1 Detection of transmission and recovery rate using Ridge regression Finite impulse response filter [50] can be used to predict the transmission and recovery rate denoted as follows r′(t) = p0+p1r(t−1)+p2r(t−2)+p3r(t−3)+...+pcr(t−c) = p0+ C X c=1 pcr(t−c) (16) a′(t) = q0+q1a(t−1)+q2a(t−2)+q3a(t−3)+...+qda(t−c) = q0+ D X d=1 qda(t−d) (17) r′(t) = p0+p1r(t−1)+p2r(t−2)+p3r(t−3)+...+pcr(t−c) = p0+ C X c=1 pcr(t−c) (16) (16) a′(t) = q0+q1a(t−1)+q2a(t−2)+q3a(t−3)+...+qda(t−c) = q0+ D X d=1 qda(t−d) (17) ( ) Where C and D are the orders of the two filters having coefficients pc(0≤c≤C) and qd(0≤d≤D) respectively and r′(t) and a′(t) are the predicted transmission and predicted recovery rate. There are various machine learning methods like partial least square, regularized least square (also called ridge regression) and original least square method to solve the following optimization problem minpc T −2 X t=C (r(t) −r′(t))2 + γ1 C X c=0 p2 c (18) (18) and and minqd T −2 X t=D (a(t) −a′(t))2 + γ2 D X d=0 q2 d (19) (19) Where γ1 and γ2 are its regularization parameters respectively. Mathematical Modelling For Decision Making of Lockdown during COVID-19 15 3.2.2 Detection of number of affected persons and recovered persons 3.2.2 Detection of number of affected persons and recovered persons Finite impulse response filter can be used to forecast the amount of affected and recovered population by previously discussed time dependent SIR model. After calculating the transmission rate and recovery rate according to (14) and (15), the ridge regression is used to solve the optimization problems of (18) and (19) keeping the constraints of (16), (17) to learn the pattern of co- efficients of finite impulse response filter. For predicting the affected and recovered population, r(t) and a(t) have been replaced by r′(t) and a′(t) in equation (17) and (18) where the predicted number of affected people and recovered people at time t = T are denoted as I′(T) and R′(T). This leads to I′(T) = (1 + r′(T −1)) −a′(T −1)I(T −1) (20) R′(T) = R(T −1) + a′(T −1)I(T −1) (21) (20) (21) When t>T the affected and recovered population can be predicted as follows I(t + 1) = (1 + r′(t) −a′(t))I(t) (22) R′(t + 1) = R(t) + a′(t)I(t) (23) I(t + 1) = (1 + r′(t) −a′(t))I(t) (22) (22) R′(t + 1) = R(t) + a′(t)I(t) (23) (23) The deterministic model for epidemic considered by us is more appropriate to large population. So, when I(t) and R(t) are comparatively trivial, the accu- racy may not be as desired, in that case, stochastic model like Markov Chain can be used to approximate and forecast the factors according to requirement in the future and more regression methods can be applied to check if any of them is giving better result. The algorithm of the prediction method is as follows. 3.2.2 Detection of number of affected persons and recovered persons ALGORITHM: Predicting time dependent discrete SIR model Input: I(t), R(t), 0≤t≤T-1, regularization parameters γ1 and γ2, two filters C and D, forecasting window F Output:r(t),a(t), 0≤t≤T-2 r’(t),a’(t),t≥T-1 I’(t),R’(t),t≥T Step 1: Calculate r(t) and a(t) using (15) and (14) respectively when 0tT-2 Step 2: Train them with ridge regression using (18) and (19) Step 3: Calculate r(T-1) and a’(T-1) using (16) and (17) Step 4: Calculate I′(T) and R′(T) for the next day T using (20) and (21) Step 5: If T≤t≤T+Fdo Calculate r′(t) and a′(t) using (16) and (17) Estimate I′(t + 1) and R′(t + 1) using (22) and (23) respectively End if Step 6: End ALGORITHM: Predicting time dependent discrete SIR model Input: I(t), R(t), 0≤t≤T-1, regularization parameters γ1 and γ2, two filters C and D, forecasting window F 16 Ahona Ghosh et al. 3.3 Steady state solutions 3.3 Steady state solutions According to the steady state solution, the probable duration of susceptibility can be defined by P(min(TL|TS)) where TL is the duration of being alive and TS is the duration of remaining in susceptible state before being affected. The probable duration can be expressed by P(min(TL|TS)) = Z ∞ o e−(µ+)xdx = 1 µ + λ (24) (24) The equilibria of steady state equilibrium states that the basic reproduction number R0 = r a (25) (25) where dI dt > 0 when R0 > 1 implies that the number of affected people increases with the increase in reproduction number and decreases with the decrease in the reproduction number. 4 Trend of disease progression with lockdown The resolved factor R depends on S and I,so here we have restricted our implementation to SI. With the progress of disease spreading over a longer time, the new births have been considered. In this scenario, the system can be represented by ds dt = λ −(1 −α)r(1 −µ)SI −αS (26) dI dt = (1 −α)r(1 −µ)SI −aI −µI (27) (26) (27) To detect the effectiveness of social distancing approach we have divided the infected population into two categories by extending the conventional SIR model. The first one is called Type I who are generally the detectable infected persons and the second one is called Type II who are undetectable infected person. If the probability of an infected people becoming detectable is denoted by p1 and becoming undetectable is p2, then To detect the effectiveness of social distancing approach we have divided the infected population into two categories by extending the conventional SIR model. The first one is called Type I who are generally the detectable infected persons and the second one is called Type II who are undetectable infected person. If the probability of an infected people becoming detectable is denoted by p1 and becoming undetectable is p2, then p1 + p2 = 1 (28) (28) If the transmission rates among Type I and Type II people are denoted by r1 and r2 respectively and the recovery rates for the same area1 and a2, then If the transmission rates among Type I and Type II people are denoted by r1 and r2 respectively and the recovery rates for the same area1 and a2, then R0 = p1 r1 a1 + p2 r2 a2 (29) (29) In practical scenario, Type II population has a higher transmission rate than that of a Type I population. The controllable measure of the disease is indi- cated by spectral radius which in turn shows that if the basic reproduction number R0 > 1, then there is some outbreak, and if it is <1, then no outbreak Mathematical Modelling For Decision Making of Lockdown during COVID-19 17 is there. Allowing all to keep their interpersonal contacts upto a fraction of normal contacts and cancelling mass gatherings, these two approaches of main- taining social distance have been considered here. 4 Trend of disease progression with lockdown The lockdown in the whole country announced by government has definitely acted as infection controller and helped India to face the challenge of COVID-19 in the desired form. 5.1 Dataset preparation and validation The proposed work for analysis and prediction of worldwide COVID-19 has been carried out using the dataset collected from worldometer[48], which is developed and maintained by an international group of researchers and devel- opers, having an aim of making world statistics available in a time relevant format and has been recognised as the best free reference website by American Library Association. Several researchers considered this for undertaking their research on COVID-19. Number of daily new cases, daily recovered popula- tion, datewise total cases and active cases have been used as the parameters for analysis and prediction of the disease transmission trend and impact of lockdown on it. 5 Experimental Result and Discussion The following section describes different aspects of our proposed work for analysis of lockdown effect on the disease transmission and discusses the con- tribution in terms of novelty and performance evaluation as well. 5.2 Analysis of lockdown effect on disease transmission There is no confusion that many factors affect the propagation of the virus, es- tablishing a dynamic propagation model from the estimated factors is a tough job, but basic estimation of some parameters like mortality and latency can take place to help people for being cautious about the transmission trend and acting accordingly. The time evolution of the outbreak has been analysed by time dependent SIR model and represented in Figure 9- Figure 11. Result of mathematical modelling in Figure 9 shows date-wise actual recovered popu- lation vs predicted recovered population using SIR model from where it is evident that our prediction results are quite near to the reality and proposed model suits its concerned domain. As it is not feasible to give the whole basic reproduction number calculation and daily new case prediction result in the paper, thus only for one country from each first, second and third world, the same are shown for the duration of ten days, which is from 20.03.2020 to 30.03.2020 in Table 3 and 4 respectively. 18 Ahona Ghosh et al. Country 21.03 22.03 23.03 24.03 25.03 26.03 27.03 28.03 29.03 30.03 France 0 2.38 0 2.122 4.24 3.32 4.51 0 1.68 5.45 China 0.17 0.08 0.16 0.12 0.12 0.14 0.11 0.15 0.037 0.19 India 0 0 0 0 0.80 0.45 0.65 0.08 0.09 0.34 Table 3 Basic reproduction number calculation from 21.03.2020 to 30.03.2020 18 Ahona Ghosh et al. Country 21.03 22.03 23.03 24.03 25.03 26.03 27.03 28.03 29.03 30.03 France 0 2.38 0 2.122 4.24 3.32 4.51 0 1.68 5.45 China 0.17 0.08 0.16 0.12 0.12 0.14 0.11 0.15 0.037 0.19 India 0 0 0 0 0.80 0.45 0.65 0.08 0.09 0.34 Table 3 Basic reproduction number calculation from 21.03.2020 to 30.03.2020 18 Ahona Ghosh et al. Table 3 Basic reproduction number calculation from 21.03.2020 to 30.03.2020 Country 21.03 22.03 23.03 24.03 25.03 26.03 27.03 28.03 29.03 30.03 Germany 2516 2509 4183 3935 4332 6615 6933 6822 4740 4450 France 1847 1559 3838 2448 2929 3922 3809 4611 2599 4376 Italy 6557 5560 4789 5249 5210 6203 5909 5974 5217 4050 USA 4848 9400 10311 11366 13451 17388 18743 19452 20065 20732 India 0 1 0 2 3 1 22 17 11 36 justification=centering Table 4 Daily new case prediction from 21.03.2020 to 30.03.2020 A free machine learning library of Python 3, Scikit-learn has been used for running the ridge regression calculation. Since the transmission rate can never be a negative one, it has been set as 0 when it is 0 and since the data before 15th February, 2020 is very small and sometimes unavailable which can result to a noise in analysing the trend, so only the data onwards 15.02.2020 has been considered here. In an epidemic model, one question always arises that whether the epidemic will end and whether a certain population may be infected from the total population due to the conversion of disease nature from epidemic to pandemic. For answering these questions, the parameter of basic reproduction number is defined as the number of infected people by an already infected person before his/her recovery or death. In the classical SIR model, it is simply the ratio of the number of persons in contact with an infected one and number of days he/she takes to recover which is modified as a function of time in the time dependent SIR model later. When the basic reproduction number is calculated as greater than 1, in that scenario, the disease is said to spread exponentially and a certain fraction of the total population is said to be affected eventually. Mathematical Modelling For Decision Making of Lockdown during COVID-19 19 Figure 9. Date-wise actual total recovered population vs predicted total recovered population in five most affected countries Figure 9. 18 Ahona Ghosh et al. Country 21.03 22.03 23.03 24.03 25.03 26.03 27.03 28.03 29.03 30.03 France 0 2.38 0 2.122 4.24 3.32 4.51 0 1.68 5.45 China 0.17 0.08 0.16 0.12 0.12 0.14 0.11 0.15 0.037 0.19 India 0 0 0 0 0.80 0.45 0.65 0.08 0.09 0.34 Table 3 Basic reproduction number calculation from 21.03.2020 to 30.03.2020 Date-wise actual total recovered population vs predicted total recovered population in five most affected countries The predicted daily new cases have been compared to the actual daily- infected population in Figure 10 for the most affected twelve countries among the fifteen countries considered by us, where it is clearly seen that SIR model has been able to predict the infection rate and infected population efficiently. Deviation is there but predicted graph is following the pattern of actual one. Ahona Ghosh et al. 20 Figure 10. Actual daily new cases vs predicted new cases Figure 10. Actual daily new cases vs predicted new cases Mathematical Modelling For Decision Making of Lockdown during COVID-19 21 The effect of lockdown in the basic reproduction number (R0) of five most af- fected countries according to our research, i.e. France, Germany, India, China and Brazil has been shown in Figure 11 where the transmission rate is con- cerned, not the infected people. After lockdown, the infected population has increased exponentially, it means it has followed its previous trend, but the basic reproduction number, which is basically the ratio of transmission rate and recovery rate, has decreased globally and the change has been marked by the black circles, so we can conclude that lockdown decision is obviously an effective one and if its more strict, then the infected population can also decrease and once the vaccine comes to the market, people will be safe. Figure 11. Change in basic reproduction number after lockdown in the five most affected countries Figure 11. Change in basic reproduction number after lockdown in the five most affected countries Therefore, the experimental results show that prediction of infected popula- tion and recovered population both have some minor error when the predicted result is compared to the actual one. Moreover, we have been able to track the transmission trend and characteristics with respect to time for fifteen coun- tries all over the world. To understand the impact of social distancing during lockdown imposed by the concerned governments, it has been seen that proper social distancing can lead to decrease in propagation rate. 6 Conclusion The role of lockdown on susceptible people and isolation of infectious people has been discussed in this paper based on the basic reproduction number of SIR model on epidemic spread in general. Three different phases of corona virus 22 Ahona Ghosh et al. spread in different countries have been considered here for the analysis: Begin- ning/ first phase of the outbreak (3rd-8th March), Intermediate stage/ second stage of the outbreak (9th-19th march), Third stage of contamination (20th march-till date). In the first stage, the cumulative growth rate was moderate where as in the second stage of the epidemic, the transmission rate gets higher and the infection rate increases drastically. In the third and the final stage, the lockdown and isolation effects have been taken into account; the transmission rate has been smaller when compared to the previous phase. Since the basic re- production number somewhere depends on the lockdown decision and actions, it has been evident that with the increasing rate of lockdown and isolation, the reproduction number has been less than 1. The challenges of predicting these kinds of parameters include: a) Lack of proper data, b) No proper lockdown imposed by governments, c) No proper tracking of active and new cases. In future, these shortcomings will be attempted to overcome for getting improved result of the scenario and experimental results show that our pro- posed approach is well suited in its concerned domain. However, the prediction will change based on some external factors like government decisions and hu- man actions, so we can’t train the data as of today to be true for a different situation in future. Our modified SIR model is not only productive than the traditional one, but also more capable of handling and adopting the current situation. References Transmission of 2019-nCoV infection from an asymptomatic contact in Ger- N E l d J l f M di i 382(10) 970 971 15. 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References Preliminary estimation of the basic reproduction number of novel coronavirus (2019-nCoV) in China, from 2019 to 2020: A data-driven analysis in the early phase of the outbreak. Int J Infect Dis. 2020; doi:10.1101/2020.01.23.916395. 31. Li Q, Guan X, Wu P, et al. Early Transmission Dynamics in Wuhan, China, ofNovel Coronavirus-Infected Pneumonia; 2020. Available from: https://doi.org/ /10.1088%2F0951-7715%2F16%2F2%2F308. 32. Zhao S, Lin Q, Ran J, Musa / SS, Yang G, Wang W, et al. Preliminary estimation of the basic reproduction number of novel coronavirus (2019-nCoV) in China, from 2019 to 2020: A data-driven analysis in the early phase of the outbreak. Int J Infect Dis. 2020; doi:10.1101/2020.01.23.916395. / 33. Wu JT, Leung K, Leung GM. Nowcasting and forecasting the poten- tial domestic and international spread of the 2019-nCoV outbreak originating in Wuhan, China: a modelling study. The Lancet. 2020;doi:10.1016/s0140- 6736(20)30260-9. ( ) 34. Caccavo, D. (2020). Chinese and Italian COVID-19 outbreaks can be cor- rectly described by a modified SIRD model. medRxiv. 35. Zareie, B., Roshani, A., Mansournia, M. A., Rasouli, M. A., Moradi, G. (2020). A model for COVID-19 prediction in Iran based on China parameters. medRxiv. 36. Siwiak, M. M., Szczesny, P., Siwiak, M. P. (2020). From a single host to global spread. The global mobilitybased modelling of the COVID-19 pan- demic implies higher infection and lower detection rates than current esti- mates. medRxiv. 37. Russo, L., Anastassopoulou, C., Tsakris, A., Bifulco, G. N., Campana, E. F., Toraldo, G., Siettos, C. (2020). Tracing DAY-ZERO and Forecasting the Fade out of the COVID-19 Outbreak in Lombardy, Italy: A Compartmental Modelling and Numerical Optimization Approach. medRxiv. 38. Nadim, S. S., Ghosh, I., Chattopadhyay, J. (2020). Short-term predic- tions and prevention strategies for COVID-2019: A model based study. arXiv preprint arXiv:2003.08150. 39. Hossain, M., Junus, A., Zhu, X., Jia, P., Wen, T. H., Pfeiffer, D., Yuan, H. Y. (2020). The effects of border control and quarantine measures on global spread of COVID-19. 40. Volpert, V., Banerjee, M., Petrovskii, S. (2020). On a quarantine model of coronavirus infection and data analysis. Mathematical Modelling of Natural Phenomena, 15, 24. 41. Fong, S.J., Li, G., Dey, N., Crespo, R.G. and Herrera-Viedma, E., 2020. Composite Monte Carlo decision making under high uncertainty of novel coro- Mathematical Modelling For Decision Making of Lockdown during COVID-19 25 navirus epidemic using hybridized deep learning and fuzzy rule induction. Ap- plied Soft Computing, p.106282. p p g, p 42. Botha, A.E. Figures Figures Figures References and Dednam, W., 2020. A simple iterative map forecast of the COVID-19 pandemic. arXiv preprint arXiv:2003.10532. 43. Toda, A.A., 2020. Susceptible-infected-recovered (sir) dynamics of covid- 19 and economic impact. arXiv preprint arXiv:2003.11221. 44. Bhattacharjee, S., 2020. Statistical investigation of relationship between spread of coronavirus disease (COVID-19) and environmental factors based on study of four mostly affected places of China and five mostly affected places of Italy. arXiv preprint arXiv:2003.11277. y 45. Hethcote, H. W. (2000). The mathematics of infectious diseases. SIAM review, 42(4), 599-653. 46. Almeida, R., da Cruz, A.M.B., Martins, N. and Monteiro, M.T.T., 2019. An epidemiological MSEIR model described by the Caputo fractional deriva- tive. International Journal of Dynamics and Control, 7(2), pp.776-784. 46. Almeida, R., da Cruz, A.M.B., Martins, N. and Monteiro, M.T.T., 2019. An epidemiological MSEIR model described by the Caputo fractional deriva- tive. International Journal of Dynamics and Control, 7(2), pp.776-784. 47. Li, M.Y. and Muldowney, J.S., 1995. Global stability for the SEIR model in epidemiolog Mathematical biosciences 125(2) pp 155 164 47. Li, M.Y. and Muldowney, J.S., 1995. Global stability for the SEIR model in epidemiology. Mathematical biosciences, 125(2), pp.155-164. 48. “COVID-19 CORONAVIRUS PANDEMIC,”May 2020. [Online]. Avail- able: https://www.worldometers. info/coronavirus 49. Z. Hu, Q. Ge, L. Jin, and M. Xiong, “Artificial intelligence forecasting of covid-19 in china,” arXiv preprint arXiv:2002.07112, 2020. covid-19 in china,” arXiv preprint arXiv:2002.07112, 2020. 50. Saramaeki, T., Mitra, S.K. and Kaiser, J.F., 1993. Finite impulse response filter design. Handbook for digital signal processing, 4, pp.155-277. 51. Pedregosa, F., Varoquaux, G., Gramfort, A., Michel, V., Thirion, B., Grisel, O., Blondel, M., Prettenhofer, P., Weiss, R., Dubourg, V. and Vanderplas, J., 50. Saramaeki, T., Mitra, S.K. and Kaiser, J.F., 1993. Finite impulse response filter design. Handbook for digital signal processing, 4, pp.155-277. 51. Pedregosa, F., Varoquaux, G., Gramfort, A., Michel, V., Thirion, B., Grisel, O., Blondel, M., Prettenhofer, P., Weiss, R., Dubourg, V. and Vanderplas, J., 2011. Scikit-learn: Machine learning in Python. the Journal of machine Learn- ing research, 12, pp.2825-2830. Figures Figure 1 Country wise total cases upto 26.04.2020 Figure 2 Figure 1 Country wise total cases upto 26.04.2020 Country wise total cases upto 26.04.2020 Figure 2 Figure 2 Country wise total cases per 1 milion population Figure 3 Figure 3 Date and country wise COVID-19 total cases Figure 4 Date and country wise daily new cases Figure 5 D d C i l i Figure 5 Figure 5 Date and Country wise total active cases Date and Country wise total active cases Figure 6 Date and Country wise total deaths ate-wise worldwide cumulative death rate and recovery rate in percentage Figure 9 Date-wise actual total recovered population vs predicted total recovered population in five most affected countries Figure 6 Date and Country wise total deaths Date and Country wise total deaths Date and Country wise total deaths Figure 7 Date and country wise daily new deaths Figure 7 Date and country wise daily new deaths Date and country wise daily new deaths Figure 8 Date-wise worldwide cumulative death rate and recovery rate in percentage Figure 11 Change in basic reproduction number after lockdown in the five most affected countries Change in basic reproduction number after lockdown in the five most affected countries Task1.xlsx Figure 9 Date-wise actual total recovered population vs predicted total recovered population in five most affected countries Date-wise actual total recovered population vs predicted total recovered population in five most affected countries Figure 10 Actual daily new cases vs predicted new cases Actual daily new cases vs predicted new cases Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. Task1.xlsx
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English
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The moral footprint of animal products
Agriculture and human values
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Agric Hum Values DOI 10.1007/s10460-012-9402-x Agric Hum Values DOI 10.1007/s10460-012-9402-x Keywords Animals  Animal products  Animal welfarists  Animal rights  Ethics of killing  Vegetarianism  Veganism Abstract Most ethical discussions about diet are focused on the justification of specific kinds of products rather than an individual assessment of the moral footprint of eating products of certain animal species. This way of thinking is represented in the typical division of four dietary attitudes. There are vegans, vegetarians, welfarists and ordinary meat-eaters. However, the common ‘‘all or nothing’’ dis- cussions between meat-eaters, vegans and vegetarians bypass very important factors in assessing dietary habits. I argue that if we want to discover a properly assessed moral footprint of animal products, we should take into consid- eration not only life quality of animals during farming or violation of their rights—as is typically done—but, most of all, their body weight, life time in farms and time efficiency in animal products acquisition. Without these factors, an assessment of animal products is much too simplified. If we assume some easily accepted premises, we can justify a thesis that, regardless of the treatment of animals during farming and slaughtering, for example, eating chicken can be 163 times morally worse than eating beef, drinking milk can be 58 times morally better than eating eggs, and eating some types of fish can be even 501 times worse than eating beef. In order to justify such a thesis there is no need to reform common morality by, for example, criticizing its speciesism. The thesis that some animal products are much worse than others can be justified on common moral grounds. Keywords Animals  Animal products  Animal welfarists  Animal rights  Ethics of killing  Vegetarianism  Veganism Introduction Most ethical discussions about diet are focused on the justification of specific kinds of products rather than on an individual moral assessment of eating products of certain animal species, which I refer to as the moral footprint of animal products. While many animal protectionists or abolitionists argue that at least people from developed countries should abandon meat eating or even the use of any animal products, their opponents criticize this claim and defend the common view of animals as producers of many goods for people. These ways of arguing are repre- sented in the typical division into four dietary attitudes. There are vegans who refuse to use any animal products; vegetarians who do not accept eating any meat but con- sume other animal products such as eggs, milk, or honey; animal welfarists who do not condemn using animals, but try to minimize unnecessary suffering in farming; and the huge mass of ordinary people who eat without remorse everything permissible by law. However, these common ‘‘all or nothing’’ discussions between meat-eaters, vegans, and vegetarians bypass some very important factors in assessing dietary habits. K. Saja (&) Department of Philosophy, University of Szczecin, ul. Krakowska 61/69, 71-017 Szczecin, Poland e-mail: krzysztofsaja@gmail.com The moral footprint of animal products Krzysztof Saja Accepted: 31 July 2012  The Author(s) 2012. This article is published with open access at Springerlink.com Accepted: 31 July 2012  The Author(s) 2012. This article is published with open access at Springerlink.com The philosophical premises of the argument g It is widely assumed that the animal well-being is an important moral factor. However, I argue that if we want to discover a properly assessed moral footprint of animal products, we need to take into consideration not only the life quality of animals during farming or violation of their rights—as is typically the case—but also their body weight and life duration in farms. If we consider such commonly neglected factors and assume some described in the next section premises, we can show that regardless of the treatment of animals during farming and slaughtering, for example eating 1 kg of chicken can be preliminarily 163 times morally worse than eating 1 kg of beef, drinking 1 average daily portion of milk can be 58 times morally better than eating 1 average daily portion of eggs, and eating 1 kg of carp can be 501 times worse than 1 kg of beef. Such conclusions undermine not only the common ‘‘all or nothing’’ discussion about animal products con- sumption, but also have very important practical implica- tions for a state policy of minimizing animal harm, which I sketch later in the paper. The first important premise of the argument is the platitude of common morality and the minimal claim of all animal activists. Roughly speaking, it says that animals are not stones and for that reason they have at least some minimal rights. I hope that only someone who believes that animals do not have any moral rights or that their suffering or killing is morally irrelevant would not accept it. However, I do not know anybody who defends such a view. Most people (at least in the USA) admit that there are morally better and worse ways of treating animals.1 The common belief that animals should have at least some minimal moral protection can be specified in different ways due to different reasons for moral status of animals or badness of harming and killing. If someone believes that animals have at least the right not to be killed without a good reason, she should accept the first version of the argument and the following simple premise: A1. It is morally wrong to kill sentient animals without important justification. Different people can give different reasons to support the above claim. 1 In a 2004 survey conducted by researchers at The Ohio State University, 92 % of Ohio residents agreed that it is important that farm animals are well cared for, and 81 % said the well-being of farm animals is just as important as the well-being of pets (Rauch and Sharp 2005). Furthermore, 95 % of respondents to a nationwide telephone survey conducted by Oklahoma State University agreed with the statement: ‘‘It is important to me that animals on farms are well cared for’’ (Lusk et al. 2007). In a 2005 survey of Michigan residents conducted by researchers at Michigan State University, 92 % of respondents rated ‘‘humane animal treatment’’ as ‘‘very important’’ or ‘‘somewhat important’’ as a factor when purchasing animal products (Conner et al. 2008). What I argue and what I don’t There are many philosophical disputes about the strength, justification, and content of animal rights. Animal activists and philosophers give different reasons why animals have 12 3 K. Saja moral status and should be treated properly. The most common and important justifications for abandoning or carefully selecting animal products lie in condemnation of unjustified killing of animals, using them merely as a means or causing suffering during farming. For these rea- sons, vegetarians refuse to eat meat and many animal welfarists fight for farming methods that increase respect for animal rights or their standard of life. common premises we should accept that there are very important moral differences between animal products from different animal species due to animal body weight and life duration in farms. 2 The animal welfare position holds that there is nothing inherently wrong with using animals for human purposes, such as food, clothing, entertainment, and research, but that it should be done in a humane way that minimizes unnecessary pain and suffering. In the UK, the Farm Animal Welfare Council was set up by the government to act as an independent advisor on animal welfare in 1979 and expresses its policy as five freedoms: from hunger and thirst; from discomfort; from pain, injury, or disease; to express normal behavior; and from fear and distress. The philosophical premises of the argument It is morally wrong to cause suffering to animals without important justification. The second version of the argument is based on a dif- ferent type of ethics of killing because, even if we agree that it is morally wrong to kill sentient animals without important justification, we can give other reasons for that thesis. We can claim that the badness of killing depends not on any intrinsic evil of death or sanctity of life, but on deprivation of possible future goods or preventing satis- faction of future interests and preferences (Marquis 2007; McMahan 1988; Nagel 1970; Rachels 1986). In this ver- sion if we try to assess the moral footprint of animal products we need to assess the loss involved in the death of an animal from a certain species. This loss is determined by how many future goods are taken by killing an animal. If someone believes in such an ethics of killing, she should accept the second version of the argument and the second version of premise A: Every animal activist defends at least one of the above assumptions. However, there is no need to be a moral revisionist—as many animal protectionists are—to accept any of them. Most people agree that if we can minimize animal suffering or deaths and still achieve the objective of having plentiful, inexpensive food, then we should do it (Rauch and Sharp 2005; Lusk et al. 2007). Therefore, due to the different possible answers to what ‘‘without impor- tant justification’’ in these contexts could mean, I assume that most of us would accept at least one of the premises (A1, A2, or A3). The second premise (premise B) is a statement of fact about the acquisition of animal products in most developed countries. B1 (B2) are important for the first two versions of the argument. A2. It is morally wrong to deprive animals of possible future goods without important justification. B1. (B2.) Breeding animals for food (meat, eggs, milk) causes them to be brought into existence and causes them to be killed. B1. (B2.) Breeding animals for food (meat, eggs, milk) causes them to be brought into existence and causes them to be killed. The third version of the argument is presented for per- sons who do not believe that killing animals is prima facie bad. The philosophical premises of the argument It corresponds with an assumption that the badness of animal products does not lie in animal death, but in harming animals through the bad conditions they experi- ence before their death. For that reason in order to assess the moral footprint of animal products we have to be able to measure the quantity of harm (or benefit) done to ani- mals during their lifetime. The most obvious types of harm are pain and suffering or unsatisfied preferences that are caused by farming (Singer 1977, 2006; Thomson 1990). The same version of the argument can be used if we believe that the wrongness done to animals lies in violating their rights not to be used as property (Francione 1995; Regan 1983). In order to assess this immoral usage we need to consider, as we also must when we are focused on suf- fering, the amount of time spent by animals in these harming conditions, and the quantity and quality of harm done during that time. However, because an aggregation of violations of rights may engender some problems, we will focus on suffering as the more common and less contro- versial reason to abandon eating animal products. If somebody thinks about the badness of animal products like animal welfarists2 or Peter Singer, she should accept the following premise: It is not practically possible to eat meat without killing an animal. Although we can imagine farming methods for non-meat products (e.g., eggs and milk) in which people are not directly involved in breeding new animals or killing those that are not economically efficient enough, almost every animal-sourced food consumed in developed coun- tries is produced in farming conditions in which people are responsible for animal life and death. If we did not produce meat, eggs, or milk, we would not bring to life and kill pigs, chickens, and dairy cows. Every kind of animal product is involved in death of respected animals directly or indirectly. In the third version of the argument, which is focused on the well-being of animals, we should consider one other premise: B3. Farming affects well-being of animals positively or negatively depending on the farming method and the time spent in farms. B3. Farming affects well-being of animals positively or negatively depending on the farming method and the time spent in farms. The philosophical premises of the argument Many just think—especially non-philos- ophers—that unjustified killing is bad as a simple, intuitive, unanalyzable moral fact that does not need any further explanation or justification. This fact is sometimes stated in terms of a moral right to life: killing is bad for the same reason that it is bad to violate a right to life without important justification (e.g., self-defense). Some other people may claim that the badness of killing lies in the death that it brings. In this case, killing is wrong because it steals a life and cannot be analyzed or reduced to the loss of possible future goods or preferences satisfaction. Such a belief is sometimes associated with the thesis about the sanctity of life. The thesis that animal products from some animal spe- cies are worse than others due to animal body weight and life duration in farms will be justified regardless of the reason for enhancing animal rights or the assumed ethics of killing. In order to broaden my argument, I show that my conclusions follow regardless of which particular ethics of protecting animals is considered. Therefore, I am not going to argue that one is sounder than any other. Instead, I present the argument in three different versions depending on the three most common answers to the question ‘‘What are the main reasons to stop using animal products?’’ These versions are entirely independent. In fact, I propose three different arguments of the same structure but treat them as one and explain jointly. Thus, for example, if we consider the first version of the argument we should take into account premises A1, B1, C, D1, E1, F1, the second ver- sion premises A2, B2, C, D2, E2, F2, and the third version premises A3, B3, C, D3, E3, F3. It is important to remember that the premises with the same letter (e.g., A1, A2, A3) are not different expressions of one and the same claim but rather different claims that play the same role in the paper. To fully justify all of them individually is beyond my scope. Therefore, I will present only a basic argument for their validity. The main thesis of the paper is only a conceptual claim, mainly that if we assume such 123 123 123 The moral footprint A3. It is morally wrong to cause suffering to animals without important justification. A3. The philosophical premises of the argument If we believe in the second reason for protecting animals (A2) and therefore focus on the second version of the argument, we need to accept: D2. It is N times morally worse to deprive n times more possible future goods (if everything else remains the same). The last version of premise D for the third version of the argument is focused on suffering or harm during farming: In addition to the above premises we also should assume that: D3. It is N times morally worse to cause n times more suffering (if everything else remains the same). C. Bringing an animal into the world is not morally better than not bringing an animal into the world. Let us imagine three cases in which you have only two options and everything else remains the same: to let 2 or 3 people die; to let 3 or 4 people die; to let 4 or 10 people die. The above premises assume that the first options are morally better and letting more rather than fewer people die is worse. If everything else remains the same, and the only thing that can be changed is the duration of feeling pain, the better option is to feel pain for as short a time as possible. The same holds true for animals. If we can test some medical research with an option that kills fewer laboratory rabbits and everything else remains the same we ought to take that option. Fewer total deaths and loss of possible future goods or pain are better than more. For every person who accepts an utilitarian ethics, the above thesis has to be perfectly obvious. In addition, most non- consequentialists would agree that if everything else remains the same, numbers morally count. On the other hand, anybody who would claim that numbers do not morally count will not accept my argument. However, only a few philosophers would defend such a view about people (Taurek 1977; Timmermann 2004) and it would be even more uncommon to claim this about animals. It could be said that farming animals for food is good for animals even if they suffer. Meat production gives animals life and without this production these animals would not exist at all. However, such a claim and its implications are quite odd because comparing existence with non-existence produces many philosophical problems that it would be better to omit here. The philosophical premises of the argument In order to build the third version of the argument we need to assume that different farming methods have dif- ferent effect on animal well-being, either positively or negatively (for the sake of simplicity I am not going to consider animals killed in the wild). Every type of farming for every animal species causes a different quality of life. On the one hand, many farmed animals, such as pigs, chickens, dairy cows, or fish in artificial tanks, are kept in Concentrated Animal Feeding Operations (CAFOs). The standard of their life in many CAFOs is probably lower than it would be in nature. Livestock is harmed by close con- finement systems (cages, crates) or lifetime confinement in 12 3 3 K. Saja K. Saja or harming animals without important justification is immoral, she will not agree with premise D either. indoor sheds; discomfort and injuries caused by inappro- priate flooring and housing; restriction or prevention of normal exercise and most natural foraging or exploratory behavior; restriction or prevention of natural maternal behavior; lack of daylight or fresh air and poor air quality in animal sheds; social stress and injuries caused by over- crowding; health problems caused by extreme selective breeding and management for fast growth and high pro- ductivity; reduced lifetime (longevity) of breeding animals (dairy cows, breeding sows); fast-spreading infections encouraged by crowding and stress in intensive conditions; de-beaking (beak amputation without pain killer) in the poultry and eggs industry to avoid pecking in overcrowded quarters; forced and overfeeding (by inserting tubes into the throats of ducks) in the production of foie gras, etc. On the other hand, some types of farming (e.g., extensive and ecological farming of beef cattle) can probably give a better standard of life than cattle could experience in the wild. If we admit that what makes animal products morally bad is a badness of killing animals (A1), which is the obvious precursor of eating meat and, most often, also a consequence of the production of eggs and milk, we need to assume the following premise: D1. It is N times morally worse to kill n times more sentient animals (if everything else remains the same). D1. It is N times morally worse to kill n times more sentient animals (if everything else remains the same). 3 The above remarks about premise C I owe to a private conversation with Jeff McMahan. Commonly missed facts about animal products In order to show that there are preliminarily very large differences in the moral footprint of animal products from different animal species, it is important to take into con- sideration some other factors that are commonly missing in discussions about the ethics of diet. They are factual in nature and represent morally important statistical data about the farming and consumption of animals for food. In the following discussion I will rely on typical Polish practices of production and consumption of animal prod- ucts such as beef, pork, chicken, carp (a traditional Christmas Eve fish in Poland), eggs, and milk. According to statistical data the average Pole consumes approximately 70 kg (154 lbs) of different meat products per year, but mostly from cattle, pigs, chicken, and fish. They also eat 200 eggs and drink 190 kg (418 lbs) of milk per year.4 If we want to assess and compare how much aggregate harm would be done to animals of certain species during an average 70 years of consumption by one person, it is useful to ignore the fact that meat eaters consume different kinds of meat products from different species. Therefore, for the sake of argument I will investigate how much aggregate harm would be done to animals of certain species if a typical meat-eater were to eat animal products from only one species for all of his or her life. The moral footprint of animal products depends also on the assumed reason for the badness of using animals. If the harm is caused by taking life or bringing death (A1), then according to premise D1 it would be n times morally worse to kill n times more animals of some kind. In this respect to assess the moral footprint of animal products we should include the quantity of animal deaths. In order to do this we need to know the average amount of animal products (meat, eggs, milk) per one ani- mal. The important numbers are in Table 1. The premise E and the above reservations show that to make a final and detailed comparison of the moral footprint of animal products, we would need to collect a lot of empirical data about the compared animals’ psychology, physiology, and farming condition in order to assess the proper amount of harm caused by killing or farming con- dition of animals from different species. This is beyond the scope of this paper. The philosophical premises of the argument Moreover, even if causing an animal to exist can benefit that animal, nothing follows about the permissibility of killing it or causing it to suffer once it exists. First, causing an animal to exist benefits it only if its life is worth living, which arguably is not always the case for animals that are kept in CAFOs. Second, causing an animal to exist might entail a responsibility to care for it, not a permission to kill it. Third, once an animal exists, it may have rights that protect it, independently of how it came to exist. Fourth, it may be that once an animal exists, its interest in continuing to live outweighs any human interest that might be served by killing it. The weighing of interests seems independent of how the animal came into existence. In short, premise C is a stronger claim than I need in the argument. My argument should be persuasive even to most of those who deny premise C because of the considerations I have just noted.3 The last conceptual premise (E) is important to allow cross-species moral comparisons. The formal structure of its more detailed versions (E1–E3) is: A fourth important premise (premise D) can be roughly stated as the claim that the quantity of victims matters morally. Its validity depends on accepting premise A, because if somebody does not believe that killing E. The badness of killing or causing suffering to an animal from species a is m times as much as the badness of killing or causing suffering to an animal from species b. Not every animal has the same right to life and the killing of any of them is not equally bad. Most people have 3 The moral footprint different attitudes toward killing parasites, pests, livestock, wild animals under protection, or pets. In Poland people much more value the lives of cats and dogs than those of pigs, chickens, and cows. We do not kill homeless domestic animals, but put them in an animal shelter. Nevertheless, we are very merciless to rats and mosquitoes. We do not think that the death of every compared animal species is equally bad. Moreover, there are natural differences in animal bodies and psychology. 4 All statistical data in this article are from the Polish Central Statistical Office (2010). The philosophical premises of the argument As Jeff McMahan (2002) argues, psychological capacity is related to the degree of psycho- logical (prudential) unity within a life and that psycholog- ical unity is relevant to the assessment of the harm or loss involved in death. This psychological capacity is also rel- evant to an animal’s capacity for well-being. Some animals are capable of much higher levels of well-being than others are capable of. Even if fish can feel pain their lost future goods are less valuable than the future goods of killed pigs, as the life of cognitively more developed animals is richer, more worth living, and worse to be shortened. Additionally, not every species has the same capability to feel pain. The nervous system of fish is not as developed as that of cows, so they could not be harmed as much by farming as cows. It can be argued that carp living in crowded tanks suffer less than pigs living alone in small steel and concrete cages. Similar treating of different animal species can produce different amounts of harm. in assessing moral footprint of animal products such as animal body weight or longevity in farms. If we accept E1– E3 we will be able to see that animal capacity of well- being, amount of suffering, and harm is not as important as it is commonly believed. Commonly missed facts about animal products Therefore, the average consumer of eggs, who eats 200 eggs per year, is responsible for the death of 1 hen and the death of 1 male chick (cocks do not give eggs, therefore, male chicks are typically killed right after their birth) ** The amount of 0.036 cows killed per year is assessed on the assumption that the average consumption of milk per year is 190 kg per person, which is the typical amount in Poland. This would give 0.009 killed cows per year of average milk consumption. However, in modern dairy farms cows are inseminated about 3 times per life for better efficiency in milk production; therefore, typical byproduct of milk acquisition are 3 calves per dairy cow. For that reason the amount of 0.036 cattle killed per 1 year of typical milk consumption is a product of killed cows (0.009) and their three calves (3 9 0.009 calves) (0.009 cow ? (3 9 0.009 calves) = 0.036 cattle per 1 year.) of meat per year by one person who would consume only products from one species. If we think that the wrongness of killing lies in the deprivation of possible future goods or preventing of sat- isfaction of future interests (A2), in order to construct the moral footprint of animal products we should take into consideration not only the quantity of animal deaths, but also how many years of life we take away from them for certain products. The important numbers are in Table 2. If we assume the consumption of eggs and milk as equal to the average consumption in Poland (200 eggs per year and 190 kg milk per year), we can show in Table 5 pre- liminary consequences of 70 years of consuming typical amounts of eggs and milk. Even if the badness of killing lies in depriving possible future goods, the important differences between animal products from different species are similar to that from Table 1. We can estimate that 70 years of eating carp would cost 135,240 aggregate years of life in comparison to 710 years of life when consuming pork. Another step in the argument requires some computed data from the above tables, i.e. assessments of the dispro- portion between the consequences of farming animals from different species. This data will vary depending on the version of the argument and the compared animal species. Commonly missed facts about animal products However, in order to justify the thesis of the article that the ethics of consuming animal products ought to consider not just animal harm but also other factors such as animal body weight or life duration in farms, then initially we need to ignore these differences and for the sake of simplicity assume that killing or harming animals of different species is important equally. For that reason let us assume that (accordingly to the version of the argument): E1. The death of every farmed animal is equally bad. E2. For every farmed animal in an equal period of time of being dead the quantity and value of a loss of possible goods are equal. E3. For every farmed animal in an equal period of time th tit d lit f ff i l If one would consume only the meat of a single species, the aggregate quantity of killed animals per 70 years of consumption would vary a lot due to the weight of each killed animal. If someone would eat only beef they would be responsible for the deaths of 15 animals, but if they were to consume only chicken or carp, then they would be responsible for the deaths of 2,450 or 3,220 animals respectively. E2. For every farmed animal in an equal period of time of being dead the quantity and value of a loss of possible goods are equal. E3. For every farmed animal in an equal period of time the quantity and quality of suffering are equal. E3. For every farmed animal in an equal period of time the quantity and quality of suffering are equal. The premises E1–E3 are inconsequential. However, if we assume them for a moment we can later show that even if we know the relative physiological capacity of animals to feel pain and the real amount of harm or benefit caused by farming, this will not override the more important factors 12 3 K. 5 The number 163 is equal to the quantity of killed chickens from Table 4 row (c) divided by the quantity of killed beef cattle (2,450/ 15 = 163). 7 The number 358 is equal to the quantity of aggregate period of suffering of carps from Table 4 row (h) divided by the quantity of aggregate period of animal suffering of beef cattle (10,733/ 30 = 358). 6 The number 501 is equal to the quantity of the aggregate years of shorten lifespan of carps from Table 4 row (g) divided by the quantity of aggregate years of shortened lifespan of beef cattle (135,240/ 270 = 501). Commonly missed facts about animal products Saja Table 1 Calculus of animal deaths Beef (beef cattle) Pork (pigs) Poultry (chickens) Fish (carps) Eggs Milk (a) Average animal products per one animal life 320 kg (704 lbs) 100 kg (220 lbs) 2 kg (4.4 lbs) 1.5 kg (3.3 lbs) 200 eggs 21,000 kg (46,200 lbs) (b) Quantity of killed animals of certain species to satisfy average animal products intake by 1 person in 1 year [70 kg (154 lbs)], who would consume only the product of a certain species (a / 70 kg) 0.22 lives 0.7 lives 35 lives 46 lives 2 lives* (assumed 200 eggs consumed per year) 0.036 lives** (assumed 190 kg (418 lbs) of milk consumed per year) (c) Quantity of killed animals per average time of consumption (70 years), if one would consume only the product of single species (b 9 70 years) 15 lives 49 lives 2,450 lives 3,220 lives 140 lives 2.5 lives All statistical data are from the Polish Central Statistical Office (2010) * One typical hen produces 200 eggs per year and lives in a farm for about 12 months. Therefore, the average consumer of eggs, who eats 200 eggs per year, is responsible for the death of 1 hen and the death of 1 male chick (cocks do not give eggs, therefore, male chicks are typically killed right after their birth) ** The amount of 0 036 cows killed per year is assessed on the assumption that the average consumption of milk per year is 190 kg per person * One typical hen produces 200 eggs per year and lives in a farm for about 12 months. Therefore, the average consumer of eggs, who eats 200 eggs per year, is responsible for the death of 1 hen and the death of 1 male chick (cocks do not give eggs, therefore, male chicks are typically * One typical hen produces 200 eggs per year and lives in a farm for about 12 months. Therefore, the average consumer of eggs, who eats 200 eggs per year, is responsible for the death of 1 hen and the death of 1 male chick (cocks do not give eggs, therefore, male chicks are typically killed right after their birth) * One typical hen produces 200 eggs per year and lives in a farm for about 12 months. 123 All statistical data are from the Polish Central Statistical Office (2010) 5 The number 163 is equal to the quantity of killed chickens from Table 4 row (c) divided by the quantity of killed beef cattle (2,450/ 15 = 163). 6 The number 501 is equal to the quantity of the aggregate years of shorten lifespan of carps from Table 4 row (g) divided by the quantity of aggregate years of shortened lifespan of beef cattle (135,240/ 270 = 501). 7 The number 358 is equal to the quantity of aggregate period of suffering of carps from Table 4 row (h) divided by the quantity of aggregate period of animal suffering of beef cattle (10,733/ 30 = 358). Commonly missed facts about animal products Farmed carp in aggregate suffer 358 times longer than farmed beef cattle.7 All of this can be summarized in Table 4, in which we compare the important consequences of eating 70 kg (154 lbs) 123 The moral footprint Table 2 Calculus of taken animals’ possible future goods Beef (beef cattle) Pork (pigs) Poultry (chickens) Fish (carps) Eggs Milk (c) Quantity of killed animals per average human time of consumption (70 years), if one would consume only the product of a single species 15 49 2,450 3,220 140 2.5 (d) Average animal lifespan in farms (months) 24 6 4.5 40 6* 30** (e) Normal average lifespan, if animals would not be killed (years) 20 15 10 45 10 20 (f) Years of shortened lifespan of one animal by killing it (e - d) 18 14.5 9.5 42 9.5 17.5 (g) Aggregate years of shortened lifespan by killing animals during 70 years of animal product consumption (c 9 f) 270 710 23,275 135,240 1,330 43.8 * An average hen lives on a farm for 1 year and male chicks are killed after their birth. Therefore, average animal lifetime in egg production is 0.5 years ** An average dairy cow lives on a farm for 4 years. However, it breeds 3 calves, which live 2 years on average. Therefore, I estimate that the average lifetime of cattle on dairy farms is 2.5 years Table 2 Calculus of taken animals’ possible future goods Table 3 Calculus of animal suffering in farming Beef (beef cattle) Pork (pigs) Poultry (chickens) Fish (carps) Eggs Milk (c) Quantity of killed animals per average human time of consumption (70 years), if one would consume only the product of a single species 15 lives 49 lives 2,450 lives 3,220 lives 140 lives 2.5 lives (d) Average animal lifespan in farms (months) 24 6 4.5 40 6 30 (h) Aggregate time of farming animals per average human lifetime (c 9 d) (years) 30 25 919 10,733 70 6.3 (i) Global well-being of animals in farming if we assume the same quality of life of different species (-1 9 h) -30 -25 -919 -10,733 -70 -6.3 Table 5 Important consequences of non-meat animal products Table 4 Important consequences of meat consumption Commonly missed facts about animal products We can propose the following factual estimations: If we are focused on the third version of the argument and believe that what is morally bad about animal products is causing animals to suffer (A3), then an important factor for assessing the moral footprint of animal products would be the aggregate product of quality of life and period of suffering in farming. If the harm lies in violating their rights not to be used or enslaved without important justi- fication, then crucial would be the aggregate product of the level of injustice and time period of wrongdoing. In either case the time spent in farms would have a significant impact. Therefore, global well-being or wrongdoing in farms depends not only on the assumed quality of life or level of injustice, but mostly on the average farming time and the quantity of killed animals. The huge impact of these factors can be shown in Table 3. F1. Eating chicken in aggregate kills 163 times more animals than eating beef.5 F2. Farming carp in aggregate takes away 501 times more animal possible future goods than farming cattle for beef.6 F3. Farmed carp in aggregate suffer 358 times longer than farmed beef cattle.7 F1. Eating chicken in aggregate kills 163 times more animals than eating beef.5 F1. Eating chicken in aggregate kills 163 times more animals than eating beef.5 F1. Eating chicken in aggregate kills 163 times more animals than eating beef.5 F2. Farming carp in aggregate takes away 501 times more animal possible future goods than farming cattle for beef.6 F3. Farmed carp in aggregate suffer 358 times longer than farmed beef cattle.7 F2. Farming carp in aggregate takes away 501 times more animal possible future goods than farming cattle for beef.6 F3. Farmed carp in aggregate suffer 358 times longer than farmed beef cattle.7 F3. The argument If we accept premises A1–F1, the table shows that, for example, eating 1 kg of carp is 214 times morally worse than eating 1 kg of beef, 1 kg of chicken is 50 times worse than 1 kg of pork, and 1 average daily egg ration is 56 times worse than 1 typical daily milk ration. B1. Breeding animals for food (meat, eggs, milk) causes them to be brought into existence and to be killed. B1. Breeding animals for food (meat, eggs, milk) causes them to be brought into existence and to be killed. C. Bringing an animal into the world is not morally better than not bringing an animal into the world. D1. It is N times morally worse to kill n times more sentient animals (if everything else remains the same). E1. The death of every farmed animal is equally bad. F1. Eating chicken in aggregate kills 163 times more animals than eating beef. C. Bringing an animal into the world is not morally better than not bringing an animal into the world. D1. It is N times morally worse to kill n times more sentient animals (if everything else remains the same). The death of every farmed animal is equally bad F1. Eating chicken in aggregate kills 163 times more animals than eating beef. Therefore, eating chicken is preliminarily 163 times morally worse than eating beef. If we think that the moral significance lies not in animal deaths, but in the suffering in farming, the reconstructed argument would be the following: In the light of the above data we can fully answer the question about the validity of premises E1–E3. Now it can be seen that the disproportions of the preliminary badness of one unit of animal product of different species (for example 1 kilogram of meat or one average daily ration of milk or eggs) presented in Table 6 are much bigger than the presumptive cognitive or moral diversities of different species. Even if there is some disproportion about the right to life, degree of psychological unity within a life, capa- bility of suffering, or farming conditions of different spe- cies, it would be very odd to claim that, for example, fish suffer 300 times less than cattle in the equal period of time or that a chicken’s life is morally worth 50 times less than a pig’s life. The argument If we choose one version of the argument and take for granted all premises A to F, we can reconstruct it in few simple steps. Its different versions, which depend on the different philosophies of the badness of killing or using animals, could be presented by changing the numbers of the appropriate premises of the argument. If we think that animals have at least the right not to be killed without a good reason, we can show the argument in the following way: A1. It is morally wrong to kill sentient animals without important justification. A1. It is morally wrong to kill sentient animals without important justification. 12 3 K. Saja Table 6 Preliminary comparison of the moral footprint of animal products* Preliminary comparison of two animal products How many times one unit of animal product is preliminarily worse than a second one due to different factors of badness of animal production (killing, taken future goods, suffering in farming) Death/killing (c) Loss of future (e) Suffering in farming (h) Carp to beef (n times worse) 214 501 358 Chicken to beef (n times worse) 163 86 30 Pork to beef (n times worse) 3 0.4 0.8 Chicken to pork (n times worse) 50 107 37 Eggs to beef (n times worse) 9 10 2 Eggs to pork (n times worse) 3 12 3 Eggs to milk (n times worse) 56 30 11 * All of the numbers in Table 6 were computed as described in Footnotes 5–7 Table 6 Preliminary comparison of the moral footprint of animal products* How many times one unit of animal product is preliminarily worse than a second one due to different factors of badness of animal production (killing, taken future goods, suffering in farming) preliminarily worse than another. The results are divided into three columns according to three different factors of animal products badness: the first shows how many times one product is preliminarily worse than another according to the quantity of deaths, the second is according to pre- venting future goods, and the third is according to the suffering in farms or violating the right not to be treated as property. The argument If someone would claim that eating 1 kg of chicken is not worse than eating 1 kg of pork because pigs can have richer and more valuable future lives than chickens, then they would have to prove that a pig’s future life is richer or more valuable at least 107 times more than a chickens. This seems false as well. Speaking more formally, if we would claim that despite the data from Table 6 the eating of one unit of animal product (e.g., meat, eggs or milk) is not worse than the eating of another product (e.g., pork or beef) because of x (e.g., cognitive abilities and degree of psy- chological unity within a life of animals), we have to prove that factor x differentiates animal species at least as much as is showed in Table 6. However, because morally sig- nificant aggregate body weight and lifespan of animals of different species can differ by a factor of a hundred or more, there is only a very low probability that other mor- ally important factors would be able to overbalance them. be to consume beef. The same is true for welfarist meat-eaters who accept common morality, believe in anthropocentrism in ethics, but think that animals have some minimal rights. If we can fulfill our dietary aims of eating meat without increasing unjustified or unnecessary animal deaths and suffering, we should do that. Everybody who accepts that mere diversity of animal products on our plates does not justify, for example, 300 times more animal deaths or suffering should change their diet. Most philosophers or animal activists, who try to change our common morality of using animals for consumption, focus on violating animal rights or the bad conditions in farming. Many developed countries try to legally restrict some harmful forms of treating animals such as long transportation for slaughter without stops, battery cages for egg-laying hens, de-beaking of chickens, confinement of sows during pregnancy and for most of their adult life, and slaughtering animals without stunning them beforehand. However, if we want to reduce animal harm, then there are more important factors than the quality of life of livestock that should be taken into consideration. The argument Even if we agreed that the life of a fish is, for example, 10 times less valuable than that of a pig because fish are 10 times less cognitively developed and can feel 10 times less pain accordingly, it would still not be enough to make the claim that eating 1 kg of fish is as bad as eating 1 kg of pork. The preliminary differences of the moral footprint of meat from carps and pigs are much bigger than 10 and they seem bigger than any cognitive disproportion between the compared vertebrates. If someone wants to prove that, for example, eating 1 kg of carp is morally equal to eating 1 kg of beef or pork because carp suffer less, they would have to show accordingly that, for A3. It is morally wrong to cause suffering to animals without important justification. B3. Farming affects well-being of animals positively or negatively depending on the farming method and the time spent in farms. C. Bringing an animal into the world is not morally better than not bringing an animal into the world. D3 It is N times morally worse to cause n times more suffering (if everything else remains the same). E3. For every farmed animal in an equal period of time the quantity and quality of suffering are equal. F3. Farmed carp in the aggregate suffer 358 times longer than farmed cattle for beef. F3. Farmed carp in the aggregate suffer 358 times longer than farmed cattle for beef. Therefore, eating carp is preliminarily 358 times mor- ally worse than eating beef. The second version of the argument can be constructed in a similar fashion. The conclusions, which are compar- ative judgments about the preliminary badness of some specified unit of different animal products (for example 1 kilogram), can be presented in one simple table. In the rows of Table 6 different animal products are compared by asking how many times one unit of a product is 123 123 The moral footprint example, carp are at least 358 times less capable of suf- fering than beef cattle. And this is probably false. The argument If my argument is correct and we want to construct a detailed moral footprint of animal products, then we should take into account not only life quality (preference satisfactions) during farming and violation of animal rights, but, first and foremost, animal body weight and lifetime animals spend in farms. Important implications The thesis that animal body weight and life duration in farms are significant but commonly neglected moral fea- tures has important practical implications. First of all, if someone tries to minimize the harm done to animals caused by producing food from them, they should consider not only supporting farming methods that increase respect for animal rights or their standard of life—as it is typically the case— but, first of all, try to promote eating beef and pork rather than chicken and fish. If minimizing harm to animals would be our aim, a much bigger impact could be achieved not by consuming only ecological, low-suffering animal products, but mostly by choosing products from bigger animals like beef cattle. Despite the strangeness of such a conclusion, choosing and promoting big body weight and efficient animal products is the most rational way of minimizing animal harm (except by being a vegan). If we would eat only meat from such animals we would prevent much more pain, death, or loss of possible future goods than by trying to consume, for example, well-farmed chickens. Even if the quality of life in farming is important, more important is animal weight and farming time. Moreover, the traditional division of diets made by food products types (vegetable, meat, non-meat animal products) is irrelevant from the ethical point of view. Not well-informed vegetarians who eat eggs produced in CAFOs can cause much more animal harm than a person who eats only red meat without eggs. Drinking milk could be much less harmful than eating eggs. Promoting ‘‘white meat’’ (poultry, fish) can be more wrong than promoting a normal meat diet. If someone cannot abandon meat eating, maybe the best ethical option would Acknowledgments I would like to extend a note of gratitude to Renata Pilarczyk, Jeff McMahan, Harvey S. James, Adriana Schetz, Urszula Zarosa, and anonymous reviewers from Agriculture and Human Values for their very helpful and valuable comments. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, dis- tribution, and reproduction in any medium, provided the original author(s) and the source are credited. Acknowledgments I would like to extend a note of gratitude to Renata Pilarczyk, Jeff McMahan, Harvey S. James, Adriana Schetz, Urszula Zarosa, and anonymous reviewers from Agriculture and Human Values for their very helpful and valuable comments. McMahan, J. 1988. Death and the value of life. Ethics 99(1): 32–61. References Central Statistical Office. 2010. Statistical Yearbook of Agriculture, Warsaw: Zaklad Wydawnictw Statystycznych. http://www.stat. gov.pl/gus/5840_4127_PLK_HTML.htm. Accessed 26 July 2012. Conner, D.S., V. Campbell-Arvai, and M.W. Hamm. 2008. Consumer preferences for pasture-raised animal products: results from Michigan. Journal of Food Distribution Research 39(2): 12–25. Francione, G.L. 1995. Animals, property, and the law. Philadelphia: Temple University Press. Lusk, J.L., F.B. Norwood, and R.W. Prickett. 2007. Consumer preferences for farm animal welfare: results of a nationwide telephone survey. Department of Agricultural Economics, Okla- homa State University. http://asp.okstate.edu/baileynorwood/ Survey4/files/InitialReporttoAFB.pdf. Accessed 26 July 2012. Marquis, D. 2007. An argument that abortion is wrong. In Ethical theory: An anthology, ed. R. Shafer-Landau, 439–450. Oxford: Blackwell. McMahan, J. 1988. Death and the value of life. Ethics 99(1): 32–61. 12 3 3 K. Saja Taurek, J.M. 1977. Should the numbers count? Philosophy and Public Affairs 6(4): 293–316. McMahan, J. 2002. The ethics of killing: Problems at the margins of life. New York: Oxford University Press. N l T 1970 D h N ˆ 4(1) 73 80 f y Nagel, T. 1970. Death. Nouˆs 4(1): 73–80. Thomson, J. 1990. The realm of rights. Cambridge: Harvard University Press. Rachels, J. 1986. The end of life: Euthanasia and morality. Oxford: Oxford University Press. Timmermann, J. 2004. The individualist lottery: how people count, but not their numbers. Analysis 64(282): 106–112. Rauch, A., and J.S. Sharp. 2005. Ohioans’ attitudes about animal welfare. A topical report from the 2004 Ohio Survey of Food, Agricultural and Environmental Issues. Columbus, OH: Social Responsibility Initiative, Department of Human and Community Resource Development, The Ohio State University. http://ohio survey.osu.edu/pdf/2004_Animal_report.pdf. Accessed 26 July 2012. Regan, T. 1983. The case for animal rights. Berkeley: University of California Press. Singer, P. 1977. Animal liberation. New York: Avon Books. Singer, P. (ed.). 2006. In defense of animals: The second wave. Oxford: Blackwell. McMahan, J. 2002. The ethics of killing: Problems at the margins of life. New York: Oxford University Press. Nagel T 1970 Death Nouˆs 4(1): 73 80 McMahan, J. 2002. The ethics of killing: Problems at the margins of life. New York: Oxford University Press. Nagel, T. 1970. Death. Nouˆs 4(1): 73–80. Rachels, J. 1986. The end of life: Euthanasia and morality. Oxford: Oxford University Press. Rachels, J. 1986. The end of life: Euthanasia and morality. Oxford: Oxford University Press. f y Nagel, T. 1970. Death. Nouˆs 4(1): 73–80. Author Biography Krzysztof Saja is an assistant professor in the Department of Philosophy at the University of Szczecin in Poland. Regan, T. 1983. The case for animal rights. Berkeley: University of California Press. Singer, P. 1977. Animal liberation. New York: Avon Books. Singer, P. (ed.). 2006. In defense of animals: The second wave. Oxford: Blackwell. 123 123
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Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pintas) di Dinas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan
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Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pintas) di Dinas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan Maria Gabriella Riberu1*, Roro Merry Chornelia Wulandary2, Dekki Umamur Ra'is3 1,2,3 Prorgram Studi Administrasi Publik, Universitas Tribhuwana Tunggadewi *Email korespondensi: mariagabriellariberu@gmail.com Abstrak Pelayanan administrasi di Indonesia masih menjadi persoalan yang perlu memperoleh perhatian dan penyelesaian yang komperhensif. Masih ada diskriminasi dalam praktik pelayanan publik bagi kelompok rentan penyandang disabilitas. Maka dari itu penyelenggara pelayanan publik harus berasaskan keadilan sosial tanpa unsur diskriminatif. Seperti yang tertera dalam Undang-Undang Nomor 25 Tahun 2009 tentang pelayanan publik yang menjadi angin segar dalam upaya peneyedian pelayanan publik yang baik. Maka diperlukan inovasi yang menjadi kebutuhan utama sebagai jawaban atas beragam persoalan yang tengah dihadapi. Penelitian ini memiliki tujuan untuk mengetahui inovasi “Jalan Pintas” di Dinas Kependudukan dan Pencatatan Sipil Kabupaten Pasuruan dan apa saja faktor yang mempengaruhi inovasi “Jalan Pintas”. Metode penelitian ini menggunakan metode kualitatif. Teknik pengumpulan data yang digunakan adalah wawancara, observasi, dan dokumentasi. Teknik penentuan informan adalah purposive sampling yaitu kepala bidang pengelolaan informasi administrasi kependudukan dan masyarakat penyandang disabilitas. Hasil penelitian peneliti menunjukan bahwa Inovasi ini dilihat dari keuntungan, kesesuaian, kerumitan, kemungkinan dicoba dan kemudian diamati memperoleh hasil bahwa inovasi ini memudahkan masyarakat penyandang disabilitas dalam memenuhi dokumen kependudukan bagi masyarakat yang mempunyai keterbatasan fisik dan mental. Hasil dalam pelayanan inovasi Jalan Pintas ini berupa Biodata, KK, dan KTP- El. Sedangkan untuk faktor pendukung yaitu Undang-Undang yang mengatur serta sarana dan prasarana. Dan faktor penghambat yaitu kesadaran masyarakat penyandang disabilitas terhadap pentingnya dokumen kependudukan dan kendala jaringan saat pelayanan. Kata Kunci: Disabibilitas; Dispendukcapil; Inovasi; Pelayanan Publil; 20 | Riberu, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pintas) di Dinas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. Abstract Administrative services in Indonesia are still an issue that needs comprehensive attention and resolution. There is still discrimination in public service practices for vulnerable groups of persons with disabilities. Therefore, public service providers must be based on social justice without discriminatory elements. As stated in Law Number 25 of 2009 concerning public services which is a breath of fresh air in efforts to provide good public services. So innovation is needed which is the main need as an answer to the various problems that are being faced. This study aims to find out the innovation of "Jalan Pintas" in the Pasuruan Regency Population and Civil Registration Office and what are the factors that influence the innovation of "Shortcuts". This research method uses qualitative methods. The data collection techniques used are interviews, observation, and documentation. The informant determination technique is purposive sampling, namely the head of the information management division of the population administration and the community with disabilities. The results of the researcher's research show that this innovation is seen from the advantages, suitability, complexity, possibility of being tried and then observed to obtain the results that this innovation makes it easier for people with disabilities to fulfill population documents for people who have physical and mental limitations. The results in this Shortcut innovation service are in the form of Biodata, KK, and KTP-El. As for the supporting factors, namely the laws that regulate and facilities and infrastructure. And the inhibiting factor is the awareness of people with disabilities on the importance of population documents and network constraints during services. Keywords: Dispendukcapik; Dissability; Innovation; Public Service; PENDAHULUAN Dinas Kependudukan dan Pencatatan Sipil merupakan instansi teknis penyelenggara pelayanan publik di bidang administrasi kependudukan mulai dari pendaftaran penduduk, pencatatan sipil, sampai dengan pengelolaan informasi administrasi kependudukan. Administrasi adalah keseluruhan proses pelaksanaan dari keputusan-keputusan yang telah diambil dan Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 pelaksanaan itu pada umumnya dilakukan oleh orang manusia atau lebih untuk mencapai tujuan yang telah ditentukan sebelumnya (Marzuki, 2023). Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 pelaksanaan itu pada umumnya dilakukan oleh orang manusia atau lebih untuk mencapai tujuan yang telah ditentukan sebelumnya (Marzuki, 2023). pelaksanaan itu pada umumnya dilakukan oleh orang manusia atau lebih untuk mencapai tujuan yang telah ditentukan sebelumnya (Marzuki, 2023). Pelayanan kepada masyarakat saat ini sudah menjadi ukuran kinerja pemerintah. Masyarakat di era pasca reformasi ini sudah kian memahami hak-haknya dan berani bersuara untuk menuntut, jika mendapat pelayanan yang tidak sesuai dengan yang dijanjikan. Begitu pula dengan pelayanan administrasi di Indonesia, masih menjadi persoalan yang perlu memperoleh perhatian dan penyelesaian yang komperhensif. Dalam pelayanan publik yang diberikan oleh penyelenggara pelayanan administrasi hingga saat ini masih kurang optimal, masih ada diskriminasi dalam pratik pelayanan publik terhadap kelompok rentan seperti masyarakat penyandang disabilitas (Rohmad, 2016). Maka dari itu penyelenggara pelayanan publik harus berasaskan keadilan sosial tanpa unsur diskriminatif. Seperti yang tertera dalam Undang-Undang Nomor 25 Tahun 2009 tentang pelayanan publik, adapun ciri pelayanan publik dikatakan baik jika meningkatkan tingkat kepuasan publik, harus sesuai dengan undang-undang, kesamaan hak, profesionalitas, partisipatif, akuntabilitas, fasilitas dan perlakuan khusus bagi kelompok rentan, serta memberikan kemudahan dan ketergantungan (Musyafaah & Wijaya, 2020). Sehingga seluruh masyarakat dapat memperoleh haknya sebagai warga negara tanpa terkeculi karena substansi pelayanan publik adalah memberi pelayanan yang berkualitas kepada masyarakat serta memenuhi visi dan misi dalam menyelenggarakan good governance. Sehingga diperlukan inovasi yang menjadi kebutuhan utama pada bidang pelayanan publik sebagai jawaban atas beragam persoalan yang tengah dihadapi. Dengan memperhatikan jumlah penduduk yang mencapai 1.622.659 jiwa pada tahun 2023, terdiri dari laki-laki sebanyak 809.060 jiwa dan perempuan sebanyak 813.599 jiwa serta pola distribusi populasi di Kabupaten Pasuruan yang memiliki 24 Kecamatan dan 365 Desa, menjadi penting bagi pemerintah untuk mengidentifikasi kebutuhan masyarakat secara lebih spesifik. 21 | Riberu, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pintas) di Dinas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. , , y, , , , ( ) g y g y g ( ) Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. PENDAHULUAN Misalnya, kecamatan Gempol dengan jumlah penduduk paling banyak sebanyak 130.033 jiwa dapat menjadi fokus untuk peningkatan dan pengembangan pelayanan publik yang lebih intensif, sementara kecamatan Tosari dengan jumlah penduduk paling sedikit sebanyak 18.416 jiwa mungkin memerlukan strategi yang berbeda dalam memenuhi kebutuhan masyarakatnya. Tabel 1. Data Penyandang Disabilitas Kabupaten Pasuruan Tabel 1. Data Penyandang Disabilitas Kabupaten Pasuruan JENIS DISABILITAS Tahun 2022 Tahun 2023 L P Jumlah L P Jumlah Cacat Fisik 39 40 79 46 52 98 Cacat Netra / Buta 33 28 61 32 30 62 Cacat Rungu/Wicara 76 76 152 78 76 154 Cacat Mental/Jiwa 352 199 551 392 223 615 Cacat Fisik dan Mental 1 1 4 8 19 27 Cacat Lainnya 20 20 80 21 22 43 JUMLAH 521 364 885 577 422 999 Sumber: Data Sekunder, Dinas Kependudukan Dan Pencatatan Sipil Kabupaten Pasuruan Tahun 2023. Sumber: Data Sekunder, Dinas Kependudukan Dan Pencatatan Sipil Kabupaten Pasuruan Tahun 2023. Berdasarkan tabel 1 tingginya data masyarakat penyandang disabilitas pada tahun 2022 sebesar 885 jiwa dan pada tahun 2023 sebesar 999 jiwa di Kabupaten Pasuruan maka perlu adanya inovasi sebagai jawaban atas beragam persoalan yang tengah dihadapi. Melihat tuntutan masyarakat ditengah perkembangan zaman untuk mendapatkan pelayanan publik bagi mereka Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 yang berkebutuhan khusus di bidang administrasi kependudukan dan pecatatan sipil sebagai bentuk pemenuhan terhadap hak asasi manusia, sehingga berjalan efektif dan efisien serta merata bagi masyarakat penyandang disabilitas harus diperlakukan sama dan tidak boleh ada unsur diskriminatif pemerintah harus hadir ditengah-tengah mareka untuk melindungi hak mareka sebagai warga Negara Indonesia. yang berkebutuhan khusus di bidang administrasi kependudukan dan pecatatan sipil sebagai bentuk pemenuhan terhadap hak asasi manusia, sehingga berjalan efektif dan efisien serta merata bagi masyarakat penyandang disabilitas harus diperlakukan sama dan tidak boleh ada unsur diskriminatif pemerintah harus hadir ditengah-tengah mareka untuk melindungi hak mareka sebagai warga Negara Indonesia. Pada tanggal 15 November 2021 Dinas Kependudukan dan Pencatatan Sipil Kabupaten Pasuruan telah mendapatkan penghargaan Top 30 Kompetisi Inovasi Pelayanan Publik (KOVABLIK) dari Gubernur Jawa Timur melalui Inovasi Kios-e PAKLADI. Setelah mendapatkan penghargaan tersebut, Dinas Kependudukan dan Pencatatan Sipil mengembangkan dan meluncurkan beberapa inovasi diantaranya inovasi JALAN PINTAS (Jaringan Pelayanan bagi Penyandang Disabilitas). 22 | Riberu, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pintas) di Dinas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. ru, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pintas) di Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. METODE PENELITIAN Jenis penelitian menggunakan kualitatif. Moleong (2014) menjelaskan bahwa penelitian kualitatif adalah penelitian yang bermaksud untuk memahami fenomena apa yang dialami oleh subjek penelitian, misalnya perilaku, persepsi, motivasi, tindakan, dan lain sebagainya, pada kondisi obyek yang alamiah, dimana peneliti sebagai instrumen kunci (Ali. M, 2015). Tempat penelitian yaitu pada Dinas Kependudukan dan Pencatatan Sipil Kabupaten Pasuruan yang di laksanakan selama 6 bulan. Fokus penelitian di tujukan pada karakteristik inovasi oleh Suwarno yang merujuk dari Rogers (Hilda, 2014). Pada teknik pengambilan sampling menggunakan purposive sampling dengan beberapa informan yang sudah diketahui peran dalam menjalankan inovasi yaitu kepala bidang pemanfaatan data dan inovasi, kepala bidang pengelolaan informasi administrasi kependudukan, petugas pelayanan dan masyarakat penyandang disabilitas. Teknik pengumpulan data dilakukan dengan interview (wawancara), observasi (pengamatan), dan dokumentasi. Teknik keabsahan data menggunakan triangulasi teknik. PENDAHULUAN Sejak inovasi ini digaungkan, Dinas Kependudukan dan Pencatatan Sipil beberapa kali memberikan layanan dokumen kependudukan bagi masyarakat penyandang disabilitas. Inovasi ini sama seperti Inovasi "Aduh Bra" yang diluncurkan oleh Dispendukcapil Kota Malang yang merupakan langkah inovatif yang diharapkan dapat memberikan kemudahan bagi masyarakat dalam mengurus segala kebutuhan administrasi kependudukan, khususnya bagi penyandang disabilitas. Penelitian terdahulu yang dilakukan oleh (Helda, etc., 2023) dengan judul "Inovasi Pelayanan Kependudukan Berbasis Braille Di Kota Malang" telah memberikan dasar untuk pengembangan inovasi tersebut. Inovasi'Aduh Bra'" merupakan bentuk pelayanan publik yang berfokus pada kepengurusan dokumen kependudukan dengan huruf braille, yang menjadi fasilitas khusus bagi penyandang disabilitas, terutama tunanetra di Kota Malang. Hal ini sesuai dengan tuntutan masyarakat akan pelayanan publik yang inklusif, terutama dalam bidang administrasi kependudukan dan pencatatan sipil. Pengembangan inovasi bagi masyarakat penyandang disabilitas juga sejalan dengan amanat Undang-Undang RI Nomor 8 Tahun 2016 tentang Penyandang Disabilitas, yang menegaskan perlunya pemerintah memastikan bahwa pelayanan publik dapat diakses secara merata oleh seluruh lapisan masyarakat, termasuk penyandang disabilitas. Dengan memberikan kemudahan akses terhadap dokumen kependudukan, inovasi "Jalan Pintas” diharapkan dapat mendukung penyandang disabilitas dalam mencapai kehidupan yang sejahtera, mandiri, dan bebas dari diskriminasi. Pemerintah hadir sebagai garda terdepan dalam melindungi hak-hak warga negara, termasuk penyandang disabilitas, dan memastikan bahwa pelayanan publik diselenggarakan secara adil dan tidak diskriminatif. Dengan demikian, penerapan inovasi seperti "Jalan Pintas" menjadi langkah konkret dalam mewujudkan prinsip-prinsip hak asasi manusia dan inklusi sosial bagi seluruh warga negara Indonesia. PENDAHULUAN Berdasarkan dengan berbagai permasalahan dalam mengurus dokumen kependudukan dan banyaknya penyandang disabilitas di wilayah Kabupaten Pasuruan; Kondisi kesehatan dan kondisi sosial masyarakat penderita disabilitas; Jarak tempuh ke kecamatan atau Dispendukcapil yang jauh maka untuk memenuhi dokumen kependudukan bagi masyarakat yang mempunyai keterbatasan fisik dan mental, dalam rangka peningkatan pelayanan Administrasi Kependudukan sejalan dengan tuntutan pelayanan Administrasi Kependudukan yang profesional, memenuhi standar teknologi informasi, dinamis, tertib, dan tidak diskriminatif dalam pencapaian standar pelayanan minimal menuju pelayanan prima yang menyeluruh untuk mengatasi permasalahan kependudukan, maka pada tanggal 15 Maret 2022 Dispendukcapil Kabupaten Pasuruan melalui kebijakan inovasi daerah dalam penyelenggaraan pemerintahan negara dengan berbagai latar belakang masalah lahirlah inovasi JALAN PINTAS ( Jaringan Pelayanan bagi Penyan dang Disabilitas ) dengan tujuan memudahkan masyarakat penyandang disabilitas dalam rangka Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 layanan dokumen kependudukan mulai dari perekaman sampai pada penertiban dokumen kependudukan Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 layanan dokumen kependudukan mulai dari perekaman sampai pada penertiban dokumen kependudukan. Teori yang digunakan dalam penelitian ini adalah persepsi dalam inovasi ini adalah 1) Keandalan yaitu kemampuan untuk melaksanakan pelayanan yang dijanjikan dengan tepat dan terpercaya. 2) Ketanggapan yaitu kemampuan untuk membantu pelanggan dan memberikan pelayanan dengan cepat. 3) Keyakinan yaitu pengetahuan dan kesopanan pegawai serta kemampuan untuk menimbulkan kepercayaan dan keyakinan atau “assurance”. 4) Empati yaitu syarat untuk peduli, memberi perhatian pribadi bagi pelanggan. 5) Berwujud yaitu penampilan fasilitas fisik, peralatan, personel, dan media komunikasi (Parasuraman,1988). 23 | Riberu, M.G., Wulandary, R.M.C., Ra is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jala Dinas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. 23 | Riberu, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pintas) di Dinas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. g g g nas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. ru, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pintas) di HASIL DAN PEMBAHASAN Memiliki keuntungan relatif (relatife atvantage) dalam hal ini adalah inovasi yang harus mempunyai keunggulan dari inovasi sebelumnya atau pelayanan sebelumnya yang selalu ada sebuah nilai baru yang melekat didalam inovasi yang menjadi ciri pembedaan dari yang lain. Dan merupakan inovasi yang memiliki keuntungan dari arti sebuah ide, program, dan sistem yang akan menghasilkan kelebihan atau keuntungan dari inovasi ini. Keuntuangan atau kelebihan pada inovasi ini agar dapat menunjukan dan memberikan pelayanan yang baik kepada masyarakat khususnya penyandang disabilitas sampai kepelosok terpencil wilayah Kabupaten Pasuruan dalam mengurus administrasi Kependudukannya. Gambar 1. Proses Perekaman Jemput Bola dalam Inovasi Jalan Pintas oleh Petugas dirumah masyarakat Disabilitas Mental Sumber: Dokumentasi pribadi, 2023. Gambar 1. Proses Perekaman Jemput Bola dalam Inovasi Jalan Pintas oleh Petugas dirumah masyarakat Disabilitas Mental Sumber: Dokumentasi pribadi, 2023. Berdasarkan gambar 1 menunjukan proses perekaman jemput bola dalam inovasi Jalan Pintas oleh petugas dirumah masyarakat penyandang disabilitas. Tujuan inovasi Jalan Pintas ini Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 adalah memberikan kemudahan bagi penyandang disabilitas dalam memiliki dokumen data kependudukan, seperti penyampaian keuntungan relatif yang diberikan sangat mudah, sebagaimana Dispendukcapil sebagai penyedia sarana dan prasarana dalam bentuk pelayanan yakni pelayanan jemput bola bagi masyarakat yang didaerah pelosok dan jauh dari kantor pemerintah. Program Inovasi ini berdampak signifikan terutama pada kelompok masyarakat yang rentan memiliki disabilitas fisik dan disabilitas mental (ODGJ), karena masyarakat tidak perlu mendatangi gerai pelayanan tetapi cukup dengan mengajukan surat permohonan kepada Dinas Kependudukan dan Pencatatan Sipil melalui pemerintah desa dengan mengetahui kecamatan untuk mendapatkan dokumen kependudukan. Penerapan sistem inovasi Jalan Pintas sudah memenuhi standar yang ada yaitu memenuhi apa yang sudah di amanatkan dalam UU RI nomor 8 tahun 2016 tentang penyandang disabilitas yang memberikan kemudahan bagi penyandang disabilitas dalam memiliki dokumen data kependudukan Untuk mewujudkan kesan dan kesempatan bagi penyandang disabilitas menuju kehidupan yang sejahtera, mandiri dan tanpa diskriminasi sehingga inovasi ini memberikan kejelasan identitas dan status penduduk bagi penyandang disabilitas dan mendapatkan kepastian hukum, perlindungan hukum dan kenyamanan bagi pemiliknya. Masyarakat penyandang disabilitas yang sebelumnya kurang mendapat perhatian dari pemerintah sudah perlahan haknya mulai terpenuhi dengan adanya Kartu Keluarga (KK), Kartu Tanda Penduduk (KTP), KIA, Akte Kelahiran dan dokumen-dokumen lain yang mempertegas status kependudukkan yang sah sebagai warga negara. 24 | Riberu, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pintas) di Dinas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. y g y g y g nas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. 4 | Riberu, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pint Dinas Kependudukan dan Pencatatan Sipil Kabupaten Pasuruan Indonesian Social Science Review 2(1) 20 30 HASIL DAN PEMBAHASAN Kepemilikan dokumen administrasi kependudukan memiliki arti penting dan strategis bagi pemerintah dan masyarakat disabilitas itu siendiri yang menjadi syarat untuk mengakses berbagai layanan publik yang disediakan oleh pemerintah, termasuk akses terhadap berbagai bantuan sosial. Oleh karena itu dengan adanya inovasi “Jalan Pintas” ini masyarakat disabilitas di ajak untuk sadar dan peduli dengan administrasi kependudukannya terkait hal-hal yang berhubungan dengan identitas pemohon. Keuntungan relatif tidak terlepas dari dimensi ekonomis, terdapat perbedaan pelayanan sebelum adanya inovasi Jalan Pintas, bahwa dalam proses pelayanan mengurus dokumen Adminduk tidak ada perbedaan dengan masyarakat pada umumnya dimana masyarakat penyandang disabilitas harus mendatangi kantor Kecamatan dan Dispendukcapil dengan jarak tempuh yang jauh, seperti halnya pada keuntungan relatif yang diberikan Dispendukcapil kepada masyarakat, mengenai pelayanan administrasi kependudukan yang menjadi efektif dan efisien karena Tim Jemput Bola Dispendukcapil yang akan mendatangi rumah pemohon. Dimana pada saat melakukan pelayanan masyarakat tidak mengeluarkan waktu maupun biaya selama proses kepengurusan dokumen kependudukan. Dengan pelayanan yang lebih efektif dan efisien tersebut terdapat kepuasan masyarakat penyandang disabilitas dalam pelayanan inovasi Jalan Pintas ini, sehingga terdapat perubahan pandangan terhadap Dispendukcapil Kabupaten Pasuruan, masyarakat penyandang disabilitas sebagai penerima layanan menghargai dan memberikan apresiasi dengan mengungkapkan kepuasan dan kesenangannya saat tim Jemput Bola Inovasi Jalan Pintas melakukan perekaman dirumah mereka, mereka merasa keberadaan dan keterbatasan yang mereka miliki dihargai dan diberi kesempatan untuk mendapatkan hak-hak dasarnya. Berdasarkan teori Rogers, atribut atau ciri-ciri kedua dari inovasi pelayanan publik yaitu Kesesuaian (Compability) diartikan sebagai inovasi mempunyai sifat kompatibel dan sesuai dengan inovasi yang digantinya agar inovasi sebelumnya tidak mubasir. Kesesuaian (Compability) Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 dari sebuah Inovasi pelayanan publik yang dibentuk harus sesuai dengan apa yang dibutuhkan oleh sasaran inovasi pelayanan publik atau tepat sasaran. Berkaitan dengan indikator kesesuaian it b h l i i “J l Pi t ” d h i d k b t h k t Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 Volume 2 Nomor 1 (2024) Pg 20 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 dari sebuah Inovasi pelayanan publik yang dibentuk harus sesuai dengan apa yang dibutuhkan oleh sasaran inovasi pelayanan publik atau tepat sasaran. 25 | Riberu, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pintas) di Dinas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. HASIL DAN PEMBAHASAN Screenshoot Survey Kepuasan Masyarakat Tahun 2023 Sumber : Dokumen Dispendukcapil Kab Pasuruan, 2023 Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 Volume 2 Nomor 1 (2024) Pg 20 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 Gambar 3. Screenshoot Survey Kepuasan Masyarakat Tahun 2023 Sumber : Dokumen Dispendukcapil Kab Pasuruan, 2023 Gambar 3. Screenshoot Survey Kepuasan Masyarakat Tahun 2023 Sumber : Dokumen Dispendukcapil Kab Pasuruan, 2023 Gambar 3. Screenshoot Survey Kepuasan Masyarakat Tahun 2023 Sumber : Dokumen Dispendukcapil Kab Pasuruan, 2023 Berdasarkan gambar 3 diatas menunjukkan survey kepuasan masyarakat pada Dinas Kependudukan dan Pencatatan Sipil Kabupaten Pasuruan memperoleh nilai 93.81 dengan indikator kesesuaian produk pelayanan mencapai 99,48 yaitu salah satunya produk pelayanan inovasi Jalan Pintas yang sudah memenuhi kebutuhan masyarakat. Dan dengan indikator penanganan pengaduan, saran dan masukan yang mempunyai nilai 99,62 menunjukkan bahwa inovasi ini memberikan dampak yang signikan sesuai dengan kebutuhan masyarakat di Kabupaten Pasuruan. Indikator kerumitan (Complexity), merupakan ciri ketiga dari inovasi pelayanan publik yang dikemukakan oleh Rogers. Kerumitan ini diartikan dengan sifatnya yang baru, maka inovasi ini tentunya memiliki tingkat kerumitan yang bisa menjadi hasil lebih dibandingkan dengan inovasi atau pelayanan sebelumnya. kerumitan yang didapatkan dari inovasi “Jalan Pintas” ini bahwa dalam memberikan pelayanan kepada masyarakat disabilitas dalam mengurus dokumen kependudukannya dibutuhkan Sumber daya manusia yang memiliki dedikasi yang tinggi baik itu Tim Dispendukcapil sebagai pemberi layanan maupun masyarakat yang menerima layanan. Di karenakan dalam penerapan sebuah inovasi oleh pemerintah tentunya tidak mudah. Karena berhadapan dengan situasi dan kondisi yang terkadang tidak sesuai dengan persiapan dan harapan. Hal ini membutuhkan kerja keras dan kerja sama yang baik dari Dispendukcapil sampai jajaran terendah pemerintah yang berhadapan langsung dengan masyarakat yakni tingkat RT, RW dan sebagainya. Selain itu terdapat kendala lainnya yaitu trauble jaringan internet, karena pelayanan inovasi Jalan Pintas ini menggunakan jemput bola dirumah pemohon dengan sistem daring perekaman yang dilakukan langsung terhubung dengan Kementerian Dalam Negeri yang tercantum dalam Peraturan Menteri Dalam Negeri No 7 Tahun 2019 Tentang Pelayanan Administrasi Kependudukan Secara Daring, dimana secara geografis di Kabupaten Pasuruan ini bukan cuman daerah perkotaan tetapi juga ada daerah pesisir dan daerah pegunungan sehingga masih terdapat beberapa titik yang blank spot, jika terjadi trauble jaringan maka akan kesulitan dalam proses perekaman pelayanan inovasi Jalan Pintas. 26 | Riberu, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pintas) di Dinas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. 26 | Riberu, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Dinas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. | y ( ) g y g y g ( Dinas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. HASIL DAN PEMBAHASAN Berkaitan dengan indikator kesesuaian yaitu bahwa pelayanan inovasi “Jalan Pintas” sudah sesuai dengan kebutuhan masyarakat penyandang disabilitas. Gambar 2. Alur Pengajuan Pelayanan Inovasi Jalan Pintas. Sumber: Data Sekunder, 2023 Gambar 2. Alur Pengajuan Pelayanan Inovasi Jalan Pintas. Sumber: Data Sekunder, 2023 Dari gambar 2 di atas menunjukkan alur pengajuan inovasi Jalan Pintas di Dinas Kependudukan dan Pencatatan Sipil Kabupaten Pasuruan. Inovasi Jalan Pintas ini telah memenuhi standar pelayanan menurut Undang undang Nomor 25 tahun 2019 tentang pelayanan publik menyebutkan bahwa penyelenggaraan berkewajiban memberikan pelayanan dengan perlakuan khusus kepada anggota masyarakat tertentu sesuai dengan ketentuan peraturan perundang- undangan. Dan memenuhi apa yang sudah di amanatkan dalam UU RI nomor 8 tahun 2016 tentang penyandang disabilitas, memberikan kemudahan bagi penyandang disabilitas dalam memiliki dokumen data kependudukan, untuk mewujudkan kesempatan bagi penyandang disabilitas menuju kehidupan yang sejahtera, mandiri dan tanpa diskriminasi. Dalam pelayanan administrasi kependudukan bagi masyarakat penyandang disabilitas sebelumnya tidak ada inovasi khusus bagi masyarakat penyandang disabilitas. sebelum adanya inovasi Jalan Pintas, Dispendukcapil Kabupaten Pasuruan telah meluncurkan Inovasi SI MAMA, yaitu siap melayani dirumah yang melayani kaum rentan tetapi inovasi ini kurang menjangkau untuk masyarakat penyandang disabilitas sehingga Dispendukcapil Kabuapten Pasuruan mengeluarkan inovasi khusus bagi masyarakat penyandang disabilitas baik itu mental maupun fisik yaitu inovasi Jalan Pintas. Dengan adanya inovasi Jalan Pintas ini sudah sesuai dengan yang diamanatkan dalam UU RI nomor 8 tahun 2016 tentang penyandang disabilitas yang memberikan kemudahan bagi penyandang disabilitas dalam memiliki dokumen data kependudukan, untuk mewujudkan kesan dan kesempatan bagi penyandang disabilitas menuju kehidupan yang sejahtera, mandiri dan tanpa diskriminasi. Dan memberikan kejelasan identitas dan status penduduk bagi penyandang disabilitas sehingga mendapatkan kepastian hukum, mendapatkan perlindungan hukum dan kenyamanan bagi pemiliknya. Dalam hal ini inovasi Jalan Pintas telah memenuhi standar pelayanan dengan tidak menimbulkan kesan yang lama dan tidak berguna tetapi memberikan nilai tambahan dan pembaruan untuk inovasi sebelumnya dapat dilihat dalam Survey Kepuasan Masyarakat pada tahun 2023 di Dinas Kependudukan dan Pencatatan Sipil Kabupaten Pasuruan. Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 Gambar 3. 6 | Riberu, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pint HASIL DAN PEMBAHASAN Indikator triability merupakan indikator keempat untuk ciri-ciri atau atribut inovasi pelayanan publik yang dikemukakan oleh Rogers. Kemungkinan dicoba (Triability) merupakan Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 inovasi yang hanya bisa diterima apabila teruji dan mampu terbukti keuntungan atau nilai lebih Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 inovasi yang hanya bisa diterima apabila teruji dan mampu terbukti keuntungan atau nilai lebih yang dihasilkan dibandingkan dengan dengan inovasi lama. Kemungkinan dicoba dalam penelitian ini diartikan sebagai inovasi yang dilakukan harus melewati masa pencobaan dan mampu meberikan pelayanan sebai-baiknya kepada masyarakat dalam lingkup Dinas Kependudukan dan Pencatatan Sipil Kabupaten Pasuruan terkhusus bagi penyandang disabilitas. Pelaksanaan pelayanan inovasi “Jalan Pintas” ini tidak dilakukan uji coba terlebih dahulu tetapi langsung diterapkan kepada masyarakat penyandang disabilitas karena sesuai dengan tuntutan masyarakat itu sendiri. Penerapan sistem ini dinilai cukup inovatif sebagai cara pemenuhan hak kaum penyandang disabilitas dengan penerbitan dokumen kenegaraan. Perlu diketahui bahwa sistem ini dinilai sangat bermanfaat bagi masyarakat disabilitas sehingga langsung diterapkan dan kenyataannya membuahkan hasil posistif demi pengembangan sistem di Dispendukcapil Kabupaten Pasuruan yang mana kewajiban dari pemerintah sesuai dengan Undang-Undang bahwa pelayanan dokumen-dokumen dari negara tidak berpihak atau berat sebelah. Selain itu, indicator lain seperti kemampuan untuk diamati adalah derajat di mana hasil suatu inovasi dapat dilihat orang lain. Semakin mudah seseorang melihat hasil suatu inovasi, semakin besar kemungkinan orang atau sekelompok orang tersebut mengadopsi. Dalam rangka meningkatkan kapasitas sumberdaya manusia, Dinas Kependudukan dan Pencatatan Sipil senantiasa setiap tahun memberikan sosialisasi dengan mengadakan bimbingan teknis terkait dengan materi yang disampaikan adalah regulasi dan aturan yang berkaitan dengan administrasi kependudukan serta pengenalan Inovasi Jalan Pintas Kepada Perangkat di 365 desa dan masyarakat di seluruh kabupaten Pasuruan. Gambar 3. Kegiatan Sosialisasi Pengenalan Inovasi Jalan Pintas Sumber : Data Primer, 2023. Gambar 3. Kegiatan Sosialisasi Pengenalan Inovasi Jalan Pintas Sumber : Data Primer, 2023. Berdasarkan gambar 3 menunjukkan kegiatan sosialisasi Dinas Kependudukan dan Pencatatan Sipil dalam memperkenalkan inovasi Jalan Pintas kepada aparatur desa dan masyarakat. Dan segala bentuk usaha pengenalan inovasi Jalan Pintas melalui Media Sosial yaitu Instagram (dukcapilkabpas), Youtube (Dispendukcapil Kabupaten Pasuruan), Tiktok (dispendukcapilkabpa), Facebook (dispencapil@pasuruankab.go.id), dan Website (http://dispendukcapil@pasuruankab.go.id). 27 | Riberu, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pintas) di Dinas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. , , y, , , , ( ) g y g y g ( ) nas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. 7 | Riberu, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pint Dinas Kependudukan dan Pencatatan Sipil Kabupaten Pasuruan Indonesian Social Science Review 2(1) 20-30 HASIL DAN PEMBAHASAN Faktor-faktor pendukung yang selalu memacu atau mendukung penerapan sistem “Jalan Pintas” sesuai dengan fakta di lapangan adalah sebagai berikut terdapat Peraturan Pemerintahan Nomor 38 Tahun 2017 tentang Inovasi Daerah, Terbitnya Undang-Undang Nomor 8 Tahun 2016 tentang penyandang disabilitas, Undang-Undang Nomor 24 tahun 2013 tentang Adiministrasi Kependudukan, Undang-Undang Nomor 25 Tahun 2009 tentang Pelayanan Publik, Peraturan Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 Menteri Dalam Negeri No 7 Tahun 2019 Tentang Pelayanan Administrasi Kependudukan Secara Daring, Peraturan Bupati Pasuruan Nomor 11 Tahun 2021 tentang Pelayanan Administrasi Kependudukan dan Pencatatan Sipil, adanya kepuasan dari masyarakat disabilitas, sarana dan prasarana memadai, tim Inovasi Jalan Pintas yang solid dengan tingkat kepedulian yang tinggi, keterlibatan pihak Kecamatan, desa, RT/RW, tetangga dan keluarga terkait menjadi faktor pendukung terlaksananya inovasi Jalan Pintas ini. Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 M t i D l N i N 7 T h 2019 T t P l Ad i i t i K d d k S Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 Menteri Dalam Negeri No 7 Tahun 2019 Tentang Pelayanan Administrasi Kependudukan Secara Daring, Peraturan Bupati Pasuruan Nomor 11 Tahun 2021 tentang Pelayanan Administrasi Kependudukan dan Pencatatan Sipil, adanya kepuasan dari masyarakat disabilitas, sarana dan prasarana memadai, tim Inovasi Jalan Pintas yang solid dengan tingkat kepedulian yang tinggi, keterlibatan pihak Kecamatan, desa, RT/RW, tetangga dan keluarga terkait menjadi faktor pendukung terlaksananya inovasi Jalan Pintas ini. Dan terdapat juga faktor penghambat yang mempengaruhi jalannya inovasi Jalan Pintas ini yaitu masih ada sebagian masyarakat yang tidak memiliki tingkat kesadaran dan kepedulian untuk mengurus dokumen kependudukan atau belum adanya kemaun dari masyarakat itu sendiri, banyak masyarakat yang belum mengetahui akan adanya Inovasi pelayanan “Jalan Pintas”, minimnya data masyarakat disabilitas di Dinas Kependudukan dan Pencatatan Sipil Kabupaten Pasuruan, gangguan Jaringan menjadi faktor penghambat utama dalam proses pelayanan perekaman inovasi Jalan Pintas dikarenakan letak geografis Kabupaten Pasuruan yang pegunungan dan pesisir sehingga masih ada blank spot di beberapa titik wilayah ini menghambat kerja inovasi ini karena dalam melakukan perekaman data langsung dikirim kepusat Kementerian Dalam Negeri untuk dicatat dalam database sehingga jika terjadi trauble jaringan maka akan mengganggu proses perekaman (Sinta & Hertati, 2023). HASIL DAN PEMBAHASAN Berdasarkan hasil penelitian terdahulu dari Julian (2024) Hasil penelitian menunjukkan bahwa implementasi program Jalan Pintas dalam pelayanan administrasi kependudukan telah berjalan dengan baik. Hal ini dibuktikan dengan puluhan kali aksi jemput bola yang menandakan bahwa program Jalan Pintas merupakan program yang tepat dan dibutuhkan oleh para penyandang disabilitas. Jika penelitian terdahulu di bandingkan dengan penelitian ini hampir memiliki persamaan dalam sistem pelayanan yang diberikan oleh instansi pemerintah yang dikhususkan kepada masyarakat disabilitas sedangkan perbedaan pada penelitian terdahulu dengan penelitian ini terletak pada rumusan masalah. Berdasarkan hasil penelitian terdahulu oleh Hilda (2014) hasil Penelitian ini menyimpulkan bahwa Layanan publik bagi penyandang disabilitas khususnya pada aspek aksesibilitas, competence dan security belum perselenggara dengan baik. Persamaan yang terdapat dari penelitian terdahulu dengan penelitian ini adalah penyelenggaraan pelayanan memberikan aksessibilitas kepada masyarakat disabilitas sedangkan perbedaan yang terdapat dari penelitian terdahulu dengan penelitian ini terletak pada rumusan masalah. 28 | Riberu, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pintas) di Dinas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. KESIMPULAN Dinas Kependudukan dan Pencatatan Sipil Kabupaten Pasuruan sebagai Institusi penyelenggara pelayanan publik terus berbenah khususnya dibidang inovasi, dan inovasi terbaru adalah “Jalan Pintas” (Jaringan Pelayanan Adminduk Bagi Penyandang Disabilitas) yang di cetuskan pada tanggal 15 Maret 2022 sampai dengan saat ini. Jaringan pelayanan bagi penyandang disabilitas merupakan salah satu inovasi unggulan dari Dinas Kependudukan dan Pencatatan sipil selain inovasi lainnya, kegiatan ini bisa melayani masyarakat sampai ke pelosok terpencil wilayah Kabupaten Pasuruan bagi masyarakat yang jauh dengan pusat layanan adminitrasi kependudukan yaitu dari kantor kecamatan atau kantor dispendukcapil. Untuk memenuhi dokumen kependudukan bagi masyarakat yang mempunyai keterbatasan fisik dan mental, pemerintah harus hadir ditengah-tengah mareka untuk melindungi hak mareka sebagai warga Negara Indonesia, dan dalam rangka peningkatan pelayanan Administrasi Kependudukan sejalan dengan tuntutan pelayanan Administrasi Kependudukan yang profesional, memenuhi Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 standar teknologi informasi, dinamis, tertib, dan tidak diskriminatif dalam pencapaian standar pelayanan minimal menuju pelayanan prima yang menyeluruh untuk mengatasi permasalahan kependudukan, maka lahirlah kegiatan Jalan Pintas (Jaringan Pelayanan Bagi Penyandang Disabilitas). Hasil dalam pelayanan inovasi Jalan Pintas ini berupa Biodata, KK, dan KTP-El. Langkah inovasi dari pemerintah Kabupaten Pasuruan merupakan suatu cara ampuh untuk memanusiakan manusia dalam berbagai aspek. Penyandang dasabiltas memiliki kewajiban yang sama seperti masyarakat lain pada umumnya. Adapun yang menjadi faktor pendukung dalam proses penerapan pelayanan inovasi “Jalan Pintas” yaitu Pada Peraturan Pemerintahan Nomor 38 Tahun 2017 tentang Inovasi Daerah dan Undang-Undang Nomor 8 Tahun 2016 tentang penyandang disabilitas, serta Undang-undang lain yang mengatur, tim Jemput Bola dalam inovasi Jalan Pintas yang solid, kompak dan sigap dalam pelayanan, faktor pendukung lainnya yaitu dari aparat desa, sarana dan prasarana yang memadai. Adapun yang menjadi faktor penghambat dalam penerapan inovasi “Jalan Pintas” yaitu masih ada sebagian masyarakat yang tidak memiliki tingkat kesadaran dan kepedulian untuk mengurus dokumen kependudukan, selain itu sistem jaringan pun menjadi salah satu faktor penghambat dalam menjalankan inovasi “Jalan Pintas” ini karena perekaman harus terhubung dengan Kementerian Dalam Negeri agar dapat tercatat dalam database. 29 | Riberu, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pintas) di Dinas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. DAFTAR PUSTAKA Ali, M. (2015). Metode Penelitian Kualitatif. Jakarta: Prenada Media. 30 | Riberu, M.G., Wulandary, R.M.C., Ra’is, D.U., (2024). Inovasi Jaringan Pelayanan Bagi Penyandang Disabilitas (Jalan Pintas) di Dinas Kependudukan dan Pencatatan Sipil, Kabupaten Pasuruan. Indonesian Social Science Review, 2(1), 20-30. Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 Sinta, A. N., & Hertati, D. (2023). Inovasi Aplikasi Klampid New Generation (KNG) Dalam Meningkatkan Pelayanan Administrasi Kependudukan Di Dinas Kependudukan Dan Catatan Sipil Kota Surabaya. Journal of Governance Innovation, 5(2), 193-209. Ali, M. (2015). Metode Penelitian Kualitatif. Jakarta: Prenada Media. Dinas Kependudukan dan Pencatatan Sipil Kabupaten Pasuruan (https://dispendukcapil.pasuruankab.go.id/), diakses 1 Oktober 2023. Helda, M. E. N., Dewi, D. A. P. S., & Setiawan, M. N. R. A. (2023). Inovasi Pelayanan “Administrasi Dukcapil Huruf Braille (Aduh Bra)” Bagi Penyandang Disabilitas Pada Dinas Kependudukan Pencatatan Sipil Kota Malang. Journal Indonesian Social Science Review (ISSR), 1(1): 10-19 Hilda. (2014). Faktor-Faktor Yang Mempengaruhi Keputusan Masyarakat Dalam Pemilihan Jenis Pelayanan Administrasi Kependudukan. Jurnal Administrasi Publik, 2(1), 32-48. Julian. (2024). Implementasi Program Jalan Pintas Dalam Pelayanan Administrasi Kependudukan Bagi Penyandang Disabilitas. Jurnal Inovasi Pelayanan Publik, 3(2), 78-92. Moleong, L. J. (2014). Metodologi Penelitian Kualitatif. PT. Remaja Rosdakarya. Marzuki, M. (2023). Pelaksanaan Fungsi Komunikasi Administrasi Di Dinas Pendidikan Kabupaten Melawi. Journal of Educational Review and Research, 6(1), 1-12. Musyafaah, N. L., & Wijaya, A. (2020). Pelayanan Publik Pada Dinas Kependudukan Dan Pencatatan Sipil di Kabupaten Lamongan dalam Perspektif Fikih Siyasah. Al-Daulah: Jurnal Hukum Dan Perundangan Islam, 10(2), 251-274. Parasuraman, A., (2014)., The Behaviorial Consequenses of Service Quality. New Jersey: Prentince Hall Rohmad, A. M. (2016). Penyelenggaraan Pelayanan Publik yang Baik dan Berkualitas. Jurnal Manajemen Publik, 1(1), 45-60. Rogers, E. M. (1983). Diffusion of Innovations. The Free Press. Rogers, E. M. (1983). Diffusion of Innovations. The Free Press. Rohmad, A. M. (2016). Manajemen Pelayanan Publik: Konsep dan Aplikasi. Yogyakarta: Gava Media. Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 Sinta, A. N., & Hertati, D. (2023). Inovasi Aplikasi Klampid New Generation (KNG) Dalam Meningkatkan Pelayanan Administrasi Kependudukan Di Dinas Kependudukan Dan Catatan Sipil Kota Surabaya. Journal of Governance Innovation, 5(2), 193-209. Indonesian Social Science Review (ISSR) Volume 2 Nomor 1 (2024) Pg 20 - 30 E-ISSN: 3025-7352 | P-ISSN: 3026-0035 Sinta, A. N., & Hertati, D. (2023). Inovasi Aplikasi Klampid New Generation (KNG) Dalam Meningkatkan Pelayanan Administrasi Kependudukan Di Dinas Kependudukan Dan Catatan Sipil Kota Surabaya. Journal of Governance Innovation, 5(2), 193-209. Sinta, A. N., & Hertati, D. (2023). Inovasi Aplikasi Klampid New Generation (KNG) Dalam Meningkatkan Pelayanan Administrasi Kependudukan Di Dinas Kependudukan Dan Catatan Sipil Kota Surabaya. Journal of Governance Innovation, 5(2), 193-209.
https://openalex.org/W2965957890
https://research.rug.nl/files/97353225/micromachines_10_00504_v2.pdf
English
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A Contactless and Biocompatible Approach for 3D Active Microrobotic Targeted Drug Delivery
Micromachines
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cc-by
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University of Groningen A Contactless and Biocompatible Approach for 3D Active Microrobotic Targeted Drug Delivery Ongaro, Federico; Niehoff, Dennis; Mohanty, Sumit; Misra, Sarthak Published in: University of Groningen A Contactless and Biocompatible Approach for 3D Active Microrobotic Targeted Drug Delivery Ongaro, Federico; Niehoff, Dennis; Mohanty, Sumit; Misra, Sarthak Published in: Received: 1 July 2019; Accepted: 27 July 2019; Published: 31 July 2019 Abstract: As robotic tools are becoming a fundamental part of present day surgical interventions, microrobotic surgery is steadily approaching clinically-relevant scenarios. In particular, minimally invasive microrobotic targeted drug deliveries are reaching the grasp of the current state-of-the-art technology. However, clinically-relevant issues, such as lack of biocompatibility and dexterity, complicate the clinical application of the results obtained in controlled environments. Consequently, in this work we present a proof-of-concept fully contactless and biocompatible approach for active targeted delivery of a drug-model. In order to achieve full biocompatiblity and contacless actuation, magnetic fields are used for motion control, ultrasound is used for imaging, and induction heating is used for active drug-model release. The presented system is validated in a three-dimensional phantom of human vessels, performing ten trials that mimic targeted drug delivery using a drug-coated microrobot. The system is capable of closed-loop motion control with average velocity and positioning error of 0.3 mm/s and 0.4 mm, respectively. Overall, our findings suggest that the presented approach could augment the current capabilities of microrobotic tools, helping the development of clinically-relevant approaches for active in-vivo targeted drug delivery. Keywords: microrobotics; surgical robotics; minimally-invasive surgery; ultrasound tracking; magnetic actuation; targeted drug delivery Micromachines 2019, 10, 504; doi:10.3390/mi10080504 University of Groningen A Contactless and Biocompatible Approach for 3D Active Microrobotic Targeted Drug Delivery Ongaro, Federico; Niehoff, Dennis; Mohanty, Sumit; Misra, Sarthak Published in: Micromachines DOI: 10.3390/mi10080504 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2019 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Ongaro, F., Niehoff, D., Mohanty, S., & Misra, S. (2019). A Contactless and Biocompatible Approach for 3D Active Microrobotic Targeted Drug Delivery. Micromachines, 10(8), Article 504. https://doi.org/10.3390/mi10080504 University of Groningen A Contactless and Biocompatible Approach for 3D Active Microrobotic Targeted Drug Delivery Ongaro Federico; Niehoff Dennis; Mohanty Sumit; Misra Sarthak Ongaro, Federico; Niehoff, Dennis; Mohanty, Sumit; Misra, Sarthak DOI: 10.3390/mi10080504 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2019 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Ongaro, F., Niehoff, D., Mohanty, S., & Misra, S. (2019). A Contactless and Biocompatible Approach for 3D Active Microrobotic Targeted Drug Delivery. Micromachines, 10(8), Article 504. https://doi.org/10.3390/mi10080504 Citation for published version (APA): Ongaro, F., Niehoff, D., Mohanty, S., & Misra, S. (2019). A Contactless and Biocompatible Approach for 3D Active Microrobotic Targeted Drug Delivery. Micromachines, 10(8), Article 504. https://doi.org/10.3390/mi10080504 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. micromachines micromachines micromachines micromachines A Contactless and Biocompatible Approach for 3D Active Microrobotic Targeted Drug Delivery Federico Ongaro 1 , Dennis Niehoff 1, Sumit Mohanty 1,* and Sarthak Misra 1,2 2 Surgical Robotics Laboratory, Department of Biomedical Engineering, University Medical Centre Gronin University of Groningen, 9713 AV Groningen, The Netherlands * Correspondence: s.mohanty@utwente.nl; Tel.: +31-534-897-596 www.mdpi.com/journal/micromachines 1. Introduction Considered a figment of imagination until a few decades ago, microbotic surgeries are steadily approaching clinically-relevant scenarios [1,2]. Over the past years technological advances in the fields of nanofabrication and microrobotic control have allowed to miniaturize and control robots as small as a few microns [3,4]. Consequently, microrobotics has been drawing significant attention for minimally invasive surgeries, such as biopsies, cytoreductions and endarterectomies, as well as cardiac and ophthalmic surgeries [5,6]. Among these interventions, microrobotic targeted drug delivery is arguably the closest to the reach of the current technology [7,8]. In point of fact, techniques have been presented to miniaturize microrobots well beyond the constraints of the main human vascular vessels [9]. Moreover, microrobots have been demonstrated to be capable of navigation in three dimensional (3D) and dynamic environs [10,11]. Therefore, drug-coated microrobots, capable of navigating in unpredictable environments, would be able to perform targeted drug delivery inside the human body. Not only would this microrobotic approach supersede the current approach in terms of effectiveness of drug delivery, but also under numerous other aspects, such as reduction of side effects as well as improved regulation of drug-intake [8]. Micromachines 2019, 10, 504; doi:10.3390/mi10080504 www.mdpi.com/journal/micromachines www.mdpi.com/journal/micromachines Micromachines 2019, 10, 504 2 of 11 However, most microrobotic research is still mainly confined to controlled environments [9–11]. In fact, the untethered and biocompatiblity requirements pose a main challenge to the development of sensors and actuators for in-vivo microrobotic testing. Particularly, a clinically-relevant microrobotic system has to possess biocompatible propulsion mechanisms (1), imaging systems (2), and it also has to be able to execute tasks beyond those required for navigation (3). The development of biocompatible microrobotic propulsion mechanisms (1) is challenging due to scale requirements. Customary battery-powered actuators cannot be used, as the current technology does not allow sufficient miniaturization of on-board power sources. Consequently, researchers have investigated the use of chemical, thermal, acoustic, and electromagnetic microactuators capable of wirelessly harnessing power stored within the material of the microrobot or its surroundings [12–14]. Nonetheless, due to power attenuation and lack of biocompatibility, a considerable number of these actuators is either incompatible or only compatible with a limited number of clinical procedures [9]. Also imaging techniques present a significant hurdle with regard to clinical compatibility (2). Historically, optical images have been used to sense the state of microrobots. 2. Materials and Methods This section presents the custom setup and the techniques used to perform clinically-relevant targeted delivery of a drug-model. From a hardware perspective an electromagnetic testbed, a generator of high-frequency magnetic fields, a clinical ultrasound machine, and an anatomically accurate phantom are used (Figure 1) [16]. Moreover, we develop software algorithms for the control of the electromagnetic testbed, as well as for control and tracking of the microrobots. 1. Introduction Yet optical cameras are not suitable for minimally invasive clinical interventions, as they would require incisions for their insertion. However, biocompatible alternatives often suffer from major drawbacks, such as limited bandwidth, low resolution, artifacts or long-term toxicity. Therefore, robust tracking procedures have to be developed for pose reconstruction. Finally, to perform minimally invasive surgery, microrobots generally have to be capable to execute actions beyond those required for navigation (3). Case in point, active drug-delivery microrobots need to perform chemical release of substances. Clearly, also the actuators for such actions have to be untethered and biocompatible, as they must not unintentionally alter physiological parameters, such as pH, temperature, or intravascular pressure. In this study, we present a fully biocompatible approach for microrobotic targeted drug delivery that addresses these challenges. Such approach exploits the physical properties of the used microrobot to develop a contactless actuation and sensing system that would only affect the targeted tissue. In particular, medical ultrasound imaging is used for position sensing, while quasi-static and high-frequency magnetic fields are used for navigation and active drug release, respectively. This approach is then validated using an anatomically accurate phantom of the human vascular network. Overall, this proof-of-concept work presents the first closed-loop active targeted drug-model delivery using a thermoresponsive coating and a fully biocompatible microrobotic system. Moreover, our results show reductions of several orders of magnitude in completion time of the task with respect to previous literature using similar technology [15]. 2.1. Electromagnetic Setup The electromagnetic setup used for motion control of the microrobots consists of nine metal-core electromagnets and a camera attached to a microscope (Figure 2). While the setup was originally presented in our previous work, several modifications have been done to address for this study [10]. Most notably, a coil (Ultraflex Power Technologies, New York, USA) for the generation of high-frequency magnetic fields has been added to the setup (Figure 1). Additionally, a linear stage (MISUMI, Tokyo, Japan) is now used to move the ultrasound probe (SIEMENS AG, Erlangen, Germany) in directions orthogonal to the imaging plane (Figure 2). The addition of this linear stage allows to use 3 of 11 Micromachines 2019, 10, 504 a traditional 2D medical ultrasound for 3D tracking of the microrobots. Conversely, the additional coil is used to generate sinusoidal electromagnetic fields used to trigger the release of the drug-model. y x z H M 60 20 Figure 1. (Left) A close-up image of the used setup depicting the ultrasound probe, as well as the coils used to generate high frequency  H  and quasi-static magnetic fields  M  . (Central inset) One of the used microrobots after 15 s of induction heating (18 mT at 126 kHz), captured using a cooled-detector thermal camera with 12 mm extension ring (FLIR Systems, Wilsonville, OR, USA). Scalebar in degree Celsius. (Right) Illustration of (sectioned) proposed microrobots navigating a blood vessel to perform targeted drug delivery. y x z H M 60 20 Figure 1. (Left) A close-up image of the used setup depicting the ultrasound probe, as well as the coils used to generate high frequency  H  and quasi-static magnetic fields  M  . (Central inset) One of the used microrobots after 15 s of induction heating (18 mT at 126 kHz), captured using a cooled-detector thermal camera with 12 mm extension ring (FLIR Systems, Wilsonville, OR, USA). Scalebar in degree Celsius. (Right) Illustration of (sectioned) proposed microrobots navigating a blood vessel to perform targeted drug delivery. M M U H x y z M M U H x y z 1 cm y x 1 cm x z Figure 2. The electromagnetic setup. The nine metal-core electromagnets  M  are capable of generating quasi-static fields of up to 10 mT while allowing access to objects smaller than a 160 mm sphere. 2.1. Electromagnetic Setup A linear stage is used to move the ultrasound probe  U  along the gravitational axis (z). Finally, the liquid-cooled high frequency coil  H  can be seen in the right end of the image. (Top-right inset) A representative B-mode ultrasound image. The position of the microrobot is marked by a green square. Due to diffraction, artifacts, and noise the footprint of microrobot in the ultrasound image is significantly larger than its real size. (Bottom-right inset) A coated microrobot navigating the phantom of vascular vessels (outlined by the colored dashed lines). U Figure 2. The electromagnetic setup. The nine metal-core electromagnets  M  are capable of generating quasi-static fields of up to 10 mT while allowing access to objects smaller than a 160 mm sphere. A linear stage is used to move the ultrasound probe  U  along the gravitational axis (z). Finally, the liquid-cooled high frequency coil  H  can be seen in the right end of the image. (Top-right inset) A representative B-mode ultrasound image. The position of the microrobot is marked by a green square. Due to diffraction, artifacts, and noise the footprint of microrobot in the ultrasound image is significantly larger than its real size. (Bottom-right inset) A coated microrobot navigating the phantom of vascular vessels (outlined by the colored dashed lines).   Finally, the liquid-cooled high frequency coil  H  can be seen in the right end of the image. (Top-right inset) A representative B-mode ultrasound image. The position of the microrobot is marked by a green square. Due to diffraction, artifacts, and noise the footprint of microrobot in the ultrasound image is significantly larger than its real size. (Bottom-right inset) A coated microrobot navigating the phantom of vascular vessels (outlined by the colored dashed lines). 4 of 11 Micromachines 2019, 10, 504 2.2. Ultrasound Tracking High-frequency acoustic waves are used for biocompatible tracking. For this purpose, an 18 MHz transducer is connected to a 2D medical ultrasound machine (SIEMENS AG, Erlangen, Germany). As the used transducer only allows two-dimensional imaging, additional procedures are required to obtain the 3D position of the microrobot. Specifically, the additional component has to be detected by allowing the ultrasound probe to move orthogonally to the image plane. An intuitive approach would be to use this motion to follow the microrobot in the workspace, maintaining it always in the image plane. However, due to the presence of noise and artifacts, changes in the imaged size of the robot do not reflect the actual footprint of the scanned section. Consequently, it is challenging to compute the gradient of the position of the microrobot, which is required to follow its movements with the ultrasound probe. Alternatively, we use a sonar-inspired approach. In this approach the ultrasound transducer is swept along the height of the workspace, while a Region Of Interest (ROI) in the surrounding of the estimated position of the microrobot is scanned for 2D detection (Figures 3 and 4). The joint variable of the stage in the point of detection, can then be used to triangulate the position of the microrobot. While this approach results in a reduced bandwidth of the tracking algorithm, it also provides a significant gain in robustness with respect to a gradient-based approach. Overall, this approach grants extremely robust tracking at a frequency of 2 Hz. Figure 3. Schematic depicting the approach to determine the out-of-plane component (z) in the tracking procedure. The ultrasound transducer continuously sweeps the workspace at a frequency of 1 Hz, hence providing two positions per second. The in-plane (x and y) components of the position of the microrobot are tracked using the procedure illustrated in Figure 4. The graph shows the dependence of the tracked blob size (blue line) on the position of the transducer. This dependence is used to triangulate the position of the microrobot using the point of maximum size (marked by the dashed red line). Figure 3. Schematic depicting the approach to determine the out-of-plane component (z) in the tracking procedure. The ultrasound transducer continuously sweeps the workspace at a frequency of 1 Hz, hence providing two positions per second. 2.2. Ultrasound Tracking The in-plane (x and y) components of the position of the microrobot are tracked using the procedure illustrated in Figure 4. The graph shows the dependence of the tracked blob size (blue line) on the position of the transducer. This dependence is used to triangulate the position of the microrobot using the point of maximum size (marked by the dashed red line). 5 of 11 Micromachines 2019, 10, 504 User-Provided Reference + +- > PID Reference Filter Hz Db Feed Forward Sampled-data Observer Disturbance Estimator Buoyancy Gravity Force to Current Map Electromagnetic Actuation Original Image Binary Filtering Position Background Subtraction ROI Tracking Navigation Inside Phantom Figure 4. Schematic of the control loop. The user provides the position reference. This is preprocessed by a fourth order filter that removes frequency components above the bandwidth of the controller and ensures continuous derivatives. This prevents the filtered reference from increasing with a dynamic that is faster than the maximum one of the controller The filtered reference is then provided to a Proportional, Integral and Derivative (PID) controller. This controller designed to minimize disturbances with frequencies higher than a decade below that of the tracker [17]. A feedforward component is added to improve the control performance. As the low-level controllers feed the currents determined by the force-to-current map, the microrobot moves. This motion is detected by the ultrasound tracking algorithm using the procedure shown in the image (combined with the approach of Figure 3). Such tracking procedure begins using a Gaussian mixture-based segmentation algorithm for background subtraction [18]. A Region Of Interest (ROI) around the estimated position of the microrobot is then selected and binarized using a variable-threshold. A dilation morphological filter is then applied to the image. Finally, the center of the largest blob is selected as the position of the microrobot. The computed position is then provided to a sampled-data observer, which provides the controller with intersample state estimations [19]. User-Provided Reference + +- > PID Reference Filter Hz Db Feed Forward Sampled-data Observer Disturbance Estimator Buoyancy Gravity Force to Current Map Electromagnetic Actuation Original Image Binary Filtering Position Background Subtraction ROI Tracking Navigation Inside Phantom Reference Filter Figure 4. Schematic of the control loop. The user provides the position reference. This is preprocessed by a fourth order filter that removes frequency components above the bandwidth of the controller and ensures continuous derivatives. 2.3. Microrobot Selection The microrobots used in the study are selected in order to satisfy a set of conditions. First, the microrobots must have a continuous and smooth surface to guarantee uniform drug release, as well as to minimize contact pressures on the vessels in case of collisions. Second, the microrobots size must be small enough to grant access to the major body vessels, while remaining sufficiently bigger than the ultrasound resolution (100 µm) to minimize artifacts. Third, the robot has to be able to withstand unaltered the temperatures required for drug release. Finally, we want the magnetic dipole moment of the microrobot to be as strong as possible. This allows to control the robot with weak magnetic fields and gradients, as the ones generated by distant electromagnets, effectively enlarging the accessible workspace. Addressing all these requirements we select Neodymium Iron Boron (NdFeB) microspheres with diameter of 800 µm for our study. In particular, we use N45 grade NdFeB, which has a Curie temperature of 80◦C and offers a remnant magnetization of 1.35 T. Moreover, NdFeB has a conductivity of 6.7×106 S/m which renders it particularly sensitive to induction heating. 2.2. Ultrasound Tracking This prevents the filtered reference from increasing with a dynamic that is faster than the maximum one of the controller The filtered reference is then provided to a Proportional, Integral and Derivative (PID) controller. This controller designed to minimize disturbances with frequencies higher than a decade below that of the tracker [17]. A feedforward component is added to improve the control performance. As the low-level controllers feed the currents determined by the force-to-current map, the microrobot moves. This motion is detected by the ultrasound tracking algorithm using the procedure shown in the image (combined with the approach of Figure 3). Such tracking procedure begins using a Gaussian mixture-based segmentation algorithm for background subtraction [18]. A Region Of Interest (ROI) around the estimated position of the microrobot is then selected and binarized using a variable-threshold. A dilation morphological filter is then applied to the image. Finally, the center of the largest blob is selected as the position of the microrobot. The computed position is then provided to a sampled-data observer, which provides the controller with intersample state estimations [19]. 2.5. Motion Modeling and Control After developing a testbed for drug release, we look at closed-loop motion control. For this purpose, we model the microrobots according to the following state space model: " ˙pi ¨pi # = " 0 1 0 −ρCDA 2M # " pi ˙pi # + " 0 −1 M # Fem,i + " 0 −1 M # di, (1) (1) where ρ ∈R is the density of the medium, and CD ∈R, A ∈R and M ∈R are the drag coefficient, cross sectional area, and mass of the sphere, respectively (Table 1) [10]. In turn, d ∈R3 collects all the modeled components that are not influenced by the state of the system or by the control inputs. Moreover, pi ∈R, Fem,i ∈R, and di ∈R are the i-th component of the position (p ∈R3), electromagnetic force (Fem ∈R3), and d, respectively. These are defined as follows: p = h x y z iT , (2) Fem = ∇(B · m), (3) d = ∆Fd + g(M −Vρ), (4) (2) (2) (3) (4) (4) where B ∈R3 is the magnetic flux density, m ∈R3 is the magnetic dipole moment of the microrobot, and ∇is the gradient operator. Further, ∆Fd ∈R3 represent the inaccuracies in the modeling of the drag forces, g ∈R3 is the acceleration due to gravity, and V ∈R is the volume of the microrobot. where B ∈R3 is the magnetic flux density, m ∈R3 is the magnetic dipole moment of the microrobot, and ∇is the gradient operator. Further, ∆Fd ∈R3 represent the inaccuracies in the modeling of the drag forces, g ∈R3 is the acceleration due to gravity, and V ∈R is the volume of the microrobot. Table 1. Values of the variables in the model of the microsphere. Table 1. Values of the variables in the model of the microsphere. Variable Value Unit ρm 9700 [kg/m3] CD 0.5 Pure number rmr 6 × 10−4 [m] Amr 1.131 × 10−6 [m2] Vmr 9.0478 × 10−10 [m3] M 4.1228 × 10−6 [kg] Based on such model, a closed-loop controller is designed to regulate the motion of the microrobots, using the quasi-static electromagnetic fields generated by the testbed (Figure 4). The reference of the controller—provided by the user—is filtered to guarantee continuous derivatives and eliminate components outside of the control bandwidth. 2.4. Induction Heating In point of fact, microrobots are heated using high-frequency magnetic fields, exploiting the phenomenon commonly known as induction. Therefore, the heat generated in the microrobot is a 6 of 11 Micromachines 2019, 10, 504 result of both hysteresis losses and eddy currents [20]. However, due to the low permeability—at the magnetic fields reported in this study—of the pre-magnetized NdFeB microrobots, the heat generated due to hysteresis losses is minimal with respect to eddy losses. Consequently, a custom-coil is designed to maximize these effects (Ultraflex Power Technologies, New York, USA). The resulting RLC circuit is capable of locking at two frequencies (126 kHz and 228 kHz) producing a field of 18 mT in amplitude. Depending on the magnetic energy stored in the electromagnetic field induced in the material, a power of up to 1.7 kW is required to generate such high-frequency fields. Part of such power is dissipated on the microrobot, while most of the remaining power is dissipated on the coil. In order to prevent overheating, such coil is designed to be hollow. This allows to run water inside the coil for cooling purposes (6.8 L/min at 3.4 bar). Finally, it is interesting to notice that the human body is not affected by these magnetic fields as it does not contain sufficient amounts of conductive or hard magnetic materials [16]. 2.6. Vascular Phantom and Drug-Model Such closed-loop motion control is performed inside an anatomically accurate model of the human vessels [22]. This phantom is fabricated using polydimethylsiloxane (PDMS), due to its tissue-mimicking and optical properties, which allow us to compare the results of ultrasound tracking with those of offline optical tracking [23]. The 20 mm ×20 mm ×20 mm phantom represents a human vessel forking into three different channels. In order to ensure the anatomical accuracy, we construct the phantom so that the sum of the cross-sectional area of the resulting vessels (10 mm2 each) is equal to that of the original vessel (30 mm2). Finally, the phantom is filled with liquid polymerized siloxane to ensure acoustic transparency to ultrasound waves. It is worth noting that, the used polymerized siloxane has a kinematic viscosity of 50 mm2/s, about ten times that of blood [24]. This means that the experiments are conducted in a medium with lower Reynold number than blood. Therefore, interventions in blood would have lower CD (1) and drag forces than those reported in this study. To further enhance the clinical relevance of the study, the microrobots are coated with a lipid-based thermoresponsive layer. This layer is embedded with a Sudan red dye that allow to identify the behavior of the coating. Moreover, the coating is designed to melt at 39 ◦C, to minimize the amount of heat necessary for drug-release in the human body. 2.5. Motion Modeling and Control The filtered reference is then processed by feed-forward and feed-back controllers.To avoid instabilities and undetermined behavior, the Proportional Integral Derivative (PID) feed-back controller is designed to minimize disturbances 7 of 11 Micromachines 2019, 10, 504 with frequencies higher than a decade below that of the tracker [17]. A feed-forward component is added to improve the control performance with an additional control action (uFF) with frequencies higher than a decade below that of the tracker [17]. A feed-forward component is added to improve the control performance with an additional control action (uFF) uFF = h xd ˙xd ¨xd i   0 −ρmCDA 2 −M  . (5) (5) Finally, the controller outputs forces that are mapped into currents at the electromagnets using a force-current map. As the setup is overactuated, we select a map that aims at minimizing the Frobenius norm of the third-order tensor collecting the Hessian matrices of each component of the field [10,21]. This choice minimizes the spacial variation of the electromagnetic gradient, and consequently, of the electromantic force (3). Overall, such map minimizes the sensitivity of the system to tracking errors. 2.6. Vascular Phantom and Drug-Model 3. Experimental Evaluation In order to validate the developed setup and techniques, we demonstrate contactless delivery of a drug-model. The intention is to mimic a targeted drug delivery application, in which a microrobot is released by a catheter, steered towards the region of interest where it delivers a drug, and finally returns to the catheter for recollection. Consequently, in the presented experiments, an 800 µm sphere—starting at the end of a channel in the vascular phantom—is steered towards the target area (the end of another channel). As the target is reached, the heating system is activated, triggering the release of the drug-model. After the delivery, the microrobot returns to the starting point where the experiment terminates. In order to guarantee clinical compatibility, the microrobot is tracked using exclusively ultrasound imaging. However, for reasons of comparison and data analysis the procedure is also recorded with a 2D color camera (FLIR Systems, Wilsonville, OR, USA) attached to a microscope (Qioptiq, St Asaph, United Kingdom). Please, refer to the accompanying video for the visualization of the experiment in the Supplementary Materials. The experiment is repeated ten times (Figure 5). The microrobots averagely complete the trials in 212 s, moving with an average velocity of 0.3 mm/s (Figure 6). Moreover, about 20 s are required for the heating process (Figure 1), which is activated as the microrobot is within 10 mm of the designated target. It is worth mentioning that, as the quasi-static and high-frequency magnetic fields can be superimposed without interference, the heating process does not affect the overall completion time. Moreover, this linear superposition, presents other significant advantages; case in point, uninterrupted closed-loop control would be fundamental in the presence of blood flow or dynamic environments. 8 of 11 Micromachines 2019, 10, 504 It can also be noticed that the root mean square value of the positioning and tracking error are 40% and 52% higher for the component normal to the imaging plane (z), respectively. This increased error is mainly caused by the diffraction of the ultrasonic wave around the edges of the microrobot. In point of fact, as the diameter of the microrobot is less than ten times the wavelength of the ultrasound, the artifacts resulting from diffraction are comparable in size to the footprint of the microrobot. 3. Experimental Evaluation This phenomenon renders it challenging to determine the exact center of the sphere, as the pixel-count gradient is minimum in the neighborhood of such center (Figure 3). These results, even if somewhat constrained to the tested setup and presenting an additional dimension, are comparable to previous two-dimensional studies regarding motion of microrobots with ultrasound feedback [25]. Figure 5. Representative timelapse of the experiments. In the first image, yellow, green, and red dashed lines mark the outline of the channels in the phantom. A yellow dashed line is also used to highlight the position of the microrobot in the frames. The blue arrows approximate the future trajectory of the microrobot. The microrobot starts from the end of the yellow-outlined channel, and navigates to the end of the green one, where it releases the drug-model. Finally, the microrobot returns to the starting point. Moreover, the changes in position of the ultrasound transducer, continuously sweeping through the workspace, can be noticed in the background. Please, refer to the accompanying video for the visualization of the experiment in the Supplementary Materials. Figure 5. Representative timelapse of the experiments. In the first image, yellow, green, and red dashed lines mark the outline of the channels in the phantom. A yellow dashed line is also used to highlight the position of the microrobot in the frames. The blue arrows approximate the future trajectory of the microrobot. The microrobot starts from the end of the yellow-outlined channel, and navigates to the end of the green one, where it releases the drug-model. Finally, the microrobot returns to the starting point. Moreover, the changes in position of the ultrasound transducer, continuously sweeping through the workspace, can be noticed in the background. Please, refer to the accompanying video for the visualization of the experiment in the Supplementary Materials. 9 of 11 Micromachines 2019, 10, 504 9 of 11 Figure 6. (Left) Representative example of the trajectory followed by a microrobot through the experiments. The blue curve shows the ultrasound tracking results. The red curve represent the same trajectory as computed offline by an optical tracker [10]. (Right) Histogram of the positioning and tracking errors over the performed ten trials. The positioning error (blue) is defined as the divergence between the filtered reference and the state as tracked by the ultrasound tracking system; i.e., the error as computed in the control loop. 3. Experimental Evaluation Conversely, the tracking error (yellow) is defined as the difference in tracked position between the ultrasound and optical (computed offline) tracker. Consequently, we can only compute the tracking error for x and z components. However, for reasons of symmetry, we expect the y component, which cannot be analyzed optically, to have similar tracking error to that of the x component. Figure 6. (Left) Representative example of the trajectory followed by a microrobot through the experiments. The blue curve shows the ultrasound tracking results. The red curve represent the same trajectory as computed offline by an optical tracker [10]. (Right) Histogram of the positioning and tracking errors over the performed ten trials. The positioning error (blue) is defined as the divergence between the filtered reference and the state as tracked by the ultrasound tracking system; i.e., the error as computed in the control loop. Conversely, the tracking error (yellow) is defined as the difference in tracked position between the ultrasound and optical (computed offline) tracker. Consequently, we can only compute the tracking error for x and z components. However, for reasons of symmetry, we expect the y component, which cannot be analyzed optically, to have similar tracking error to that of the x component. Figure 6. (Left) Representative example of the trajectory followed by a micr 4. Conclusions Video S1: the visualization of the experiment. Author Contributions: Conceptualization: F.O. and S.M. (Sarthak Misra); methodology: F.O., D.N. and S.M. (Sumit Mohanty); software: F.O. and D.N.; validation: F.O.; formal analysis, F.O.; investigation, F.O., D.N. and S.M. (Sumit Mohanty); resources, F.O., D.N. and S.M. (Sumit Mohanty); data curation, F.O.; writing—original draft preparation: F.O.; writing—review and editing: F.O., S.M. (Sumit Mohanty), and S.M. (Sarthak Misra); visualization: F.O. and D.N.; supervision: S.M. (Sarthak Misra); project administration: S.M. (Sarthak Misra); funding acquisition: S.M. (Sarthak Misra). Funding: This research has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation programme (Grant Agreement #638428—project ROBOTAR: Robot-Assisted Flexible Needle Steering for Targeted Delivery of Magnetic Agents). Conflicts of Interest: The authors declare no conflict of interest. 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Ghosh, A.; Yoon, C.; Ongaro, F.; Scheggi, S.; Selaru, F.M.; Misra, S.; Gracias, D.H. Stimuli-Responsive Soft Untethered Grippers for Drug Delivery and Robotic Surgery. Front. Mech. Eng. 2017, 3, 7. [CrossRef] 6. Ghosh, A.; Yoon, C.; Ongaro, F.; Scheggi, S.; Selaru, F.M.; Misra, S.; Gracias, D.H. Stimuli-Responsive Soft Untethered Grippers for Drug Delivery and Robotic Surgery. Front. Mech. Eng. 2017, 3, 7. [CrossRef] 7. Chen, X.Z.; Hoop, M.; Shamsudhin, N.; Huang, T.; Özkale, B.; Li, Q.; Siringil, E.; Mushtaq, F.; Di Tizio, L.; Nelson, B.J.; et al. Hybrid magnetoelectric nanowires for nanorobotic applications: fabrication, magnetoelectric coupling, and magnetically assisted in vitro targeted drug delivery. Adv. Mater. 2017, 29, 1605458. [CrossRef] [PubMed] 8. Jang, D.; Jeong, J.; Song, H.; Chung, S.K. 4. Conclusions This proof-of-concept work presents a biocompatible system for targeted delivery of a drug-model. In order to render the approach fully biocompatible, magnetic fields are used for motion control, a medical ultrasound system is used for imaging, and induction heating is used for active drug release. The presented system is validated in a 3D phantom of human vascular network. In this validation, we simulate a scenario in which the microrobot has been released in a vessel. We navigate such microrobot toward the targeted area, where we trigger the active drug-model release. Finally, we return the robot to the drop-off point, after the release is complete. Compared to previous approaches using induction heating—requiring up to an hour for drug-model release—this approach has an average completion time of 212 s for a release within half a millimeter of the targeted point. In spite of this, a comparison with previous literature using optical feedback shows the motion control performance is clearly constrained by the limited bandwidth of the ultrasound feedback [10,26,27]. Ultrasound scanners with higher refresh-rate that allow higher scanning frequencies could be used to address this issue. Overall, the promising results of this approach, as well as its fast and fully biocompatible nature, render it interesting for further investigation, especially in ex-vivo and in-vivo environments. Future work will address the limitations of this work to develop a path toward clinical application. For this purpose, we will investigate hardware and smart materials solutions to improve the ultrasound scanning frequency. This will allow to extend the workspace of the quasi-static and high-frequency electromagnetic systems, permitting interventions in the larger parts of the body. Navigation in channels of other size and in the presence of flow will also be investigated. Further, we will test hardware solutions that allow to improve the scanning frequency, therefore increasing the workspace and control bandwidth. Such improvements in hardware and methodology could enable an extensive quantitative analysis with increased number of trials, thereby providing a statistical means to evaluate a myriad of clinically relevant constructs. Finally, we will analyze the use of 3D ultrasound transducers for targeted drug delivery using both individual microrobots as well as swarms. Computer vision and fusion algorithms that allow to address obstructions, such as bones and inhomogeneous tissues, will also be investigated. 10 of 11 10 of 11 Micromachines 2019, 10, 504 Supplementary Materials: The following is available online at http://www.mdpi.com/2072-666X/10/8/504/s1. References Targeted drug delivery technology using untethered microrobots: A review. J. Micromech. Microeng. 2019, 29. [CrossRef] 9. Ongaro, F.; Scheggi, S.; Ghosh, A.; Denasi, A.; Gracias, D.H.; Misra, S. Design, characterization and control of thermally-responsive and magnetically-actuated micro-grippers at the air-water interface. PLoS ONE 2017, 12, e0187441. [CrossRef] 10. Ongaro, F.; Pane, S.; Scheggi, S.; Misra, S. Design of an Electromagnetic Setup for Independent Three-Dimensional Control of Pairs of Identical and Nonidentical Microrobots. IEEE Trans. Robot. 2019, 35, 174–183. [CrossRef] 11. Ongaro, F.; Scheggi, S.; Yoon, C.; Van den Brink, F.; Oh, S.H.; Gracias, D.H.; Misra, S. Autonomous planning and control of soft untethered grippers in unstructured environments. J. Micro-bio Robot. 2017, 12, 45–52. [CrossRef] 12. Chowdhury, S.; Švec, P.; Wang, C.; Seale, K.T.; Wikswo, J.P.; Losert, W.; Gupta, S.K. Automated cell transport in optical tweezers-assisted microfluidic chambers. IEEE Trans. Autom. Sci. Eng. 2013, 10, 980–989. [CrossRef] 13. Guo, F.; Mao, Z.; Chen, Y.; Xie, Z.; Lata, J.P.; Li, P.; Ren, L.; Liu, J.; Yang, J.; Dao, M.; et al. Three-dimensional manipulation of single cells using surface acoustic waves. Proc. Natl. Acad. Sci. USA 2016, 113, 1522–1527. [CrossRef] [PubMed] 11 of 11 11 of 11 Micromachines 2019, 10, 504 14. Ongaro, F.; Jin, Q.; Siciliani de Cumis, U.; Ghosh, A.; Denasi, A.; Gracias, D.H.; Misra, S. Force characterization and analysis of thin film actuators for untethered microdevices. AIP Adv. 2019, 9, 055011. [CrossRef] 15. Liu, M.; Pan, L.; Piao, H.; Sun, H.; Huang, X.; Peng, C.; Liu, Y. Magnetically actuated wormlike nano for controlled cargo release. Acs Appl. Mater. Interfaces 2015, 7, 26017–26021. [CrossRef] [PubMed] 16. Litvak, E.; Foster, K.; Repacholi, M. Health and safety implications of exposure to electromagnetic fields in the frequency range 300 Hz to 10 MHz. Bioelectromagn. J. Bioelectromagn. Soc. Soc. Phys. Regul. Biol. Med. Eur. Bioelectromagn. Assoc. 2002, 23, 68–82. [CrossRef] [PubMed] g 17. Zhuang, M.; Atherton, D. Automatic tuning of optimum PID controllers. Iee Proc. Control. Theory Appl. 1993, 140, 216–224. [CrossRef] 18. KaewTraKulPong, P.; Bowden, R. An improved adaptive background mixture model for real-time tracking with shadow detection. In Video-Based Surveillance Systems; Springer: Boston, MA, USA, 2002; pp. 135–144. 19. Karafyllis, I.; Kravaris, C. From continuous-time design to sampled-data design of observers. IEEE Trans. Autom. Control. 2009, 54, 2169–2174. [CrossRef] 20. Lee, J.H.; Jang, J.T.; Choi, J.S.; Moon, S.H.; Noh, S.H.; Kim, J.W.; Kim, J.G.; Kim, I.S.; Park, K.I.; Cheon, J. References Exchange-coupled magnetic nanoparticles for efficient heat induction. Nat. Nanotechnol. 2011, 6, 418. [CrossRef] [PubMed] 21. Ongaro, F.; Heunis, C.M.; Misra, S. Precise Model-Free Spline-Based Approach for Magnetic Field Mapping. IEEE Magn. Lett. 2018, 10, 1–5. [CrossRef] 22. Stergiopulos, N.; Young, D.; Rogge, T. Computer simulation of arterial flow with applications to arterial and aortic stenoses. J. Biomech. 1992, 25, 1477–1488. [CrossRef] 23. Avigo, C.; Di Lascio, N.; Armanetti, P.; Kusmic, C.; Cavigli, L.; Ratto, F.; Meucci, S.; Masciullo, C.; Cecchini, M.; Pini, R.; et al. Organosilicon phantom for photoacoustic imaging. J. Biomed. Opt. 2015, 20, 046008. [CrossRef] 24. Baskurt, O.; Boynard, M.; Cokelet, G.; Connes, P.; Cooke, B.M.; Forconi, S.; Liao, F.; Hardeman, M.; Jung, F.; Meiselman, H.; et al. New guidelines for hemorheological laboratory techniques. Clin. Hemorheol. Microcirc. 2009, 42, 75–97. [PubMed] 25. Scheggi, S.; Chandrasekar, K.K.T.; Yoon, C.; Sawaryn, B.; van de Steeg, G.; Gracias, D.H.; Misra, S. Magnetic Motion Control and Planning of Untethered Soft Grippers using Ultrasound Image Feedback. In Proceedings of the IEEE International Conference on Robotics and Automation (ICRA), Singapore, 29 May–3 June 2017. 26. Ongaro, F.; Pacchierotti, C.; Yoon, C.; Prattichizzo, D.; Gracias, D.H.; Misra, S. Evaluation of an electromagnetic system with haptic feedback for control of untethered, soft grippers affected by disturbances. In Proceedings of the IEEE International Conference on Biomedical Robotics and Biomechatronics (BioRob), Enschede, The Netherlands, 26–29 June 2016; pp. 900–905. 27. Ongaro, F.; Yoon, C.; van den Brink, F.; Abayazid, M.; Oh, S.H.; Gracias, D.H.; Misra, S. Control of untethered soft grippers for pick-and-place tasks. In Proceedings of the IEEE International Conference on Biomedical Robotics and Biomechatronics (BioRob), Enschede, The Netherlands, 26–29 June 2016; pp. 299–304. c⃝2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Outcomes of kidney transplant recipients admitted to the intensive care unit: a retrospective study of 200 patients
BMC anesthesiology
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Open Access Abstract Background: Risk of over-immunosuppression or immunization may mitigate the overall and long-term renal outcomes of kidney transplant recipients (KTR) admitted to the ICU in the modern era but remain poorly described. Thus, there is an unmet need to better characterize the survival of KTR admitted to the ICU, but also the renal and immunological outcomes of survivors. Methods: Retrospective observational study that included 200 KTR admitted between 2010 and 2016 to the ICU of a teaching hospital (median age 61 years [IQR 50.7–68]; time from transplantation 41 months [IQR 5–119]). Survival curves were compared using the Log-rank test. Results: Mortality rates following admission to the ICU was low (26.5% at month-6), mainly related to early mortality (20% in-hospital), and predicted by the severity of the acute condition (SAPS2 score) but also by Epstein Barr Virus proliferation in the weeks preceding the admission to the ICU. Acute kidney injury (AKI) was highly prevalent (85.1%). Progression toward chronic kidney disease (CKD) was observed in 45.1% of survivors. 15.1% of survivors developed new anti-HLA antibodies (donor-specific antibodies 9.2% of cases) that may impact the long- term renal transplantation function. Conclusions: Notwithstanding the potential biases related to the retrospective and monocentric nature of this study, our findings obtained in a large cohort of KTR suggest that survival of KTR admitted in ICU is good but in- ICU management of these patients may alter both survival and AKI to CKD transition, as well as HLA immunization. Further interventional studies, including systematic characterization of the Epstein Barr virus proliferation at the admission (i.e., a potential surrogate marker of an underlying immune paralysis and frailty) will need to address the optimal management of immunosuppressive regimen in ICU to improve survival but also renal and immunological outcomes. Keywords: Renal transplantation, HLA immunization, Intensive care unit, Outcomes (ARF) and septic shock, followed by cardiovascular compli- cations, acute kidney injury (AKI), drug-related complica- tions and neoplasia [4, 10]. Outcomes of kidney transplant recipients admitted to the intensive care unit: a retrospective study of 200 patients Damien Guinault1, Arnaud Del Bello1, Laurence Lavayssiere1, Marie-Béatrice Nogier1, Olivier Cointault1, Nicolas Congy2, Laure Esposito1, Anne-Laure Hebral1, Olivier Roques1, Nassim Kamar1,3,4 and Stanislas Faguer1,3,5* © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Guinault et al. BMC Anesthesiology (2019) 19:130 https://doi.org/10.1186/s12871-019-0800-0 Guinault et al. BMC Anesthesiology (2019) 19:130 https://doi.org/10.1186/s12871-019-0800-0 Background T (http://creativecommons.org/publicdomain/zero/1.0/) applies to the d * Correspondence: stanislas.faguer@inserm.fr 1Département de Néphrologie et Transplantation d’organes, Unité de Réanimation, Hôpital Rangueil, Centre Hospitalier Universitaire de Toulouse, 1, avenue Jean Poulhes, 31059 Toulouse, France 3Université Paul Sabatier, Toulouse III, F-31000 Toulouse, France Full list of author information is available at the end of the article * Correspondence: stanislas.faguer@inserm.fr 1Département de Néphrologie et Transplantation d’organes, Unité de Réanimation, Hôpital Rangueil, Centre Hospitalier Universitaire de Toulouse, 1, avenue Jean Poulhes, 31059 Toulouse, France 3Université Paul Sabatier, Toulouse III, F-31000 Toulouse, France Full list of author information is available at the end of the article * Correspondence: stanislas.faguer@inserm.fr 1Département de Néphrologie et Transplantation d’organes, Unité de Réanimation, Hôpital Rangueil, Centre Hospitalier Universitaire de Toulouse, 1, avenue Jean Poulhes, 31059 Toulouse, France 3Université Paul Sabatier, Toulouse III, F-31000 Toulouse, France Full list of author information is available at the end of the article Background About 10% of kidney transplant recipients (KTR) experi- ence a life-threatening disease requiring admission in an in- tensive care unit (ICU) [1–7]. Most admissions occur more than 6 months after the renal transplantation [3–5, 8, 9]. The main causes of admission were acute respiratory failure In-hospital mortality after admission to the ICU is mostly related to the cause of admission and the number of organ failures at presentation, whereas the character- istics of the renal transplantation are not associated with the outcomes [3, 4, 6]. Whether these findings hold true in the modern era characterized by an increase of trans- plantations at high risk of surgical and immunological p the outcomes [3, 4, 6]. Whether these findings hold in the modern era characterized by an increase of tr plantations at high risk of surgical and immunolo © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: stanislas.faguer@inserm.fr 1Département de Néphrologie et Transplantation d’organes, Unité de Réanimation, Hôpital Rangueil, Centre Hospitalier Universitaire de Toulouse, 1, avenue Jean Poulhes, 31059 Toulouse, France 3Université Paul Sabatier, Toulouse III, F-31000 Toulouse, France Full list of author information is available at the end of the article the outcomes [ in the modern plantations at © The Author(s). 2019 Open Access This article is distributed under t International License (http://creativecommons.org/licenses/by/4.0/), w reproduction in any medium, provided you give appropriate credit to the Creative Commons license, and indicate if changes were made. Patients and methods In this retrospective single-center study, we included all KTR admitted between January 2010 and June 2016 to the ICU of the Department of Nephrology and Organ Transplantation at the University Hospital of Toulouse (France), a 10-bed tertiary care ICU backed by a 30 beds-unit of solid organ transplantation, and with 24-h- a-day intensivist. Immunological tests Immunological tests Assessment of anti-HLA immunization was performed according to our institutional protocol (i.e. every 12 months or after events at risk of anti-HLA immunization like RBC transfusion, acute rejection), with the Luminex° technique. A baseline value of > 500 was considered positive. Page 2 of 9 Guinault et al. BMC Anesthesiology (2019) 19:130 Guinault et al. BMC Anesthesiology (2019) 19:130 Guinault et al. BMC Anesthesiology Page 2 of 9 (University Hospital of Toulouse – Office of Research, Development and Innovation), the need for written con- sent was waived. complications and with widespread use of prophylaxis for opportunistic infections remain to be addressed. KTR are at higher risk of severe AKI in the ICU, com- pared to unselected critically ill patients [3, 11], and up to 40% of KTR will required renal replacement therapy (RRT). AKI is now recognized as a cause of chronic kid- ney disease (CKD), and estimated glomerular filtration rate (eGFR) before the injury is a strong predictive factor of progression toward CKD [12]. In old studies, the renal outcome was poor, ranging from 20 to 30% of patients with eGFR decline after ICU stay. KTR that develop AKI have a relative risk of graft loss of 3.2, and up to 20% of patients will ultimately lose their renal graft [13]. The primary objective of the study was to identify the predictive factors of death in the hospital of KTR admitted to the ICU. Secondary objectives were the characterization of the risk to develop AKI to CKD transition and to ac- quire HLA immunization. Clinical characteristics l l d b l Clinical and biological data were collected from the computerized charts of the patients. The following pa- rameters related to transplantation were collected: age at transplantation, cause of renal disease, immunosuppres- sive regimen, episodes of biopsy-proven antibody or T- cell-mediated rejection (ABMR and TCMR), EBV or CMV replication in blood or BK virus shedding in urine within the 6 months preceding the admission to the ICU. The parameters related to the ICU stay included age at admission, gravity scores, causes for admission, infections, organ failures, RBC transfusions, and im- munosuppression management. Changes in immunosup- pression were not standardized in our ICU and were left at the discretion of each physician. CKD was assessed at month 1 and 6 in survivors according to the CKD KDIGO staging [17]. Similarly to unselected critically ill patients, renal out- come of KTR results from the combination of the under- lying CKD (i.e, basal eGFR), the use of nephrotoxicants in the ICU, and episodes of ischemic, hemodynamic or septic AKI [3, 4, 14]. In the setting of renal transplantation, im- munological injuries may also promote the progression of graft dysfunction observed after ICU admission. Indeed, the withdrawal of immunosuppressive drugs and/or red blood cells (RBC) transfusion may lead to the develop- ment of de novo donor-specific antibodies during or after the stay in the ICU [15, 16]. This may reduce the graft sur- vival in ICU survivors and reduce the access to a subse- quent transplantation in patients who lost their graft function. To date, no study accurately assessed the im- munological outcome in KTR admitted in ICU. In this study, which included a large cohort of 200 KTR admitted in ICU over a 6 years period, we aimed to identify the predictive factors for in-hospital mortality, to characterize the predictive factors of progression from AKI of CKD, and to assess the risk of anti-HLA immunization, two factors associated with long-term survival. Characteristics in the ICU At admission, median SOFA gravity score was 6 [IQR 4–8]. Main causes of admission were ARF (27.5%), septic shock (26.5%), post-operative period (peritonitis, hemorrhage, 23%), acute neurological disorder (6%), AKI requiring RRT (5%) or cardiac arrest (1%). Statistical analyses b Continuous variables are reported as their median and interquartile ranges (IQR), and discontinuous var- iables as numbers and percentages. Univariate ana- lyses of in-hospital mortality were performed using the Mann–Whitney or Fischer’ exact tests, as appro- priate. Multivariate analyses were performed using a step-by-step logistic regression model. All variables associated with in-hospital survival by univariate ana- lysis (p < 0.1) were included in the multivariate ana- lysis. Survival curves were drawn according to the Kaplan-Meier method and compared with the Log- Rank test (univariate analysis). Statistical significance was assumed at p < 0.05. Statistical analyses were per- formed using the GraphPad Prism6 (San Diego, CA, USA) and Xlstat softwares (Addinsoft, Paris, France). To be included in this study, patients met the follow- ing criteria: (i) over 18 years of age, (ii) to have received a renal transplantation before the admission, and (iii) an admission to the ICU for acute conditions. Patients ad- mitted for a close monitoring just after the renal trans- plantation, and those with known irreversible graft failure, were excluded from the analysis. Only the first admission was reported. According to the French law related to retrospective observational studies and our Institutional Review Board Page 3 of 9 Guinault et al. BMC Anesthesiology (2019) 19:130 Guinault et al. BMC Anesthesiology (2019) 19:130 Results TCMR). When available within the 6 months preceding the admission to the ICU, CMV and EBV replication in the blood were observed in 16/142 patients (11.3%), 39/ 127 (30.7%), respectively. TCMR). When available within the 6 months preceding the admission to the ICU, CMV and EBV replication in the blood were observed in 16/142 patients (11.3%), 39/ 127 (30.7%), respectively. From January 2010 to June 2016, 286 KTR were admit- ted at least once to our ICU, including 200 that met the inclusion criteria (median age 61 years [IQR 50.7–68]; male to female ratio 126/74; time from transplantation 41 months [IQR 5–119]; Fig. 1). During this period 1240 patients received renal transplantation in our transplant- ation unit, and 95 out of these 1240 (7.7%) were admitted to the ICU. Main characteristics of the transplantation and at the admission are summarized in Tables 1 and 2. Transplantation characteristics Altogether, 114/200 admissions (57%) were related to an infection (lungs (28.5%), urine (13%), gut (12%) or other (3.5%)). Pyogenes-related infections were diag- nosed in 101 patients, whereas aspergillosis, candidemia, pneumocystosis and CMV infections were identified in 8, 8, 7 and 5 patients, respectively. Multiple infections were identified in 29 patients (14.5%). Immunosuppressive regimen included induction therapy in 162 patients. At admission, patients received a com- bination of calcineurin inhibitors (81.3%; tacrolimus 74%), antimetabolites (82.3%; mycophenolic acid 79.3%), mTOR inhibitors (19.2%) and/or steroids (92.9%). Over- all, 123 patients received an immunosuppressive regimen including steroids, mycophenolic acid and calcineurins inhibitors. Median time of hospitalization in the ICU was 5 days [IQR 2–10]. Among the 200 individuals, 107 (53.5%) re- quired mechanical ventilation, and 97 (48.5%) required vasopressive drugs. One hundred and seventy-one pa- tients (85.5%) developed AKI, including 113 (56.5%) with Twenty-six (13%) and 34 (17%) out of the 200 patients had presented with an ABMR and TCMR before the ad- mission in the ICU (12 (6%) developed both ABMR and Fig. 1 Flowchart of the study Guinault et al. BMC Anesthesiology (2019) 19:130 Page 4 of 9 Guinault et al. Transplantation characteristics BMC Anesthesiology (2 Table 1 Characteristics of the 200 kidney transplant recipients before admission in ICU Characteristics Total N = 200 Survivors N = 160 In-hospital death N = 40 p Age (n, %) 61 [51–68] 60 [49–66] 65 [58–70] 0.07 Male gender (n, %) 126 (63) 97 (60.6) 29 (72.5) 0.20 Comorbidities (n, %) Diabetes mellitus 71 (35.5) 56 (35) 15 (37.5) 0.81 Heart disease (left ventricular systolic function < 45%) 97 (48.5) 73 (45.5) 24 (60) 0.11 Peripheral arterial disease 23 (11.5) 60 (37.5) 24 (60) 0.01 Solid cancer 11 (5.5) 6 (3.8) 5 (12.5) 0.05 Active hematological malignancy 9 (4.5) 6 (3.8) 3 (7.5) 1.00 Transplantation characteristics (n, %) Deceased donor 15 (7.5) 14 (8.7) 1 (2.5) 0.31 First kidney transplantation 166 (83) 136 (85) 30 (75) 0.20 Induction No 30 (15) 23 (14.4) 7 (17.5) 0.62 Polyclonal antibodies 90 (45) 74 (46.3) 16 (40) 0.58 IL2R blocking agents 72 (36) 57 (35.6) 15 (37.5) 0.85 Immunosuppressive regimen Steroids 184 (92) 148 (92.5) 36 (90) 1.00 CNI 161 (80.5) 131 (81.8) 30 (75) 0.49 Tacrolimus 123 (61.5) 104 (65) 19 (4.8) 0.97 Antimetabolites 163 (81.5) 137 (85.6) 26 (65) 0.008 MMF mTOR inhibitors 36 (18) 29 (18.1) 7 (17.5) 1.00 Belatacept 8 (4) 6 (3.8) 2 (5) 0.65 CNI, MPA, Steroids association xx xx xx History of acute rejection Antibody mediated rejection 26 (13) 19 (11.8) 7 (17.5) 0.43 T-cell mediated rejection 34 (17) 28 (17.5) 6 (15) 0.82 Viral status before the admission # (n, %) Blood Cytomegalovirus detection (n = 142) 16 (11.3) 9/107 (8.4) 7/35 (20) 0.07 Blood Epstein-Barr virus detection (n = 127) 39 (30.7) 24/96 (25) 15/31 (48) 0.02 Urine BK virus shedding (n = 115) 28 (24.3) 18/90 (20) 10/25 (40) 0.04 Immunological status before the admission in ICU (n, %) Anti-HLA immunization (n = 188) 71 (38) 59/152 (38) 12/36 (33) 0.57 Donor-specific antibodies 23 (12.2) 19 (12.5) 4 (11.1) 1.00 Abbreviations: IL-2R, interleukin-2-receptor; CNI, calcineurin inhibitors; MPA, mycophenolic acid; mTOR, mammalian target of rapamycin; ICU, intensive care unit Table 1 Characteristics of the 200 kidney transplant recipients before admission in ICU KDIGO stage 3 AKI and 103 (51.5%) that required RRT. One hundred and forty-two out of the 200 patients (71%) benefitted from RBC transfusion. KDIGO stage 3 AKI and 103 (51.5%) that required RRT. One hundred and forty-two out of the 200 patients (71%) benefitted from RBC transfusion. Transplantation characteristics patients, including 35 with septic shock. Calcineurin in- hibitors were withdrawal in 40 patients (20%) and concen- tration targets were reduced in the others (tacrolimus residual 5–8 ng/mL; cyclosporine-A residual (70–220 ng/ mL). Antimetabolites were stopped in 61/163 patients (37.4%), including 19 with sepsis. In 48 out of these 61 patients, the dose of steroids was increased. Over- all, the immunosuppressive regimen was modified in 155 patients (77.5%). patients, including 35 with septic shock. Calcineurin in- hibitors were withdrawal in 40 patients (20%) and concen- tration targets were reduced in the others (tacrolimus residual 5–8 ng/mL; cyclosporine-A residual (70–220 ng/ mL). Antimetabolites were stopped in 61/163 patients (37.4%), including 19 with sepsis. In 48 out of these 61 patients, the dose of steroids was increased. Over- all, the immunosuppressive regimen was modified in 155 patients (77.5%). Management of immunosuppression in the ICU At the admission, median residual concentration of tacro- limus and ciclosporine-A were 6.6 ng/mL [IQR 4.6–11.2] and 127 ng/mL [IQR 77–223], respectively. Prednisolone was switched to hydrocortisone (150 to 300 mg/day) in 42 Page 5 of 9 Guinault et al. BMC Anesthesiology (2019) 19:130 Guinault et al. BMC Anesthesiology Table 2 Characteristics of the 200 kidney-transplant recipients at the admission in ICU and during ICU stay Characteristics Total N = 200 Survivors N = 160 In-hospital death N = 40 p Time since kidney transplantation (months) 40 [5–119] 37 [4–118] 54 [12–122] 0.14 Causes of admission (n, %) Acute respiratory failure 55 (27.5) 42 (26.3) 13 (32.5) 0.43 Septic shock 53 (26.5) 42 (26.3) 11 (27.5) 0.84 Unplanned surgery 46 (23) 40 (25) 6 (15) 0.21 Cardiogenic shock 18 (9) 12 (7.5) 6 (15) 0.21 Acute neurological condition 12 (6) 9 (5.6) 3 (7.5) 0.71 Acute kidney injury 10 (5) 10 (6.3) 0 (0) 0.22 Others 6 (3) 5 (3.1) 1 (2.5) 1.00 Sepsis at the admission (n, %) Overall 114 (57) 89 (55.6) 25 (63) 0.48 Lung 57 (50) 47 (52.9) 10 (40) 0.70 Urinary 26 (22.8) 23 (25.8) 3 (7.5) 0.30 Peritonitis 24 (21) 14 (15.7) 10 (25) 0.01 Others 7 (6.1) 5 (5.1) 2 (1.4) 0.64 Organ failures (n, %) SOFA score 6 [4–8] 6 [4–8] 8 [7–10] < 0.001 SAPS2 score 50 [39–63] 48 [37–59] 67[57–77] < 0.001 Acute kidney injury (KDIGO) stage 1 49 (24.5) 42 (26.3) 7 (17.5) stage 2 8 (4) 6 (3.8) 2 (5) stage 3 113 (56.5) 86 (53.8) 27 (67.5) 0.008 Renal replacement therapy 103 (51.5) 76 (47.5) 27 (67.5) 0.03 Mechanical ventilation 107 (53.5) 78 (48.8) 29 (72.5) 0.008 Vasopressive drugs 97 (48.5) 68 (42.5) 29 (72.5) 0.001 Normalized prothrombin time < 50% 11 (5.5) 5 (3.1) 6 (15) 0.01 RBC transfusion 142 (71) 109 (68.1) 33 (82.5) 0.08 Immunosuppressive regimen (n, %) No change 45 (22.5) 45 (28.1) 0 (0) 0.02 Increased dose of steroids 136 (68) 100 (62.5) 36 (90) 0.2 Withdrawal of CNI 40 (20) 25 (15.6) 15 (37.5) 0.004 Withdrawal of antimetabolites 61 (30) 48 (30) 13 (32.5) 0.85 cs of the 200 kidney-transplant recipients at the admission in ICU and during ICU stay Discussion In this study, we reported the predictive factors of in- hospital mortality in a large cohort of 200 KTR ad- mitted to the ICU. Risk of transition from AKI to CKD, and anti-HLA immunization, were also re- ported. Notwithstanding the monocentric status of this study and the inherent biases of ICU admission and management, our ICU is the referral center for KTR requiring admission to the ICU (except for neurological and cardiac surgery) in a large region in the south-west of France (~ 2.3 million inhabitants). Thus, the studied cohort accurately described the causes of KTR admission to the ICU, characteristics at presentation, and specific outcomes. available virology data (n = 127), the two factors that in- dependently predicted the risk of death in hospital were the gravity SAPS2 score at admission (OR 1.07, IC95% [1.03–1.11], p = 0.001) and a positive EBV viremia in the 6 months preceding the admission (OR 4.35, IC95% [1.52–12.5], p = 0.006). Predictive factors of in-hospital mortality without antimetabolites at admission, high gravity scores at day 1, organ failure (mechanical ventilation; vasopres- sive drugs; RRT; normalized prothrombin time < 50% at admission), and withdrawal of CNI in the ICU. In pa- tients with available data, a history of positive EBV viremia or BK viruria was also predictive of death in hospital. Of the 200 patients, 25 (12.5%), 40 (20%) and 45 (22.5%) died in the ICU, the hospital, or before day-90, respect- ively. After a median follow-up of 19 months [IQR 3–45], median survival was not reached and survival at month 80 was estimated at 60% (Fig. 2). Main causes of death in the ICU were sepsis in 16 patients, cardiogenic shock in 4, cerebrovascular stroke in 2, cancers in 2, and fulminant hepatitis in 1. By multivariate analysis, SAPS2 score (OR 1.06, IC95% [1.03–1.08], p < 0.0001) and CNI withdrawal in ICU (OR 2.65, IC95% [1.11–6.32], p = 0.027) were the only predict- ive factors of death during the hospital stay. When the analysis was conducted in the sub-group of patients with By univariate analysis, predictive factors of in-hospital death were the following (Tables 1 and 2): history of per- ipheral arterial disease, immunosuppressive regimen Page 6 of 9 Guinault et al. BMC Anesthesiology (2019) 19:130 Guinault et al. BMC Anesthesiology Fig. 2 Survival curves following admission to the ICU. a. Overall population. b. Survival according to EBV replication before admission hospitalization in the ICU (p = 0.002). A progression of at least one stage in the CKD KDIGO classification was observed in 34 (30.1%) and 51 (45.1%) patients at 1 and 6 months, respectively (Fig. 3b). Staging of the CKD be- fore the admission (stage 4–5, 58% vs. stage 1–3 28.1%, p = 0.006) and the severity of AKI (stage 3 55.4% vs. stage 0–2 34.2%, p = 0.04) were significantly associated with a progression of the CKD stage at month 6. Immunological outcomes Among the 119 patients with anti-HLA immuno- logical status available before and 6 months after the admission to the ICU, 18 (15.1%) developed anti-HLA antibodies after the ICU stay, including 11 with donor-specific antibodies (DSA; 9.2%). Among these 18 patients, 6 had no detectable anti-HLA antibodies before admission, 7 had anti-HLA antibodies, and 5 had DSA. Two patients had an active ABMR, and two others stopped immunosuppressive drugs before admission. During the stay in ICU, 13 patients re- ceived RBC transfusions, and 4 needed transplant re- moval (urinoma, renal vein thrombosis, graft intolerance syndrome or emphysematous pyeloneph- ritis). Two additional patients benefitted from trans- plant removal in the next 6 months. Patients that did not acquire HLA immunization during ICU had a significantly poorer renal prognosis (62 vs. 90% func- tional graft at 12 months; p = 0.06). Fig. 2 Survival curves following admission to the ICU. a. Overall population. b. Survival according to EBV replication before admission Renal graft outcome Multiple causes of ARF were identified in 11% of patients, confirming that manage- ment of KTR should follow a dedicated workup (trans- plantation history, immunosuppressive regimens, underlying chronic disorders) [10, 22]. Here, 14.5% of the individuals admitted to the ICU for infection had at least two concomitant infections. antigens [28], associated with an increased risk of mor- tality or nosocomial infections in patient admitted in ICU for sepsis, burns or traumas [29]. Chronic EBV rep- lication may thus be a surrogate marker of frailty associ- ated with a higher risk of mortality in ICU. Further studies are warranted to confirm that EBV status at the time of admission in ICU may help to identify patients at high risk of death. In our cohort, AKI was highly prevalent (81.5%) and 51.5% of patients required RRT, contrasting with unse- lected critically ill patients (19 to 57% and 4.5 to 13.5%, respectively) [30, 31]. In addition to known risk factors for the development of AKI in the ICU, the use of cal- cineurin inhibitors, the previous episodes of AKI and the underlying CKD, all increased the risk of AKI in KTR with acute condition [13, 14, 32, 33]. Moreover, we showed that progression of CKD after admission to the ICU is highly prevalent (30% at 1 month and 45% at 6 months in our series, compared to 12–20% at 3 months in older studies [3, 4, 11] and was well predicted by the basal CKD stage and the severity of the AKI. The high incidence of transition toward CKD in KTR, which con- tinues beyond month 3, also points to additional and persistent renal injuries specifically encountered in this population. Whether specific management in ICU re- garding the immunosuppressive regimen [10], prophy- laxis of cytomegalovirus proliferation [34], and RBC transfusion policies [15] may help to overcome the risk to develop anti-HLA antibodies after admission to the ICU (15.1% of survivors in our series) in solid organ transplantation recipients need to be tested in prospect- ive trials, because our retrospective study cannot lead to specific recommendations. Contrasting to older studies [5, 6, 21], the mortality rate in our study (i.e., 20% in the hospital and 26.5% 6 months after the ICU stay) was lower than in unselected critically ill patients [23]. Of note, severity scores at the admission predicted mortality in hospital between 15 and 50% [24, 25]. Renal graft outcome Over the study period, the estimated incidence of ICU admission (7.7%) was closed to the one reported in re- cent studies (4.5 to 10.2%) [2–5, 7]. Median time from renal transplantation to ICU admission was longer (41 months vs. 4.4 to 25.2 months [1–5, 7–9, 18]) except in one study [19]. The mortality rate was not influenced by the timing of ICU admission, thus confirming survival in the ICU is not associated to the characteristics of the transplantation [2, 5, 20, 21]. As a secondary objective, we characterized the risk to develop AKI to CKD transition. Graft survival is shown in Fig. 3a. Median graft survival was 10 months. To bet- ter characterize the renal outcome and progression from AKI toward CKD in KTR admitted to the ICU, we stud- ied the sub-group of patients with stable renal function at admission (i.e. admitted more than 1 month after the renal transplantation) and still alive 6 months after the admission to the ICU. In these 113 patients, median eGFR at month 6 was 35 mL/min/1.73 m2 [IQR 11–55], compared to 44 mL/min/1.73m2 [IQR 27–61] before Our series confirms that ARF and sepsis are the main causes of ICU admission in KTR [1, 3–6, 9, 18, 20, 21]. Guinault et al. BMC Anesthesiology (2019) 19:130 Page 7 of 9 Guinault et al. BMC Anesthesiology Fig. 3 Renal outcome of the 200 kidney transplant recipients admitted to the ICU. a. Graft survival curve. b. Progression of chronic kidney disease following admission to the ICU (according to the CKD KDIGO stage) Fig. 3 Renal outcome of the 200 kidney transplant recipients admitted to the ICU. a. Graft survival curve. b. Progression of chronic kidney disease following admission to the ICU (according to the CKD KDIGO stage) Lung infection was the main cause of ARF with acute pulmonary edema ranking second, pointing to the high risk of sodium overload in KTR (steroids, underlying CKD or heart disease). Multiple causes of ARF were identified in 11% of patients, confirming that manage- ment of KTR should follow a dedicated workup (trans- plantation history, immunosuppressive regimens, underlying chronic disorders) [10, 22]. Here, 14.5% of the individuals admitted to the ICU for infection had at least two concomitant infections. Lung infection was the main cause of ARF with acute pulmonary edema ranking second, pointing to the high risk of sodium overload in KTR (steroids, underlying CKD or heart disease). Funding None 11. Badin J, Longuet H, Guillon A, Barbet C, Halimi JM, Lebranchu Y, et al. Renal function of renal transplantation patients after hospitalization in an intensive care unit. Transplant Proc. 2012;44:2792–4. https://doi.org/10.1016/ j.transproceed.2012.09.027. 11. Badin J, Longuet H, Guillon A, Barbet C, Halimi JM, Lebranchu Y, et al. Renal function of renal transplantation patients after hospitalization in an intensive care unit. Transplant Proc. 2012;44:2792–4. https://doi.org/10.1016/ j.transproceed.2012.09.027. ABMR: Antibodies-Mediated Rejection; AKI: Acute Kidney Injury; CKD: Chronic Kidney Disease; CMV: Cytomegalovirus; CNI : Calcineurin Inhibitors; DSA: Donor-Specific Antibodies; EBV: Epstein Barr Virus; GFR: Glomerular Filtration Rate; HLA: Human Leukocytes Antigens; ICU: Intensive Care Unit; KTR: Kidney Transplant Recipients; RBC: Red Blood Cells; RRT: Renal Replacement Therapy; TCMR: T-Cells Mediated Rejection Received: 25 January 2019 Accepted: 5 July 2019 Received: 25 January 2019 Accepted: 5 July 2019 Received: 25 January 2019 Accepted: 5 July 2019 References l 1. Kirilov D, Cohen J, Shapiro M, Grozovski E, Singer P. The course and outcome of renal transplant recipients admitted to a general intensive care unit. Transplant Proc. 2003;35:606 http://www.ncbi.nlm.nih.gov/ pubmed/12644065. Accessed 26 Jan 2018. 2. Klouche K, Amigues L, Massanet P, Garrigue V, Delmas S, Szwarc I, et al. Outcome of renal transplant recipients admitted to an intensive care unit: a 10-year cohort study. Transplantation. 2009;87:889–95. https://doi. org/10.1097/TP.0b013e31819a688a. 3. Bige N, Zafrani L, Lambert J, Peraldi M-N, Snanoudj R, Reuter D, et al. Severe infections requiring intensive care unit admission in kidney transplant recipients: impact on graft outcome. Transpl Infect Dis. 2014;16:588–96. https://doi.org/10.1111/tid.12249. In summary, we showed that survival of KTR follow- ing admission to the ICU is predicted by the severity of the acute condition but also by viral replication (i.e., the underlying immune defense status). ICU admission is as- sociated with a very high risk of AKI and progression to- ward CKD, as well as a significant risk of HLA immunization. 4. Canet E, Osman D, Lambert J, Guitton C, Heng A-E, Argaud L, et al. Acute respiratory failure in kidney transplant recipients: a multicenter study. Crit Care. 2011;15:R91. https://doi.org/10.1186/cc10091. 5. Mouloudi E, Massa E, Georgiadou E, Iosifidis E, Kydona C, Sgourou K, et al. Course and outcome of renal transplant recipients admitted to the intensive care unit: a 20-year study. Transplant Proc. 2012;44:2718– 20. https://doi.org/10.1016/j.transproceed.2012.09.097. 6. Arulkumaran N, West S, Chan K, Templeton M, Taube D, Brett SJ. Long- term renal function and survival of renal transplant recipients admitted to the intensive care unit. Clin Transpl. 2012;26:E24–31. https://doi.org/1 0.1111/j.1399-0012.2011.01520.x. 6. Arulkumaran N, West S, Chan K, Templeton M, Taube D, Brett SJ. Long- term renal function and survival of renal transplant recipients admitted to the intensive care unit. Clin Transpl. 2012;26:E24–31. https://doi.org/1 0.1111/j.1399-0012.2011.01520.x. Consent for publication Not applicable 14. Filiponi TC, Requião-Moura LR, Tonato EJ, Carvalho de Matos AC, e Silva- Filho AP, de Souza Durão Junior M. Hospital admission following acute kidney injury in kidney transplant recipients is associated with a negative impact on graft function after 1-year. PLoS One. 2015;10:e0138944. https:// doi.org/10.1371/journal.pone.0138944. Competing interests 15. Ferrandiz I, Congy-Jolivet N, Del Bello A, Debiol B, Trébern-Launay K, Esposito L, et al. Impact of early blood transfusion after kidney transplantation on the incidence of donor-specific anti-HLA antibodies. Am J Transplant. 2016;16:2661–9. https://doi.org/10.1111/ ajt.13795. 15. Ferrandiz I, Congy-Jolivet N, Del Bello A, Debiol B, Trébern-Launay K, Esposito L, et al. Impact of early blood transfusion after kidney transplantation on the incidence of donor-specific anti-HLA antibodies. Am J Transplant. 2016;16:2661–9. https://doi.org/10.1111/ ajt.13795. Author details 1Département de Néphrologie et Transplantation d’organes, Unité de Réanimation, Hôpital Rangueil, Centre Hospitalier Universitaire de Toulouse, 1, avenue Jean Poulhes, 31059 Toulouse, France. 2Laboratoire Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. 12. Hsu RK, Hsu C-Y. The role of acute kidney injury in chronic kidney disease. Semin Nephrol. 2016;36:283–92. https://doi.org/10.1016/j. semnephrol.2016.05.005. 12. Hsu RK, Hsu C-Y. The role of acute kidney injury in chronic kidney disease. Semin Nephrol. 2016;36:283–92. https://doi.org/10.1016/j. semnephrol.2016.05.005. Abbreviations b d ABMR: Antibodies-Mediated Rejection; AKI: Acute Kidney Injury; CKD: Chronic Kidney Disease; CMV: Cytomegalovirus; CNI : Calcineurin Inhibitors; DSA: Donor-Specific Antibodies; EBV: Epstein Barr Virus; GFR: Glomerular Filtration Rate; HLA: Human Leukocytes Antigens; ICU: Intensive Care Unit; KTR: Kidney Transplant Recipients; RBC: Red Blood Cells; RRT: Renal Replacement Therapy; TCMR: T-Cells Mediated Rejection 7. Ulas A, Kaplan S, Zeyneloglu P, Torgay A, Pirat A, Haberal M. Acute respiratory failure in renal transplant recipients: a single intensive care unit experience. Exp Clin Transplant. 2015;13(Suppl 3):44–7. https://doi.org/10.6 002/ect.tdtd2015.O37. 7. Ulas A, Kaplan S, Zeyneloglu P, Torgay A, Pirat A, Haberal M. Acute respiratory failure in renal transplant recipients: a single intensive care unit experience. Exp Clin Transplant. 2015;13(Suppl 3):44–7. https://doi.org/10.6 002/ect.tdtd2015.O37. 8. Shorr AF, Abbott KC, Agadoa LY. Acute respiratory distress syndrome after kidney transplantation: epidemiology, risk factors, and outcomes. Crit Care Med. 2003;31:1325–30. https://doi.org/10.1097/01.CCM. 0000053645.38356.A6. 8. Shorr AF, Abbott KC, Agadoa LY. Acute respiratory distress syndrome after kidney transplantation: epidemiology, risk factors, and outcomes. Crit Care Med. 2003;31:1325–30. https://doi.org/10.1097/01.CCM. 0000053645.38356.A6. Ethics approval and consent to participate This retrospective observational study was conducted according to our Institutional Review Board instructions (University Hospital of Toulouse – Office of Research, Development and Innovation). 13. Mehrotra A, Rose C, Pannu N, Gill J, Tonelli M, Gill JS. Incidence and consequences of acute kidney injury in kidney transplant recipients. Am J Kidney Dis. 2012;59:558–65. https://doi.org/10.1053/j.ajkd.2011.11.034. Renal graft outcome This mortality rate, close to the one observed in two recent French studies, suggests that usual gravity scores may be undermined in KTR. Man- agement of KTR in ICUs specialized in the field of trans- plantation and immunocompromised patients may also improves the outcome of these patients [3, 4]. Prognosis of solid organ transplant recipients with sepsis may be better than expected in unselected critically ill patients after adjustments on gravity scores, causes of admission, and number of organ failures [26]. Immunosuppressive drugs with reduction of the risk of hyper-inflammation state and subsequent refractory acute respiratory distress syndrome or shock may account for this discrepancy. Interestingly, we showed for the first time that EBV replication in the months preceding the admission of KTR to the ICU was associated with a poorer outcome. EBV replication per se is not associated with a poorer outcome in unselected KTR [27]. However, chronic EBV replication may be the trigger or the consequence of T- cell exhaustion, an acquired immune paralysis observed in patients with chronic exposure to viral or cancer Several limitations may be underlined. First, the retro- spective design of the study prevented the assessment of EBV viremia in all the KTR admitted to the ICU during Page 8 of 9 Guinault et al. BMC Anesthesiology (2019) 19:130 Page 8 of 9 Page 8 of 9 Guinault et al. BMC Anesthesiology (2019) 19:130 Page 8 of 9 the inclusion period. Nonetheless, this parameter was available in a significant number of patients (n = 127). Second, management of immunosuppressive regimen may vary during the ICU stay according to the patient status. Here, we discriminate patients according the maintain or the withdrawal of immunosuppressive drugs but how long patients had reduced immunosuppressive regimen was unknown in most patients. This limitation may also have modulated the risk of HLA immunization. Last, we reported here a large cohort of KTR admitted to the ICU in order to describe the different clinical sce- narios that can lead to ICU admission in these patients. However, the various causes of admission introduced heterogeneity in term of both mortality and risk of immunization that need to be taken in account. It also prevents to draw firm conclusions about the optimal im- munosuppressive management. the inclusion period. Nonetheless, this parameter was available in a significant number of patients (n = 127). Authors’ contributions SF d DG d d h 9. de Carvalho MA, Freitas FGR, Silva Junior HT, Bafi AT, Machado FR, Pestana JOM. Mortality predictors in renal transplant recipients with severe Sepsis and septic shock. PLoS One. 2014;9:e111610. https://doi. org/10.1371/journal.pone.0111610. 9. de Carvalho MA, Freitas FGR, Silva Junior HT, Bafi AT, Machado FR, Pestana JOM. Mortality predictors in renal transplant recipients with severe Sepsis and septic shock. PLoS One. 2014;9:e111610. https://doi. org/10.1371/journal.pone.0111610. SF and DG designed the study; SF, DG and NKD analyzed the patient data; SF, NK and DG wrote the manuscript; SF, LL, MBN, OC, NK, ADB, DG, ALH, OR and LE followed patients; NC preformed immunological analyses. All authors read and approved the final manuscript. SF and DG designed the study; SF, DG and NKD analyzed the patient data; SF, NK and DG wrote the manuscript; SF, LL, MBN, OC, NK, ADB, DG, ALH, OR and LE followed patients; NC preformed immunological analyses. All authors read and approved the final manuscript. 10. Canet E, Zafrani L, Azoulay É. The critically ill kidney transplant recipient: a narrative review. Chest. 2016;149:1546–55. https://doi.org/10.1016/j.chest.2 016.01.002. 10. Canet E, Zafrani L, Azoulay É. The critically ill kidney transplant recipient: a narrative review. Chest. 2016;149:1546–55. https://doi.org/10.1016/j.chest.2 016.01.002. Renal graft outcome Second, management of immunosuppressive regimen may vary during the ICU stay according to the patient status. Here, we discriminate patients according the maintain or the withdrawal of immunosuppressive drugs but how long patients had reduced immunosuppressive regimen was unknown in most patients. This limitation may also have modulated the risk of HLA immunization. Last, we reported here a large cohort of KTR admitted to the ICU in order to describe the different clinical sce- narios that can lead to ICU admission in these patients. However, the various causes of admission introduced heterogeneity in term of both mortality and risk of immunization that need to be taken in account. It also prevents to draw firm conclusions about the optimal im- munosuppressive management. d’Immunologie, Hôpital Rangueil, Centre Hospitalier Universitaire de Toulouse, F-31000 Toulouse, France. 3Université Paul Sabatier, Toulouse III, F-31000 Toulouse, France. 4Institut National de la Santé et de la Recherche Médicale, U1043, IFR–BMT, CHU Purpan, Toulouse, France. 5Institut National de la Santé et de la Recherche Médicale, Institut des Maladies Métaboliques et Cardiovasculaires, U1048 (Renal Fibrosis lab), and French Intensive care Renal Network (F.I.R.N), Toulouse, France. Author details 1Dé d Page 9 of 9 Page 9 of 9 Page 9 of 9 Guinault et al. BMC Anesthesiology (2019) 19:130 Guinault et al. BMC Anesthesiology Guinault et al. BMC Anesthesiology (2019) 19:130 33. Nakamura M, Seki G, Iwadoh K, Nakajima I, Fuchinoue S, Fujita T, et al. Acute kidney injury as defined by the RIFLE criteria is a risk factor for kidney transplant graft failure. 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Use of the SOFA score to assess the incidence of organ dysfunction/failure in intensive care units: results of a multicenter, prospective study. Working group on &quot;sepsis-related problems&quot; of the European Society of Intensive Care Medicine. Crit Care Med. 1998;26:1793–800 http://www.ncbi.nlm.nih.gov/pubmed/ 9824069. Accessed 26 Jan 2018. 26. Kalil AC, Syed A, Rupp ME, Chambers H, Vargas L, Maskin A, et al. Is Bacteremic Sepsis associated with higher mortality in transplant recipients than in nontransplant patients? A matched case-control propensity-adjusted study. Clin Infect Dis. 2015;60:216–22. https://doi. org/10.1093/cid/ciu789. 27. Morton M, Coupes B, Roberts SA, Johnson SL, Klapper PE, Vallely PJ, et al. Epstein-Barr virus infection in adult renal transplant recipients. Am J Transplant. 2014;14:1619–29. https://doi.org/10.1111/ajt.12703. 28. Schietinger A, Greenberg PD. Tolerance and exhaustion: defining mechanisms of T cell dysfunction. Trends Immunol. 2014;35:51–60. https:// doi.org/10.1016/j.it.2013.10.001. 29. van Vught LA, Klein Klouwenberg PMC, Spitoni C, Scicluna BP, Wiewel MA, Horn J, et al. Publisher’s Note Incidence, risk factors, and attributable mortality of secondary infections in the intensive care unit after admission for Sepsis. JAMA. 2016; 315:1469. https://doi.org/10.1001/jama.2016.2691. 30. Hoste EAJ, Bagshaw SM, Bellomo R, Cely CM, Colman R, Cruz DN, et al. Epidemiology of acute kidney injury in critically ill patients: the multinational AKI-EPI study. Intensive Care Med. 2015;41:1411–23. https:// doi.org/10.1007/s00134-015-3934-7. 30. Hoste EAJ, Bagshaw SM, Bellomo R, Cely CM, Colman R, Cruz DN, et al. Epidemiology of acute kidney injury in critically ill patients: the multinational AKI-EPI study. Intensive Care Med. 2015;41:1411–23. https:// doi.org/10.1007/s00134-015-3934-7. 31. Bouchard J, Acharya A, Cerda J, Maccariello ER, Madarasu RC, Tolwani AJ, et al. A prospective international multicenter study of AKI in the intensive care unit. Clin J Am Soc Nephrol. 2015;10:1324–31. https://doi.org/10.2215/CJN. 04360514. 31. Bouchard J, Acharya A, Cerda J, Maccariello ER, Madarasu RC, Tolwani AJ, et al. A prospective international multicenter study of AKI in the intensive care unit. Clin J Am Soc Nephrol. 2015;10:1324–31. https://doi.org/10.2215/CJN. 04360514. 32. Nagarajan M, Ramanathan S, Dhanapriya J, Dineshkumar T, Subramaniyan TB, Gopalakrishnan N. Impact of acute kidney injury on renal allograft survival. Ren Fail. 2017;39:40–4. https://doi.org/10.1080/ 0886022X.2016.1244076.
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Signalling to Experts
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Zurich Open Repository and Archive Year: 2021 Signalling to experts Kurlat, Pablo ; Scheuer, Florian Kurlat, Pablo ; Scheuer, Florian DOI: https://doi.org/10.1093/restud/rdaa068 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-195318 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at: Kurlat, Pablo; Scheuer, Florian (2021). Signalling to experts. Review of Economic Studies, 88(2):800-850. DOI: https://doi.org/10.1093/restud/rdaa068 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-195318 Journal Article Published Version Th f ll i g k i li d d C ti C Att ib ti 4 0 I t Originally published at: Kurlat, Pablo; Scheuer, Florian (2021). Signalling to experts. Review of Economic Studies, 88(2):800-850. DOI: https://doi.org/10.1093/restud/rdaa068 Originally published at: Kurlat, Pablo; Scheuer, Florian (2021). Signalling to experts. Review of Economic Studies, 88(2):800-850. DOI: https://doi.org/10.1093/restud/rdaa068 MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES Review of Economic Studies (2020) 0, 1–50 Review of Economic Studies (2020) 0, 1–50 doi:10.1093/restud/rdaa068 © The Author(s) 2020. Published by Oxford University Press on behalf of The Review of Economic Studies Limited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contactjournals.permissions@oup.com Advance access publication 20 October 2020 First version received February 2018; Editorial decision August 2020; Accepted October 2020 (Eds.) We study competitive equilibria in a signalling economy with heterogeneously informed buyers. In terms of the classic Spence (1973, The Quarterly Journal of Economics, 87, 355––374) model of job market signalling, firms have access to direct but imperfect information about worker types, in addition to observing their education. Firms can be ranked according to the quality of their information, i.e., their expertise. In equilibrium, some high-type workers forgo signalling and are hired by better informed firms, which make positive profits. Workers’ education decisions and firms’ use of their expertise are strategic complements, allowing for multiple equilibria that can be Pareto ranked. We characterize wage dispersion and the extent of signalling as a function of the distribution of expertise among firms. Our model can also be applied to a variety of other signalling problems, including securitization, corporate financial structure, insurance markets, or dividend policy. Key words: Asymmetric information, expertise, signaling, competitive equilibrium. ymmetric information, expertise, signaling, competitive equilibriu 1. Throughout, we refer to a signalling rather than a screening problem. Traditionally, which term is used depends on which party proposes contract terms. Since in our setup there are markets for all possible contracts, the distinction vanishes. The editor in charge of this paper was Veronica Guerrieri. JEL Codes: D5, D8, G1, G3, J3, L1, M5 Signalling to Experts PABLO KURLAT University of Southern California and FLORIAN SCHEUER University of Zurich REVIEW OF ECONOMIC STUDIES 2 private information about their own productivity; education is purely wasteful but is more costly for less productive workers so it can be used to signal; and firms have heterogeneous expertise in directly assessing workers’ productivities, in addition to verifying their education. For instance, firms have access to such direct information through tests, interviews, referrals or trial periods, and differ in their ability to extract accurate predictions from them. We ask how differences in recruiting expertise across firms affect the equilibrium: what wages do more- versus less-expert firms offer, which workers do they hire, how much profit do they make, what education levels do they require, and what are the implications for social welfare? While we present our setup and results in terms of this application, our model is general and can be used to answer these basic questions for many signalling and screening problems. How do investors’ abilities to directly assess a company’s profitability affect IPO prices, incentives for insiders to retain undiversified shareholdings, and the payment of dividends? What are firms’ incentives to engage in costly brand-building or to offer warranties if consumers have heterogeneous ability to find out about product quality directly, e.g., by studying product reviews? How does the use of different risk assessment models across insurance companies affect equilibrium deductibles and premiums? What are the effects of asset managers’ heterogeneous pricing techniques on the design and tranching of asset- backed securities? Returning to labour markets, we focus on the most parsimonious setting with two worker types and consider configurations for firms’ direct information that allow us to rank firms by their expertise, i.e., their probability of making mistakes: the “false positives” case where firms may observe good signals from low-productivity workers and the opposite case, with “false negatives.” We assume that each firm hires a single worker; such capacity constraints are crucial to rule out trivial solutions where the most-expert firms hire all workers. Our first task is to define a notion of competitive equilibrium that applies to this environment. We assume that each combination of a wage and an education level defines a separate market. Any worker is allowed to apply for a job in any market (provided he acquires the level of education prescribed by that market) and any firm can recruit in any market. RESTUD: The Review of Economic Studies 1. INTRODUCTION We study competitive markets with the following features: sellers are privately informed about their own type; they can take a publicly observable action that is differentially costly for different types; buyers can directly observe imperfect information about sellers’ types; and the quality of this information is heterogeneous across buyers. The first two features define a standard signalling environment.1 Our objective is to move beyond the special case, studied extensively, where buyers are completely uninformed and rely exclusively on the public signal to form beliefs about sellers’ types. Instead, we investigate the effect of adding the third and fourth features, buyers’ heterogeneous direct information, on equilibrium prices and allocations. Our running example is an extension of the canonical Spence (1973) model of job market signalling: workers have 1 Page: 1 1–50 [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES REVIEW OF ECONOMIC STUDIES [19:28 2/11/2020 OP-REST200072.tex] KURLAT & SCHEUER SIGNALLING TO EXPERTS KURLAT & SCHEUER SIGNALLING TO EXPERTS SIGNALLING TO EXPERTS 3 For the benchmark where firms have no direct information, our definition ensures that the least-cost separating allocation is the unique equilibrium. Our refinement implies that pooling is inconsistentwithequilibrium:atslightlyhighereducationlevelsthanaputativepoolingallocation, firms must believe that they will only encounter high type workers because they are the ones most willing to choose higher education, and therefore firms could profitably deviate. For the false positives case, the following “partial signalling” pattern emerges. Low worker types get no education and high types get either no education or enough education to fully separate. Firms with sufficiently accurate information recruit zero-education workers at a wage wP that leaves high-productivity workers indifferent between signalling and not signalling, and make positive profits. These firms face both high- and low-productivity applicants, so they can only profit if they are able to reject a sufficient proportion of low types. Firms with less accurate information recruit either educated workers at a wage equal to the high types’ productivity, or zero-education workers at a wage equal to low types’ productivity, and make zero profits in either case. Two simple conditions summarize any equilibrium: an indifference condition that requires the marginal firm to make zero profits by hiring zero-education workers at wage wP, and a market-clearing condition requiring high-type workers who forgo education to indeed find jobs at wage wP. This tractable structure allows us, for instance, to study comparative statics. We find that signalling decreases if the cost is higher, if the demand for workers increases, or if firms’ expertise improves, intuitive properties that, somewhat unappealingly, cannot be obtained in the standard signalling model with uninformed firms. Our model features strategic complementarities between high-quality workers’ signalling decisions and firms’ recruiting decisions. If enough high productivity workers forgo education, the pool of applicants in zero-education markets improves. This induces less-expert firms to recruit zero-educated workers, which in turn allows more high-type workers to forgo education. As a result, the model may feature multiple equilibria, each with different proportions of high types choosing to forgo education. The least cost-separating allocation, where all high types get enough education to separate, is always one of these equilibria: if all high types signal, there is no hope to hire them without requiring the signal, and therefore firms’ expertise is useless—an extreme form of coordination failure. More generally, when there are multiple equilibria, they can be Pareto ranked. REVIEW OF ECONOMIC STUDIES For workers, markets are partially exclusive: naturally, they commit to a single education level but can apply for jobs at many different wages. When hiring, firms need not hire randomly from the pool of applicants: they can reject some applicants and only hire from among those they find acceptable, but only to the extent that their own direct information allows them to tell workers apart. Markets do not necessarily clear: in any given market, workers can apply for jobs and not get them and firms may not find acceptable workers. Equilibrium requires that workers’ expectations of their chances of finding work in each market and firms’ beliefs about what workers they will encounter in each market be consistent with each other and with firm recruiting and worker education decisions. As is common in signalling models, the set of equilibria depends on what beliefs agents can entertain regarding markets where in equilibrium there is no trade. A crucial technical contribution of this paper is to construct restrictions on these out-of-equilibrium beliefs that deliver a unique and plausible equilibrium in the familiar uninformed-buyers benchmark, yet still guarantee equilibrium existence and tractability in the general case of heterogeneous expertise. We propose the following conditions: first, for any market where a firm has well-defined beliefs about what acceptable workers it would encounter, these beliefs can only place weight on workers who would find it (weakly) optimal to apply to that market. Second, if a firm does not have well- defined beliefs about acceptable workers it would encounter, we impose that any workers that would be acceptable to the firm must expect that, if they were to apply for a job in that market, they would get one for sure. Page: 2 1–50 [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies MANUSCRIPT CATEGORY: Article Copyedited by: ES RESTUD: The Review of Economic Studies [19:28 2/11/2020 OP-REST200072.tex] 1.1. Related literature This article introduces heterogeneous expertise among buyers into the canonical competitive signalling and screening environments due to Spence (1973) and Rothschild and Stiglitz (1976). To this purpose, we develop a notion of equilibrium that builds on concepts proposed by Gale (1996), Guerrieri et al. (2010), Guerrieri and Shimer (2014), and Kurlat (2016), all of which are based on the idea that different prices define different markets and the probability of trade is the market-clearing variable. This allows us to naturally incorporate capacity constraints among buyers and to study the extensive margin of trade, which is crucial in many relevant settings, such as labour or financial markets. The way in which we model heterogeneity of information on the buyers’ side—and hence their ability to distinguish between sellers based on their own direct assessment rather than just the publicly observable signal or screening device—is borrowed directly from Kurlat (2016). However, Kurlat (2016) studies a single-dimensional environment, where the set of contracts is just the set of prices, so public signalling is ruled out. Our paper instead incorporates a second dimension, allowing us to capture signalling or screening through, for example, education, underinsurance, equity retention, dividends, or advertising. On a technical side, incorporating signalling requires us to model buyers’ beliefs associated with off-equilibrium actions, a challenge that we tackle here but that is not present in Kurlat (2016). Similar to our article, Gale (1996) and Guerrieri et al. (2010) also allow for general, multidimensional contracts. Relative to them, however, our contribution is to relax the assumption that buyers are completely and uniformly uninformed, by introducing heterogeneous information for buyers. The refinement on beliefs that we impose is closely related to the D1 criterion proposed by Cho and Kreps (1987), the condition for a refined equilibrium proposed by Gale (1996), and the conditions on beliefs imposed by Guerrieri et al. (2010) for contracts that are not traded in equilibrium. It is based on the idea that, in markets with zero supply in equilibrium, buyers anticipate that, if they were to place demand there, they would only attract the sellers (among those they do not reject based on their direct assessment) who are willing to accept the lowest probability of trade. This is the natural generalization of the infinite-tightness condition imposed by Guerrieri et al. (2010) to our framework with heterogeneous information. REVIEW OF ECONOMIC STUDIES 4 of wage dispersion among equally productive (and educated) workers based on how easy it is to evaluate their productivities. It also predicts that higher demand for workers leads to polarization in signalling: fewer high types signal, but those who signal do so more intensely. Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rdaa068/5931860 by University of Zurich user on 05 January 2021 KURLAT & SCHEUER SIGNALLING TO EXPERTS The signal is a pure deadweight cost, and the equilibrium with less signalling is preferred by everyone. One feature of the classic signalling and screening model that has been criticized is a discontinuity as the buyers’ prior becomes degenerate. The symmetric information case involves no signalling, but in the presence of even a minimal mass of low types, the high types must emit a non-trivial signal to separate. Our model offers a natural way to smooth out this stark property: there always exists an equilibrium that continuously approaches the full information limit, both as the share of low types vanishes and as buyers’ direct information becomes perfect. A similar discontinuity arises in the standard signalling model when the signalling costs of the two types converge: whenever the costs differ, there is a discrete amount of signalling, but no signalling when they are equal. We show that our model overcomes this discontinuity as well. Finally,wecharacterizeequilibriuminthefalsenegativescase,whichweshowtobeessentially unique. Productive workers now make different choices depending on how transparent they are, that is, how many firms are able to identify them as high types. Those most easily identified forgo education and are paid their productivity. Less transparent workers also forgo education but now earn a range of lower wages. They are hired in part by non-selective firms in markets where low types also apply, so wages must be low enough to allow these non-selective firms to break even on whatever pool of applicants they face. The least transparent productive workers instead resort to education in order to separate from low types. Therefore, our model provides a novel theory Page: 3 1–50 [19:28 2/11/2020 OP-REST200072.tex] MANUSCRIPT CATEGORY: Article Copyedited by: ES REVIEW OF ECONOMIC STUDIES RESTUD: The Review of Economic Studies KURLAT & SCHEUER SIGNALLING TO EXPERTS KURLAT & SCHEUER SIGNALLING TO EXPERTS 5 SIGNALLING TO EXPERTS Daley and Green (2014) also study an environment where the possibility of signalling coexists with direct information (“grades”) and find conditions such that the equilibrium features either partial or complete pooling. They assume that grades are equally observable by all firms, so they have no role for expertise on the firm side. Feltovich et al. (2002) also consider an environment with (homogeneous) direct information in addition to signalling, and find that—in a model with three types—the highest types may refrain from signalling to distinguish themselves from the medium types, a behaviour they refer to as “countersignalling.” A similar feature emerges in our model in the false negatives case, where some high types separate through signalling while others pool with low types in terms of the signal they emit, relying instead on expert buyers to identify them. Fishman and Parker (2015), Bolton et al. (2016), and Kurlat (2019) study environments where buyers can differ in the quality of their information but where sellers do not have a way to signal. Their focus is on the efficiency of buyers’ information acquisition decision. Board et al. (2017) share our interest in the idea that firms differ in their ability to tell apart high- and low-quality job applicants. In their setup, however, workers do not make any decisions, so whether or not they know their own productivity does not matter. This rules out any way in which workers may signal their private information, or be screened other than through firms’ direct assessment of them. Instead, in our model, workers can emit a publicly observable signal, such as education, that can be used to convey information about their productivity. In addition, Board et al. (2017) assume that firms’ direct information is independent across firms, whereas we work with a nested information structure where more-expert buyers know strictly more than less-expert ones. The rest of this article is organized as follows. Section 2 introduces the model and briefly illustrates a number of well-known applications. Section 3 provides our equilibrium definition andSection4showsthatitgivesrisetoauniqueequilibriuminthestandardsignallingenvironment where firms are uninformed. In Section 5, we characterize the set of equilibria with false positives and in Section 6 the case of false negatives. Finally, Section 7 concludes. Various extensions and all proofs are relegated to the Appendix. 2. THE ECONOMY Our model is intended to capture a generic signalling setting. For clarity, we present our results in terms of Spence’s original job market signalling model. However, the only critical assumptions are perfect competition, heterogeneous information, and the existence of some action (the signal) that is inefficient from a first-best point of view but involves different costs for different sellers. Our results therefore apply to any setting with these features, and we provide some alternative interpretations of the model below. 1.1. Related literature The refinement eliminates the traditional reasons for multiplicity that emerge in signalling games when out-of- equilibrium beliefs are left unrestricted. By contrast, the multiplicity we find in the false positives case is due to an entirely orthogonal force, namely the strategic complementary between signalling and the use of expertise, which vanishes in the classic no-information benchmark. More broadly, our work relates to the literature that followed Rothschild and Stiglitz (1976) on competition in multidimensional contracts with asymmetric information (see e.g. Miyazaki, 1977; Wilson, 1977; Dubey and Geanakoplos, 2002; Bisin and Gottardi, 2006; Netzer and Scheuer, 2014; Azevedo and Gottlieb, 2017). Similarly, there is an extensive literature that has applied the Spence (1973) signalling model to various settings, including corporate finance (Leland and Pyle, 1977; Ross, 1977), dividend policy (Bhattacharya, 1979; Bernheim, 1991), security design (DeMarzo and Duffie, 1999; DeMarzo, 2005), and brand-building (Nelson, 1974; Kihlstrom and Riordan, 1984; Milgrom and Roberts, 1986), to name a few. None of these two strands of literature, however, have attempted to move beyond the polar case where sellers are informed and buyers are uninformed. Our article provides a general analysis of how heterogeneous information affects equilibrium in all these situations. Page: 4 1–50 [19:28 2/11/2020 OP-REST200072.tex] Copyedited by: ES MANUSCRIPT CATEGORY: Article RESTUD: The Review of Economic Studies Page: 5 1–50 REVIEW OF ECONOMIC STUDIES REVIEW OF ECONOMIC STUDIES 6 Workers can choose a publicly observable level of education e, which has no effect on their productivity. If worker i chooses a level of education e and gets a job at a wage w, his utility is w−c(i)e, where w−c(i)e, where c(i)=  cL if i<λ cH if i≥λ. c(i)=  cL if i<λ cH if i≥λ. We assume cL >cH, so low types experience a higher utility cost of obtaining education We assume cL >cH, so low types experience a higher utility cost of obtaining education Up to here, the model coincides with the Spence (1973) signalling model. Our innovation is to introduce firms’ heterogeneous information about the workers they encounter. Formally, there is a continuum of firms of measure greater than one, indexed by θ ∈[0,1]. The measure of firms over [0,1] is denoted by F. When firm θ analyses worker i, it observes a direct signal x(i,θ)= 0 if i<θ 1 if i≥θ. (2) (2) If θ =λ, this signal allows the firm to perfectly infer the worker’s productivity. If θ <λ, the firm makes “false positive” mistakes: it observes positive signals from a subset of the low-type workers. If θ >λ, the firm makes “false negative” mistakes. We assume that firms can be perfectly ranked by their expertise, so one of two cases applies: either F has support in [0,λ] or it has support in [λ,1]. For instance, firms can be interpreted as being “bold” in the first and “cautious” in the second case.2 Clearly, (2) is a restrictive model of how well informed firms are: in general, firms could make both kinds of mistakes in arbitrarily correlated ways. This formulation has the advantage of providing a natural measure of a firm’s expertise since the closer θ is to λ, the better the firm is at correctly identifying a worker’s productivity. Each firm can hire at most one worker. Equivalently, we could assume that buyers have limited funds (and are unable to borrow) to leverage their expertise, which may be more natural in some of our financial market applications sketched below. Either way, some form of capacity constraints are needed to keep the problem interesting by preventing the best-informed buyers from implementing all trades. If a firm hires worker i at wage w, its profits are q(i)−w. 3. Whenever there is no heterogeneity across firms (so the support of F is concentrated at a single value of θ), our model collapses back to the standard signalling problem. If θ <λ, then all workers i∈[0,θ) are fully identified as low types, and all i∈[θ,1] look indistinguishable to all firms. Hence, the former group of workers get their first-best outcome, and a standard signalling model without expertise applies to the latter population, with a share of low types equal to (λ−θ)/(1−θ). Similarly, if θ >λ, we obtain a standard signalling environment where the share of low types is λ/θ. REVIEW OF ECONOMIC STUDIES Thus, our key innovation compared to the canonical signalling model is that buyers have access to direct, even though imperfect, information about sellers, rather than relying exclusively on self-selection. Moreover, the quality of this information is heterogeneous.3 For example, some managers have better judgement in assessing the talent of job applicants, as in Board et al. (2017), or recruiters may run tests or interviews (see e.g. Guasch and Weiss, 1980; Lockwood, 1991). Another channel of direct information about workers is through referrals. For example, Beaman and Magruder (2012) and Burks et al. (2015) show empirically that better employees make more and better referrals, and that firms differ in the degree to which their employees can predict the performance of their referrals. 2. See Farboodi and Kondor (2018) for a model that links these two cases to the business cycle. RESTUD: The Review of Economic Studies 2.1. Job market signalling There is a unit measure of workers indexed by i, uniformly distributed in the interval [0,1]. Each worker is endowed with a single unit of labour. Worker i’s productivity is q(i)=  qL if i<λ qH if i≥λ (1) (1) with qL <qH. Workers with i<λ and i≥λ are low and high types, respectively. A worker’s index i is private information. Workers of the same type but different indices i all have the same productivity; they differ only in terms of how easy it is for firms to identify them, as specified below. RESTUD: The Review of Economic Studies Page: 5 1–50 [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies [19:28 2/11/2020 OP-REST200072.tex] Page: 5 1–50 RESTUD: The Review of Economic Studies Page: 5 1–50 MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES SIGNALLING TO EXPERTS KURLAT & SCHEUER SIGNALLING TO EXPERTS by letting u(e,w)=w−c(i)e and computing the marginal rate of substitution: −∂u(e,w)/∂e ∂u(e,w)/∂w =c(i), whichishigherforlowtypes.Therearemanyothersignallingsettingsthatareformallyisomorphic to our baseline model. We briefly describe four of them. by letting u(e,w)=w−c(i)e and computing the marginal rate of substitution: −∂u(e,w)/∂e ∂u(e,w)/∂w =c(i), whichishigherforlowtypes.Therearemanyothersignallingsettingsthatareformallyisomorphic to our baseline model. We briefly describe four of them. Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rdaa068/5931860 by University of Zurich user on 05 January 2021 Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rd 2.2.1. Securitization. Consider first the security design problem of DeMarzo and Duffie (1999).Acontinuumi∈[0,1]oforiginatorseachownapoolofassetsthatgeneratefuturecashflow y. The distribution of these cash flows is privately known to the originators, and given by GL(y) if i<λ and GH(y) if i≥λ, where GH first-order stochastically dominates GL, and they have common support. The originators prefer receiving cash over holding their risky assets, for instance because they have access to other profitable investment opportunities, or because they have superior ability in valuing assets and therefore want to raise cash to fund new asset purchases. Formally, they value future cash flows from their unissued assets at discount factor α <1. They face a pool of small, heterogeneously informed, buyers who do not discount, so the efficient allocation calls for selling all assets. Of course, due to their private information, the originators face a lemons problem when selling their assets. To raise cash, they therefore issue a limited-liability security backed by their assets. DeMarzo and Duffie (1999) show that, under general conditions, it is optimal to sell a high-quality, senior claim to the assets (i.e. debt) and retain the remaining, risky equity tranche as “skin in the game,” i.e. a signal of asset quality. Let Y denote the upper bound of Gk, k =L,H; let Y −e denote the face value of the debt tranche, and w denote its price per unit of face value. Then issuer i’s payoff is u(e,w)=(Y −e)w+α  max{y+e−Y,0}dGk(y), with k =L if i<λ and k =H if i≥λ. The marginal rate of substitution is −∂u(e,w)/∂e ∂u(e,w)/∂w = w−α[1−Gk(Y−e)] Y−e . By first-order stochastic dominance (FOSD), this is higher for low types and therefore satisfies single crossing. Finally, suppose each buyer demands one unit of face value of the asset-backed security. RESTUD: The Review of Economic Studies 2.2. Other interpretations As is common to signalling models, the crucial feature is that the signal e is costly and satisfies a single-crossing property. For the job market signalling application, single crossing can be verified [19:28 2/11/2020 OP-REST200072.tex] Page: 6 1–50 MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES 7 KURLAT & SCHEUER REVIEW OF ECONOMIC STUDIES information among investors could be the result of differential experience in this particular industry, differential contacts with company insiders, or differential access to analyst reports, which make some investors better than others at distinguishing good from bad projects.4 Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rdaa068/5931860 by University of Zurich user on 05 January 2021 2.2.3. Insurance. Our model can also be mapped into the Rothschild and Stiglitz (1976) insurance problem. A continuum i∈[0,1] of risk-averse households each have wealth X and will suffer a loss of d with probability 1−q(i). q(i) is given by (1) and is privately known to the household. They face a pool of small, risk-neutral, heterogeneously informed insurance companies, so the efficient allocation calls for households to be fully insured. Insurance companies offer policies that cover the loss d minus a deductible e, in exchange for an up-front premium (1−w)(d−e), so that 1−w is the implicit probability of loss that makes the insurance contract actuarially fair. If a household gets contract (e,w), its utility is u(e,w)=qv(X −(1−w)(d−e))+(1−q)v(X −(1−w)(d−e)−e), where v(·) is the household’s von Neumann–Morgenstern utility function. The marginal rate of substitution is −∂u(e,w)/∂e ∂u(e,w)/∂w = 1 d−e  w−q  q+(1−q) v′(X−(1−w)(d−e)−e) v′(X−(1−w)(d−e)) −1 . It is straightforward to show −∂u(e,w)/∂e ∂u(e,w)/∂w = 1 d−e  w−q  q+(1−q) v′(X−(1−w)(d−e)−e) v′(X−(1−w)(d−e)) −1 . It is straightforward to show  that this is decreasing in q and therefore satisfies single crossing. If an insurance company covers one unit of losses from household i at an implicit probability 1−w, then its profits are 1−w−(1−q(i))=q(i)−w.5 Heterogeneous information among insurance companies could be the result of some of them having larger actuarial databases or more sophisticated predictive models that allow them to tell apart riskier from safer types.  that this is decreasing in q and therefore satisfies single crossing. If an insurance company covers one unit of losses from household i at an implicit probability 1−w, then its profits are 1−w−(1−q(i))=q(i)−w.5 Heterogeneous information among insurance companies could be the result of some of them having larger actuarial databases or more sophisticated predictive models that allow them to tell apart riskier from safer types. 2.2.4. Dividend policy. Finally, consider the dividend puzzle, which observes that firms pay dividends even though their tax treatment is less favourable than that of share repurchases. 4. Leland and Pyle (1977) model the cost of retention as risk-bearing by a risk-averse entrepreneur, rather than reduced investment by an entrepreneur who can reinvest his proceeds from selling the project at an above-market rate of return r =1/α−1>0, as we do here following DeMarzo and Duffie (1999). Though the interpretation is similar, the mechanics in Leland and Pyle’s model are therefore closer to the Rothschild and Stiglitz (1976) insurance application we sketch below. 5. Since each contract covers d−e losses, covering one unit of losses means that the insurance company enters into 1/(d−e) contracts. SIGNALLING TO EXPERTS Then the buyer’s payoff is qk(e)−w just like in our baseline model, where qk(e)≡  min  y Y−e,1  dGk(y), because each unit of the security has face value Y −e, so buying one unit of face value means buying 1/(Y −e) securities. Our model thus captures the equilibrium in this classic tranching problem with the additional feature that buyers are heterogeneously informed about the quality of the asset-backed security. This may involve differential knowledge of aspects of the underlying asset pool or, more importantly, special expertise in the pricing of these securities (such as proprietary pricing models). For instance, Bernardo and Cornell (1997) provide empirical evidence for significant variation in valuations of mortgage-backed securities (with the winning bid exceeding the median bid by over 17% on average) even though all buyers in their data were sophisticated investors or intermediaries. They conclude that this variability is due to differences in pricing technology (see also Eisfeldt et al., 2019). Mattey and Wallace (2001) document heterogeneity of this variability across different mortgage-backed securities, suggesting that some securities are easier to price than others. 2.2.2. Financial structure of firms. Our next example is a variant of the corporate finance problem studied by Leland and Pyle (1977). Each entrepreneur i owns a project whose future payoff, privately known, is given by (1). As in the previous example, entrepreneurs are impatient, so their own valuation for their project’s return is αq(i), and they wish to sell their project to heterogeneously informed investors. To signal the quality of their project, entrepreneurs can publicly announce that they will retain a fraction e of the equity of their firm. If an entrepreneur sells a fraction 1−e of his firm at a price per unit of w then his utility will be w(1−e)+αq(i)e. The marginal rate of substitution is −∂u(e,w)/∂e ∂u(e,w)/∂w = w−αq(i) 1−e which, again, is higher for low types. If an investor buys one unit of firm i at a unit price w, his profits are q(i)−w. Heterogeneous RESTUD: The Review of Economic Studies Page: 7 1–50 [19:28 2/11/2020 OP-REST200072.tex] MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES REVIEW OF ECONOMIC STUDIES 8 where ¯w(e,i)=  wdµ(w;e,i) is the expected wage. We denote the choice of worker i by ei. where ¯w(e,i)=  wdµ(w;e,i) is the expected wage. We denote the choice of worker i by ei. KURLAT & SCHEUER SIGNALLING TO EXPERTS 9 3. EQUILIBRIUM We adopt a Walrasian approach similar to the notion of competitive search equilibrium. There are many (non-exclusive) markets open simultaneously, each defined by a required signal e∈R+ and a wage w∈[0,qH], and there is no guarantee for either workers or firms of finding a counterparty in a market they visit. REVIEW OF ECONOMIC STUDIES The dividend signalling hypothesis (going back to Bhattacharya, 1979) explains this corporate payout policy by viewing dividends as a costly signal to convey private information about profitability (see e.g. Bernheim and Wantz, 1995, for empirical evidence). Formally, suppose a continuum i∈[0,1] of firms will each produce a random, i.i.d. stream of cash flows {yt}∞ t=1. The distribution of y is privately known to the incumbent shareholder and given by GL(y) if i<λ and GH(y) if i≥λ, where GH first-order stochastically dominates GL. The conditional means are Ei(y)=rq(i), where r is the interest rate and q(i) is given by (1). The incumbent shareholder announces a dividend e to be paid at t =1 and then sells all its shares (cum-dividend) to heterogeneously informed outside investors. Dividends are taxed at a rate τ. Furthermore, following Bhattacharya (1979), if the cash flow y1 is less than the announced dividend e, the incumbent agrees to provide the firm with a loan to finance the shortfall, at a cost β(e−y1). Letting w−τe denote the price paid by investors, the payoff for the incumbent shareholder is u(e,w)=w−τe−β  e 0 (e−y)dGk(y) with k =L if i<λ and k =H if i≥λ. The marginal rate of substitution is −∂u(e,w)/∂e ∂u(e,w)/∂w =τ +βGk(e). By FOSD, this is higher for low types and thus satisfies single crossing. An outside investor’s profit is given by the net present value of the firm’s cash flows q(i) minus the dividend tax τe minus the price paid w−τe, for a total of q(i)−w, just like in the benchmark model. [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies Page: 8 1–50 Copyedited by: ES MANUSCRIPT CATEGORY: Article 3.1. Worker’s problem Worker i first chooses a signal e and then applies for jobs. This aligns well with the natural timing, where education is determined before entering the labour market. Similarly, in the corporate finance applications, it corresponds to situations where the design of the security (the size of the junior tranche), the financial structure of the firm (the retained equity) or the amount of dividends to be paid out are determined first, and then the securities or firm shares are offered, potentially in multiple markets with different unit prices. A worker is allowed to apply to all the markets that require his chosen signal e. We assume that, for any given signal e, markets at different wages clear sequentially, starting from the highest wage, as in the “buyer’s equilibrium” studied by Wilson (1980). Therefore, a worker starts by applying to market (e,qH) and, as long as he has not been hired, continues to apply to lower- wage markets. Eventually, he gets hired in market (e,w), and does not apply to markets with lower wages. The worker understands that each choice of e is associated with some probability distribution over wage offers, with c.d.f. denoted by µ(·;e,i). The worker’s problem is therefore: max e ¯w(e,i)−c(i)e, RESTUD: The Review of Economic Studies 3.2. Firm’s problem When a firm observes applicants, it may use its information to select which ones to hire, to the extent that it can tell them apart. A feasible hiring rule for firm θ is a function χ :[0,1]→{0,1} that is measurable with respect to its information set, that is, χ (i)=χ i′ whenever x(i,θ)= x i′,θ . A firm will reject applicants with χ (i)=0 and hire workers (which we describe as χ-acceptable) from the set Iχ ={i∈[0,1]:χ (i)=1}. Let X denote the set of possible hiring rules. A firm must decide what market to hire from and what hiring rule to apply (it is without loss of generality to assume the firm hires only in one market and uses only one rule). To make this decision, the firm needs to form beliefs G(·;e,w,χ) about what workers it will be drawing from should it choose to hire in market (e,w) with hiring rule χ. If the firm thinks it will find χ-acceptable workers in market (e,w), then G(·;e,w,χ) is a probability measure on Iχ; otherwise beliefs integrate to zero: G Iχ;e,w,χ =0. Let g denote the density or p.m.f. of G, which we assume is well defined. Firm θ’s problem is: max e,w,χ q(i)−w  dG(i;e,w,χ) s.t. χ feasible for θ. Note that a firm has the choice not to hire workers by simply directing its search to a market/hiring rule where G Iχ;e,w,χ =0. We denote the choices of firm θ by (eθ,wθ,χθ). Page: 9 1–50 [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies [19:28 2/11/2020 OP-REST200072.tex] MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES 3.3. Consistency of wage distributions 3.3. Consistency of wage distributions We define demand as a measure on the set of wages, signals and hiring rules 0,qH  ×R+×X. For any set of wages W0 ⊆ 0,qH  , signals E0 ⊆R+ and hiring rules X0 ⊆X, demand is the total number of firms who make those choices: D(E0,W0,X0)≡ I(eθ ∈E0)I(wθ ∈W0)I(χθ ∈X0)dF(θ). (3) (3) We then impose the following consistency condition on firms’ hiring and the distribution of wage offers received by workers: REVIEW OF ECONOMIC STUDIES REVIEW OF ECONOMIC STUDIES 10 6. Note that equation (4) and Condition 1 rule out a situation with excess demand for a type of worker because it would imply a job-finding probability higher than one. In other words, in any given active market, the firm-to-worker ratio (or market tightness), which generally differs across worker types, never exceeds one. RESTUD: The Review of Economic Studies Condition 1 Page: 10 1–50 [19:28 2/11/2020 OP-REST200072.tex] MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES KURLAT & SCHEUER SIGNALLING TO EXPERTS Firm θ α β γ Measure f (θ) 0.5 1 1.5 Wage wθ wH wH wL Hiring rule χθ (A) 1 1 1 χθ (B) 0 1 1 χθ (C) 0 0 1 Beliefs g(A;e,wθ,χθ) 1 0.5 0 g(B;e,wθ,χθ) 0 0.5 1/3 g(C;e,wθ,χθ) 0 0 2/3 KURLAT & SCHEUER SIGNALLING TO EXPERTS 11 Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rda Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rdaa068/5931860 by University of Zurich user on 05 January 2021 Firms of type α and β hire at wage wH. Type-α firms only hire i=A workers while type-β firms hire type i=A and i=B workers. Type-γ firms hire at wage wL, and they accept everyone. For now, we take firms’ beliefs simply as given and will show below how they are derived. Given beliefs and firms’ demand decisions, we can compute workers’ probabilities of being hired at wages wH and wL using (3) and (4): dµ(wH;e,A)=g(A;e,wH,χα)    1 D(e,wH,χα)    0.5 +g A;e,wH,χβ    0.5 D e,wH,χβ    1 =1 dµ(wH;e,B)=g(B;e,wH,χα)    0 D(e,wH,χα)    0.5 +g B;e,wH,χβ    0.5 D e,wH,χβ    1 =0.5 dµ(wL;e,B)=g B;e,wL,χγ    1 3 D e,wL,χγ    1.5 =0.5 dµ(wL;e,C)=g C;e,wL,χγ    2 3 D e,wL,χγ    1.5 =1. In words, type-A workers get hired for sure at wage wH, type-B workers get hired with probability 0.5 at wage wH and probability 0.5 at wage wL, and type-C workers get hired for sure at wage wL. Condition 1 imposes no constraints on µ when I(ei =e)=0, i.e., no constraints on i-workers’ chances of being hired in markets where there are no i-applicants. For these markets, we impose the condition: RESTUD: The Review of Economic Studies Condition 1 µ(w;e,i)I(ei =e)= ˜w≤w,X g(i;e, ˜w,χ)dD(e, ˜w,χ) for all e,w,i. The indicator I(ei =e) takes the value 1 if worker i chooses signal e and zero otherwise; µ(w;e,i) is his probability of getting a wage at most w. Hence, the left-hand side of Condition 1 is the total number of i-type workers with signal e who will obtain wages at most w. Moreover, since beliefs are rational, a firm imposing hiring rule χ in market (e,w) will hire g(i;e,w,χ) workers of type i. Adding these hires across all hiring rules and wages below w using the demand measure results in the right-hand side of Condition 1, which is the total number of i-type workers hired in markets with signal e and wages up to w. Condition 1 simplifies when i-type workers choose signal e (so I(ei =e)=1), and they have strictly positive probability of finding a job at wage w, so the c.d.f. µ makes a discrete step of size dµ(w;e,i). Then, Condition 1 can be written as: dµ(w;e,i)=  X g(i;e,w,χ)dD(e,w,χ) I(ei =e) . (4) (4) This is the standard rationing rule under frictionless matching, by which the probability dµ for an i-type worker of finding a job at wage w is equal to the ratio of i-type workers demanded by firms in that market over their supply, which is equal to 1.6 The more general formulation of Condition 1 also deals with cases where µ may increase continuously over some interval of wages, so the probability of being hired in any single market is zero but there is an associated probability density. Both situations will occur in the equilibria we find below. Example 1 To illustrate the meaning of equations (3) and (4), consider the following example. There are three types of workers i∈{A,B,C}, each of mass one, who choose signal e, and three firm types θ ∈{α,β,γ } who hire in markets that require e. The measures of each type of firm, the wage at which they recruit, their hiring rules, and beliefs are: 6. Note that equation (4) and Condition 1 rule out a situation with excess demand for a type of worker because it would imply a job-finding probability higher than one. In other words, in any given active market, the firm-to-worker ratio (or market tightness), which generally differs across worker types, never exceeds one. 3.4. Consistency of beliefs 3.4. Consistency of beliefs Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rdaa068/5931860 by University of Zurich user on 05 January 2021 Consider a firm that hires in market (e,w) with hiring rule χ. The pool of workers available for hire in this market includes i-type workers only if they choose education e and have not already been hired at higher wages. Therefore, it includes I(ei =e)µ(w;e,i) i-type workers. If firms simply chose at random from the χ-acceptable subset of this pool, then Bayes’ Rule would imply that rational beliefs should be: g(i;e,w,χ)= I(ei =e)χ (i)µ(w;e,i)  iI(ei =e)χ (i)µ(w;e,i)di, (5) (5) However, if firms with different hiring rules hire sequentially in the same market, firms that hire earlier skew the pool that later firms face, so rational beliefs depend on the order in which firms hire within a market. Kurlat (2016) assumes that there exist separate markets for each wage combined with each possible way of ordering rules, and firms and workers choose which markets to trade in, making the order endogenous. He shows that under “false positives” information, less selective firms hire first. This implies that no one’s sample is skewed by earlier firms, so it is as if all firms were drawing from the entire pool of χ-acceptable applicants, and (5) applies. However, if firms with different hiring rules hire sequentially in the same market, firms that hire earlier skew the pool that later firms face, so rational beliefs depend on the order in which firms hire within a market. Kurlat (2016) assumes that there exist separate markets for each wage combined with each possible way of ordering rules, and firms and workers choose which markets to trade in, making the order endogenous. He shows that under “false positives” information, less selective firms hire first. This implies that no one’s sample is skewed by earlier firms, so it is as if all firms were drawing from the entire pool of χ-acceptable applicants, and (5) applies. Example 2 Continuing on Example 1, we illustrate how to derive firms’ beliefs using (5) and workers’ hiring probabilities computed above. In market (e,wH), both firms of type α and β hire, but β-firms get to pick first. Since all three worker types apply in this market but firms of type β reject type-C workers, their beliefs are g(i;e,wH,χβ)= 1 1+1+0 =0.5 for i=A,B, g(C;e,wH,χβ)= 0 1+1+0 =0. REVIEW OF ECONOMIC STUDIES REVIEW OF ECONOMIC STUDIES 12 Condition 2 µ is weakly decreasing in i Condition 2 says that higher-i workers expect higher wages in a FOSD sense. This rules out low types being more optimistic than high types about the wages they would obtain for some off-equilibrium signals. This condition can be derived from more primitive assumptions. Suppose workers believe that firms which hire in markets with off-equilibrium signals use optimal hiring rules. These are weakly monotonic: no firm finds it optimal to accept worker i while rejecting worker i′ >i. If firms draw workers randomly from those that satisfy their hiring rule (as discussed in the next section), applying Condition 1 to off-equilibrium signals implies that higher types will be hired at weakly higher rates than lower types, resulting in FOSD higher wages. Page: 11 1–50 [19:28 2/11/2020 OP-REST200072.tex] MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES 3.4. Consistency of beliefs This means that the unit measure of type-β firms hire a total of half a type-A and half a type-B worker. Firms of type α hire next in this market, and they reject all but type-A workers, so their beliefs are trivially: g(A;e,wH,χα)= 1 1+0+0 =1, g(i;e,wH,χα)= 0 1+0+0 =0 for i=B,C. Hence, the measure 0.5 of type-α firms hires the remaining half type-A worker. Finally, firms of type γ hire in market (e,wL) accepting all applicants. Since type-A workers have been hired for sure at the higher wage wH, they are no longer applying at wL, so g(A;e,wL,χγ )=0. γ -firms thus face an applicant pool of half a type-B worker and one type-C worker, so Bayes’ rule implies beliefs g(B;e,wL,χγ )= 1 2 0+ 1 2 +1 = 1 3, g(C;e,wL,χγ )= 1 0+ 1 2 +1 = 2 3. Condition 3 If  iI(ei =e)χ (i)µ(w;e,i)di>0, then Condition 3 If  iI(ei =e)χ (i)µ(w;e,i)di>0, then Condition 3 If  iI(ei =e)χ (i)µ(w;e,i)di>0, then Condition 3 If  iI(ei =e)χ (i)µ(w;e,i)di>0, then Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rdaa068/5931860 by University of Zurich user on 05 January 2021 i q(i)dG(i;e,w,χ)=  iq(i)I(ei =e)χ (i)µ(w;e,i)di  iI(ei =e)χ (i)µ(w;e,i)di . Condition 3 says that beliefs must be such that the average productivity that firms expect to get if they hire in market (e,w) with hiring rule χ must be the same as if they were drawing from the entire pool. For the false positives case, it holds because it is implied by (5). For the false negatives case, it holds because the only cases where (5) might not hold are when firms only accept high type workers. Rather than explicitly allowing for endogenous ordering of firms’ trades and re- deriving these results, we incorporate them directly into our definition of equilibrium by imposing Condition 3. Example 3 Continuing further on Examples 1 and 2, suppose that workers of type A and B have productivity qH while worker type C has productivity qL. If less selective firms hire first, as assumed in Example 2, then by Bayes’ rule, firms α and β expect average quality qH, while firm γ expects average quality (2qL +qH)/3, so Condition 3 applies. Suppose now, by contrast, that more selective firms hire first, so firm α gets to pick workers before firm β in market (e,wH). Since firm α hires half a type-A worker first, this skews firm β’s remaining applicant sample, and hence its beliefs are as follows: g(A;e,wH,χβ)= 1 2 0+ 1 2 +1 = 1 3, g(B;e,wH,χβ)= 1 0+ 1 2 +1 = 2 3, which violates equation (5). Nonetheless, since both workers A and B have quality qH, firm β expects average quality qH, so Condition 3 remains satisfied. g(A;e,wH,χβ)= 1 2 0+ 1 2 +1 = 1 3, g(B;e,wH,χβ)= 1 0+ 1 2 +1 = 2 3, which violates equation (5). Nonetheless, since both workers A and B have quality qH, firm β expects average quality qH, so Condition 3 remains satisfied. which violates equation (5). Nonetheless, since both workers A and B have quality qH, firm β expects average quality qH, so Condition 3 remains satisfied. Condition 3 only applies to markets where the denominator is positive, i.e., where there are χ-acceptable workers. 7. This issue does not arise in Kurlat (2016). He only studies unidimensional contracts, where the price is the only contract dimension. This rules out signalling, and thus there are no markets corresponding to off-equilibrium signals, and no need to specify how beliefs react to these off-equilibrium signals. In his setup, beliefs must satisfy Condition 3, but there is no equivalent to the condition we impose in the following. Condition 4 For any i in the support of G(·;e,w,χ): RESTUD: The Review of Economic Studies As a result, the 1.5 measure of type-γ firms hire all the remaining workers. As a result, the 1.5 measure of type-γ firms hire all the remaining workers. As a result, the 1.5 measure of type-γ firms hire all the remaining workers. Instead, under “false negatives” information, Kurlat (2016) shows that there may be markets where more selective firms hire first, and (5) does not apply. However, this possibility only arises among firms who only accept high types. Therefore, even if early firms skew the pool, later firms still hire only high types, just as if they had been the first in line. Therefore, the following weaker condition still holds: [19:28 2/11/2020 OP-REST200072.tex] Page: 12 1–50 Page: 12 1–50 RESTUD: The Review of Economic Studies MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES KURLAT & SCHEUER SIGNALLING TO EXPERTS KURLAT & SCHEUER SIGNALLING TO EXPERTS 13 We summarize the above discussion in the following equilibrium definition: We summarize the above discussion in the following equilibrium definition: Definition 1 An equilibrium consists of (i) a signal ei for each worker i; (ii) a hiring decision (eθ,wθ,χθ) for each firm θ; (iii) wage distributions µ(·;e,i); and (iv) beliefs G(·;e,w,χ) that satisfy: 1. Worker optimization. ei solves worker i’s problem, taking µ as given. 1. Worker optimization. ei solves worker i’s problem, taking µ as given. p i p , g µ g 2. Firm optimization. (eθ,wθ,χθ) solves firm θ’s problem, taking G as given. m optimization. (eθ,wθ,χθ) solves firm θ’s problem, ta 3. Consistency. µ, G, (eθ,wθ,χθ) and ei satisfy Conditions 1 to 5. REVIEW OF ECONOMIC STUDIES 14 2. e solves worker i’s problem 3. µ(w;e,i)>0 The alternative is that a firm is certain that it cannot find χ-acceptable workers in market (e,w). We impose that a firm can only reach that conclusion if guaranteeing χ-acceptable workers a job with a wage at least w is not enough to persuade them to choose signal e. Formally, we impose: Condition 5 If G Iχ;e,w,χ =0, then µ(w;e,i)=0 for all i such that χ (i)=1. Conditions 4 and 5 are closely related to the infinite-tightness condition in Guerrieri et al. (2010) and Guerrieri and Shimer (2014). In their setup, for every market there either is at least one worker type who finds that market optimal, or the market tightness is infinite. In the first case, this allows firms to have well-defined beliefs about which workers they would encounter; in the second, workers would match for sure. Conditions 4 and 5 generalize this idea by imposing it separately for each χ-acceptance group. For each χ, it has to be the case that either some χ-acceptable worker finds visiting this market optimal (in which case this worker can be in the support of well-defined beliefs) or all χ-acceptable workers are guaranteed jobs. Within a given market, which of these possibilities applies can be different across different hiring rules χ. Condition 3 If  iI(ei =e)χ (i)µ(w;e,i)di>0, then The key challenge in constructing a tractable equilibrium notion is how to discipline firms’ beliefs in markets that are empty of χ-acceptable workers. We propose a refinement which guarantees equilibrium uniqueness in the no-information benchmark, and at the same time preserves equilibrium existence throughout.7 For markets in which no χ-acceptable workers apply, there are two possibilities: either the firm nevertheless believes it could find χ- acceptable workers and G(·;e,w,χ) is a well-defined probability measure, or the firm believes the market is empty and G Iχ;e,w,χ =0. χ For the first case, we require that beliefs only place weight on χ-acceptable workers that would in fact be willing to look for a job in market (e,w). In other words, a firm can never expect to find in market (e,w) a worker who could obtain higher utility by choosing a different signal, or who can find a job for sure with the same signal but a higher wage. Formally, we require: 1. χ (i)=1 1. χ (i)=1 Page: 13 1–50 [19:28 2/11/2020 OP-REST200072.tex] MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES REVIEW OF ECONOMIC STUDIES RESTUD: The Review of Economic Studies 4. PURE SIGNALLING We now characterize equilibrium for the case where F is a point mass at θ =0 (or equivalently at θ =1), i.e. when all firms are completely uninformed. This corresponds to the classic signalling environment. For this case, the least-cost separating allocation emerges as the unique equilibrium. In this allocation, low types get no education, high types get just enough education to separate with: e∗= qH −qL cL , (6) (6) and each type is paid their own productivity, as illustrated in Figure 1. Proposition 1 If F is a point mass at θ =0, there is a unique equilibrium, given by: 1. Worker decisions: ei =  0 if i<λ e∗if i≥λ (7) ei =  0 if i<λ e∗if i≥λ (7) 1. Worker decisions: (7) 2. Firm decisions: e=e∗,w=qH,χ (i)=1 for a measure λ of firms e=0,w=qL,χ (i)=1 for a measure 1−λ of firms (8) e=e∗,w=qH,χ (i)=1 for a measure λ of firms e=0,w=qL,χ (i)=1 for a measure 1−λ of firms (8) (8) RESTUD: The Review of Economic Studies Page: 14 1–50 RESTUD: The Review of Economic Studies Page: 14 1–50 [19:28 2/11/2020 OP-REST200072.tex] Page: 14 1–50 Page: 14 1–50 MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES KURLAT & SCHEUER SIGNALLING TO EXPERTS 15 KURLAT & SCHEUER Figure 1 The least-cost separating allocation. Figure 1 The least-cost separating allocation. 3. Wage distributions: 3. Wage distributions: butions: µ(w;e,i)=I w≥min  qL +cLe,qH +cH e−e∗  (9) g(i;e,w,χ)= ⎧ ⎪⎪⎨ ⎪⎪⎩ 1 λI(i<λ) if e<e∗,w≥qL +cLe 1 1−λI(i≥λ) if e≥e∗,w≥qH +cH e−e∗ 0 otherwise (10) (9) 4. Beliefs: 4. Beliefs: (10) The equilibrium is constructed by setting the distribution µ as a point mass at the lower envelope of the indifference curves of both types, which makes low types indifferent between any e∈ 0,e∗ and high types indifferent between any e≥e∗. Therefore, e=0 for low types and e=e∗for high types is indeed optimal. This is then sustained by firms’ belief that in the range 0,e∗ they will only encounter low types above the lower envelope and no one at all below, and similarly for high types above e∗. Hence there are no profits in any market, and firms are trivially optimizing. A measure F(1)−1 remain inactive, for instance by choosing a market with e=0 and w<qL (and selection rule χ(i)=1 for all i). The key step in establishing uniqueness is to rule out pooling, i.e. 8. Rosenthal and Weiss (1984) and Dasgupta and Maskin (1986) show that mixed-strategy equilibria do exist. RESTUD: The Review of Economic Studies REVIEW OF ECONOMIC STUDIES 16 if a small firm tries to hire in this market, it will not attract any high types, because they know that they will be competing with all the low types for an infinitesimal chance to be hired and will have to settle for w=qL if they are not. Formally, this is captured by the assumption that beliefs do not depend on whether a firm decides to recruit in a particular market. This is the same logic that leads to existence and uniqueness in Guerrieri et al. (2010). 5. FALSE POSITIVES We now consider the case where F has full support on [0,λ] and is continuous. We start by characterizing the equilibrium when workers do not have a way of signalling (“no signalling”). Next, we characterize a class of possible equilibria (“partial signalling”) that involve signalling by a fraction of the high types. We then show that any equilibrium must be either the pure signalling equilibrium described in Section 4, a no signalling equilibrium or a partial signalling equilibrium, and find conditions for each of them to arise. 5.1. No signalling We now characterize the equilibrium for the case where workers are constrained to choose e=0, which is the case studied by Kurlat (2016). Let θN and wN be defined as the solutions to: λ θN 1 1−θ dF(θ)=1. (11) (11) θN = wN −(λqL +(1−λ)qH) wN −qL (12) (12) In equilibrium, all high-type workers (and some low-type workers) are hired at some wage wN, and the low-type workers who fail to find a job at wage wN are hired at wage qL. To understand the meaning of conditions (11) and (12), observe first that if firm θ hires at wage wN and imposes hiring rule χθ (i)=I(i≥θ), it hires randomly from the interval [θ,1]. Therefore it ends up hiring a low type with probability λ−θ 1−θ and a high type with probability 1 θ 1−λ 1−θ . Its expected profits will be: (θ)= (λ−θ)qL+(1−λ)qH 1−θ −wN. Profits are increasing in θ: firms whose information enables them to screen out a higher proportion of low types will be hiring from a better pool of workers. Only firms that are sufficiently confident in their ability to tell workers apart will be willing to hire in this market; they will make profits if and only if they are above the cutoff θN defined by equation (12), which satisfies (θN)=0. 1 θ 1−λ 1−θ . Its expected profits will be: (θ)= (λ−θ)qL+(1−λ)qH 1−θ −wN. Profits are increasing in θ: firms whose information enables them to screen out a higher proportion of low types will be hiring from a better pool of workers. Only firms that are sufficiently confident in their ability to tell workers apart will be willing to hire in this market; they will make profits if and only if they are above the cutoff θN defined by equation (12), which satisfies (θN)=0. For all 1−λ high-type workers to be hired at wage wN, it must be that there are enough firms in the range θN,λ  to hire all of them. Given that in expectation firm θ hires 1−λ 1−θ high-type workers, this means that θN must satisfy (11).9 9. Note that low-type workers do not guarantee themselves a job at wage wN since some of the firms hiring in this market will reject them; only firms θ ∈ θN,i  hire in market at wage wN and accept worker i. 9. Note that low-type workers do not guarantee themselves a job at wage wN since some of the firms hiring in this market will reject them; only firms θ ∈ θN,i  hire in market at wage wN and accept worker i. Therefore, his probability of finding a job at wage wN is 4. PURE SIGNALLING markets with positive supply of both high and low types. This follows the standard logic based on single crossing. If there was pooling at a level of education e′, then high types would require a lower wage than low types to be willing to choose e=e′+ǫ. Hence firms that consider hiring in a market with e=e′+ǫ and a wage that leaves high types indifferent must believe that they will only encounter high types, which for small ǫ must be more profitable than hiring at e′. The types of deviations to pooling contracts that may lead to non-existence of a pure-strategy equilibrium in Rothschild and Stiglitz (1976) are not profitable because each firm perceives itself to be small.8 A job with e=0 and w=qH −cHe∗+ǫ is strictly preferred to the equilibrium by all workers and if a firm was large and could hire the entire population it could break the equilibrium by offering to hire everyone in this market, which would be profitable for low values of λ. Here, [19:28 2/11/2020 OP-REST200072.tex] Page: 15 1–50 RESTUD: The Review of Economic Studies MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES REVIEW OF ECONOMIC STUDIES RESTUD: The Review of Economic Studies Page: 16 1–50 KURLAT & SCHEUER SIGNALLING TO EXPERTS KURLAT & SCHEUER SIGNALLING TO EXPERTS 17 The following result, proved by Kurlat (2016), establishes that this is the unique equilibrium. Proposition 2 If workers are constrained to choose e=0, there is a unique equilibrium where: 1. Firms with θ ≥θN hire at wage wN and other firms are indifferent between hiring at wage qL or not hiring. 1. Firms with θ ≥θN hire at wage wN and other firms are indifferent between hiring at wage qL or not hiring. 2. High types are hired at wN with probability 1. 3. Beliefs follow (5) for all w≥qL and are zero for lower wages. Conditions (11) and (12) are the analogues of conditions (19) and (20) in Kurlat (2016). There are four minor differences. First, Kurlat (2016) assumes that assets are divisible and the law of large numbers applies, so he has exact pro-rata rationing instead of probabilistic rationing. Under risk neutrality, this distinction does not matter. Second, he allows some sellers to have a positive value for retaining the good, while we assume it to be zero so workers sell all their labour endowment in equilibrium. Third, Kurlat (2016) assumes qL =0, so the one-price equilibrium he finds is equivalent to the two-price equilibrium we have, where some low types trade at price qL. Finally, he models buyers’ capacity constraints in terms of dollars rather than in terms of quantities, so the price appears in the market-clearing condition. Note that as F approaches a point mass at θ =0, then equations (11)–(13) imply that θN →0, wN →λqL +(1−λ)qH and dµ wN;i →1. If firms are uninformed and workers cannot signal, then all workers get hired for sure at a wage equal to the average productivity. This is the pure Akerlof (1970) outcome: all workers have the same reservation wage (zero) so there is no adverse selection at the pooled price. 5.1. No signalling Therefore, his probability of finding a job at wage wN is i dµ wN;i = i θN 1 1−θ dF(θ) for i∈ θN,λ  . (13) (13) This probability is increasing in i since higher-i low types mislead more firms into hiring them at wage wN. It is equal to zero for workers i<θN since no firm that would accept them hires at wage wN. RESTUD: The Review of Economic Studies Page: RESTUD: The Review of Economic Studies Page: 16 1–50 Page: 16 1–50 [19:28 2/11/2020 OP-REST200072.tex] Page: 16 1–50 MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES 5.2. Partial signalling In a partial signalling equilibrium, low-type workers choose e=0. They are hired with some probability in at wage wP, defined by: In a partial signalling equilibrium, low-type workers choose e=0. They are hired with some probability in at wage wP, defined by: wP =qH −cHe∗. (14) (14) and otherwise at wage qL. High-type workers choose either e=0 (and are hired for sure at wage wP) or e=e∗(and are hired for sure at wage qH, which gives them the same utility). Let πP be the fraction of high types that choose e=0. If firm θ hires in market 0,wP with hiring rule χθ (i)=I[i≥θ], it will hire a high type with probability πP(1−λ) λ−θ+πP(1−λ), so its profits will be (θ)= (λ−θ)qL+πP(1−λ)qH λ−θ+πP(1−λ) −wP. This defines a cutoff θP such that firms can make profits in market 0,wP if and only if θ >θP: θP =λ−πP(1−λ) qH −wP wP −qL . (15) (15) Firms with θ <θP are indifferent between hiring in market (0,qL) (with low-type applicants only), in market e∗,qH (with high-type applicants only), or not hiring at all, since they make zero profits in any case. The calculations above assume that some high types are indeed willing to apply to market 0,wP . For this to be true, it must be the case that they are sure they will find a job, since they can always guarantee themselves the same utility by choosing e=e∗and getting a job that pays w=qH. This means that there must be enough firms above θP to hire all πP(1−λ) high types RESTUD: The Review of Economic Studies Page: 17 1–50 [19:28 2/11/2020 OP-REST200072.tex] Page: 17 1–50 RESTUD: The Review of Economic Studies Page: 17 1–50 MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES REVIEW OF ECONOMIC STUDIES 18 Figure 2 Indifference and market-clearing conditions for the false positives case. Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rda Figure 2 Indifference and market-clearing conditions for the false positives case. who forgo education and apply to market 0,wP . By the arguments above, each firm θ ≥θP hires πP(1−λ) λ−θ+πP(1−λ) high types, so in equilibrium we need λ θP 1 λ−θ +πP(1−λ)dF(θ)=1. (16) (16) By the same reasoning as in the no-signalling case, low types are hired in market 0,wP with probability dµ wP;0,i =  i θP 1 λ−θ+πP(1−λ)dF(θ). RESTUD: The Review of Economic Studies KURLAT & SCHEUER SIGNALLING TO EXPERTS KURLAT & SCHEUER 19 KURLAT & SCHEUER SIGNALLING TO EXPERTS 19 Figure 3 Active markets in each class of equilibrium. Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rdaa068/5931860 by University of Zurich user on 05 January 2021 Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rda Active markets in each class of equilibrium. Active markets in each class of equilibrium. Note that a no-signalling equilibrium corresponds to a situation where the market-clearing condition is above the indifference condition at π =1, as in Figure 2. This means that if π =1 (no high types signal) there are more firms willing to hire at wP than the total mass of workers they would accept. As a result, high-θ firms “bid up” the wage to wN >wP, leading some firms to drop out until the number of firms willing to pay this wage equals the number of high- type workers. Moreover, a pure signalling equilibrium is a special case of a partial signalling equilibrium, with πP =0 and θP =λ. Figure 3 shows which markets are active in each class of equilibrium. RESTUD: The Review of Economic Studies 5.2. Partial signalling λ θ+π (1 λ) Theindifferencecondition (15)andthemarket-clearingcondition (16)definetworelationships between the cutoff firm θP and the fraction of high types πP that forgo signalling. Both of these relationships are downward sloping, as shown in Figure 2. The indifference condition (15) is downward-sloping because if more high types decide to forgo education, they improve the pool of workers available for hire in market 0,wP , allowing less-informed firms to earn profits. The same is true for the market-clearing condition (16) because if more high types decide to forgo education, they can only find jobs in market 0,wP if additional firms decide to hire there. In other words, there is a complementarity between entry into market 0,wP by firms and by high-type workers. The more high types forgo education, the more profitable it is for any given firm to hire in 0,wP ; the more firms hire in 0,wP , the more high-type workers can refrain from signalling. The strategic complementarity implies that there can be multiple intersections of (15) and (16), and possibly multiple partial signalling equilibria. This source of multiplicity is different from the forces that may lead to multiplicity in Akerlof (1970) (where adverse selection depends on the price) or in canonical signalling models (where different off-equilibrium beliefs can be self- sustaining). Indeed, with our refinement on beliefs, the uninformed-firms benchmark has a unique equilibrium (Proposition 1), as does the no-signalling case (Proposition 2). The multiplicity we identify here relies on the presence of both signalling and heterogeneous information among firms. Page: 18 1–50 [19:28 2/11/2020 OP-REST200072.tex] MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES KURLAT & SCHEUER SIGNALLING TO EXPERTS KURLAT & SCHEUER SIGNALLING TO EXPERTS 5.3. Candidate equilibria The following result establishes that any equilibrium must belong to one of the three cases described above. Proposition 3 Any equilibrium is of one of the three following types: Proposition 3 Any equilibrium is of one of the three following types: 1. Pure signalling. Low types choose e=0 and high types choose e=e∗. 2. No signalling. All workers choose e=0. Firms hire in market 0,wN if and only if θ ≥θN. θN and wN satisfy (11) and (12); and wN ≥wP. P 3. Partial signalling. Low types choose e=0; a fraction πP of high types choose e=0 and the rest choose e=e∗. Firms hire in market 0,wP if and only if θ ≥θP. πP and θP satisfy (15) and (16). 3. Partial signalling. Low types choose e=0; a fraction πP of high types choose e=0 and the rest choose e=e∗. Firms hire in market 0,wP if and only if θ ≥θP. πP and θP satisfy (15) and (16). The key to proving Proposition 3 is to establish that high and low types cannot coexist at any level of education other than e=0, so there is no pooling at positive signalling levels. The logic is similar, though somewhat subtler, to that in the uninformed-firms benchmark. Suppose that low and high types coexisted in some market (e,w) with e>0, as illustrated in Figure 4. With differentially informed firms, the standard argument that rules this out by considering market e′,w′ does not go through. Some firms hiring in market (e,w) may be screening out low types, so low types’ expected wage with signal e could be lower than that of high types. Thus, it is possible that both high and low types find e′,w′ more attractive than (e,w). Instead, we can rule out pooling in market (e,w) by contradiction, as follows. There are two possibilities: either the highest firm that hires in market (e,w) has θ =λ (i.e. it can tell workers Page: 19 1–50 [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES 20 REVIEW OF ECONOMIC STUDIES Figure 4 Ruling out pooling at e>0. apart perfectly), or the highest firm to hire in this market has θ <λ. In the first case, we arrive at a contradiction by considering the beliefs of firm θ =λ about market 0,w′′ . 5.3. Candidate equilibria This firm’s beliefs can only include high types so it only cares about the wage it pays. Therefore this firm finds market 0,w′′ preferable over market (e,w), leading to a contradiction. If instead the highest firm that hires in market (e,w) has θ <λ, then any low-type worker in the range i∈(θ,λ) has the same chance of getting a job in market (e,w) as a high type. If this is so, then firm θ can apply the standard cream-skimming deviation by hiring in market e′,w′ . Firm θ can reject all low types who prefer e′,w′ over (e,w), so it can guarantee itself high types by hiring in this market, which contradicts the premise that it hires in market (e,w). 0 REVIEW OF ECONOMIC STUDIES Figure 4 Ruling out pooling at e>0. REVIEW OF ECONOMIC STUDIES REVIEW OF ECONOMIC STUDIES 20 Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rda Figure 4 Ruling out pooling at e>0. apart perfectly), or the highest firm to hire in this market has θ <λ. In the first case, we arrive at a contradiction by considering the beliefs of firm θ =λ about market 0,w′′ . This firm’s beliefs can only include high types so it only cares about the wage it pays. Therefore this firm finds market 0,w′′ preferable over market (e,w), leading to a contradiction. If instead the highest firm that hires in market (e,w) has θ <λ, then any low-type worker in the range i∈(θ,λ) has the same chance of getting a job in market (e,w) as a high type. If this is so, then firm θ can apply the standard cream-skimming deviation by hiring in market e′,w′ . Firm θ can reject all low types who prefer e′,w′ over (e,w), so it can guarantee itself high types by hiring in this market, which contradicts the premise that it hires in market (e,w). RESTUD: The Review of Economic Studies Page: 20 1–50 5.4. Existence At education levels higher than eD θ , firm θ can only believe that it will encounter exclusively high types, because high types would be willing to choose these education levels for a lower wage than worker i=θ. Hi i i k t lik D D i i il t th ki i d i ti th t d t Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rdaa068/5931860 by University of Zurich user on 05 January 2021 encounter low types in e-markets. The reason is that since u(θ)<wP, worker i=θ will be willing to choose e for a lower wage than high types would. Hence, one can specify beliefs such that firm θ does not want to recruit at education level e. However, for large e that is no longer the case because education is more costly for low types. eD θ ,wD θ is defined by the intersection of the equilibrium indifference curves of worker i=θ and high types. At education levels higher than eD θ , firm θ can only believe that it will encounter exclusively high types, because high types would be willing to choose these education levels for a lower wage than worker i=θ. encounter low types in e-markets. The reason is that since u(θ)<wP, worker i=θ will be willing to choose e for a lower wage than high types would. Hence, one can specify beliefs such that firm θ does not want to recruit at education level e. However, for large e that is no longer the case because education is more costly for low types. eD θ ,wD θ is defined by the intersection of the equilibrium indifference curves of worker i=θ and high types. At education levels higher than eD θ , firm θ can only believe that it will encounter exclusively high types, because high types would be willing to choose these education levels for a lower wage than worker i=θ. g g Hiring in a market like eD θ ,wD θ is similar to the cream-skimming deviations that are used to break putative pooling equilibria in Rothschild and Stiglitz (1976) and related models, including the uninformed-firms benchmark of Section 4. In candidate equilibria where some high types choose e=0, they end up being hired in market 0,wP , where they are pooled with low types. 5.4. Existence So far, we have described the possible candidates for equilibrium but we have not proved that any of them is actually an equilibrium. We now show that the candidate equilibria described above may or may not actually be equilibria. We construct a class of possible deviations and derive an easy-to-verify condition to determine whether these deviations are profitable. We then show that checking this condition is sufficient to establish an equilibrium, and prove that at least one equilibrium always exists. Consider first a candidate partial signalling equilibrium. Define eD θ ,wD θ as the lowest-wage market where equilibrium requires that the beliefs of firm θ ∈ θP,λ only include high types. A necessary condition for equilibrium is that firm θ cannot increase its profits by recruiting in market eD θ ,wD θ instead of market 0,wP . The location of market eD θ ,wD θ is illustrated in Figure 5. Worker i=θ is the lowest-i low-type worker that firm θ cannot filter out. In equilibrium, this worker obtains expected utility: u(θ)= qL +  θ θP 1 πP(1−λ)+λ−t dF(t) wP −qL (17) (17) by getting a wage of either wP or qL with the equilibrium probabilities. For small but positive levels of education e, it is consistent with equilibrium for firm θ to believe that it will only RESTUD: The Review of Economic Studies Page: 20 1–50 [19:28 2/11/2020 OP-REST200072.tex] [19:28 2/11/2020 OP-REST200072.tex] Page: 20 1–50 Page: 20 1–50 MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES KURLAT & SCHEUER SIGNALLING TO EXPERTS 21 KURLAT & SCHEUER SIGNALLING TO EXPERTS 2 Figure 5 Beliefs for firm θ KURLAT & SCHEUER SIGNALLING TO EXPERTS 21 Figure 5 Beliefs for firm θ encounter low types in e-markets. The reason is that since u(θ)<wP, worker i=θ will be willing to choose e for a lower wage than high types would. Hence, one can specify beliefs such that firm θ does not want to recruit at education level e. However, for large e that is no longer the case because education is more costly for low types. eD θ ,wD θ is defined by the intersection of the equilibrium indifference curves of worker i=θ and high types. [19:28 2/11/2020 OP-REST200072.tex] 5.4. Existence Just like in the benchmark, the possible deviation involves peeling off high types by requiring an action that is more costly for low types than for them. However, there are two important differences. First, unlike in the Rothschild and Stiglitz (1976) model, purely local deviations do not work. A firm cannot cream-skim the high types off a pooling contract by requiring a small amount of signalling. Since low types are hired from the 0,wP pool at lower rates than high types, they obtain lower utility. Therefore, they find deviations more attractive than high types as long as they involve only a small amount of extra signalling. In order to repel the low types, the deviating firm must require a sufficiently larger signal. Second, in order to profit, the deviating firm must use both sources of information in combination: direct assessment and signalling. A completely uninformed firm cannot profitably deviate because in order to repel the lowest-i low types (who cannot get jobs at 0,wP at all) it must require e=e∗and therefore pay at least qH to attract high types, at which point the deviation is no longer profitable. In order to profitably deviate, a firm must possess sufficient expertise to be able to reject the lowest-i low types directly and then rely on the signal to screen out the higher-i low types. y A candidate partial pooling equilibrium can only be an equilibrium if, for every θ ∈(θP,λ), the profits firm θ can obtain in market eD θ ,wD θ by hiring only high types are weakly lower than Page: 21 1–50 [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES 22 REVIEW OF ECONOMIC STUDIES Figure 6 Candidate equilibria that do or do not satisfy condition (19). REVIEW OF ECONOMIC STUDIES 22 Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rda Figure 6 Figure 6 Figure 6 Candidate equilibria that do or do not satisfy condition (19). those it obtains in market 0,wP by hiring a mixture of workers at a lower wage. A similar logic applies to the case of a no-signalling equilibrium. The following result determines when this condition is satisfied in either case and establishes that checking against this possible deviation is a sufficient condition for equilibrium existence. those it obtains in market 0,wP by hiring a mixture of workers at a lower wage. 5.4. Existence A similar logic applies to the case of a no-signalling equilibrium. The following result determines when this condition is satisfied in either case and establishes that checking against this possible deviation is a sufficient condition for equilibrium existence. Proposition 4 1. The pure signalling candidate equilibrium described in Proposition 3 part 1 is always an equilibrium. 2. Suppose θN and wN ≥wP satisfy equations (11) and (12) for a no-signalling candidate equilibrium. Then the worker and firm decisions described in Proposition 3 part 2 are part of an equilibrium if and only if: λ−θ 1−θ ≤ cH cL −cH 1−λ 1−θN λ θ 1 1−t dF(t) for all θ ∈  θN,λ  . (18) (18) 3. Suppose πP and θP satisfy equations (15) and (16) for a partial signalling candidate equilibrium. Then the worker and firm decisions described in Proposition 3 part 3 are part of an equilibrium if and only if 3. Suppose πP and θP satisfy equations (15) and (16) for a partial signalling candidate equilibrium. Then the worker and firm decisions described in Proposition 3 part 3 are part of an equilibrium if and only if λ−θ πP(1−λ)+λ−θ ≤cH cL λ θ 1 πP(1−λ)+λ−t dF(t) for all θ ∈ θP,λ . (19) (19) RESTUD: The Review of Economic Studies Page: 22 1–50 Page: 22 1–50 [19:28 2/11/2020 OP-REST200072.tex] Page: 22 1–50 MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES KURLAT & SCHEUER SIGNALLING TO EXPERTS GNALLING TO EXPERTS 23 23 Figure 7 Types of equilibria depending on parameters Figure 7 Types of equilibria depending on parameters Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rda Figure 7 Types of equilibria depending on parameters In sum, the pure signalling equilibrium, which coincides with the no-information benchmark, always exists in our model. The reason is that πP =0, θP =λ always satisfies equations (15) and (16) and condition (19) holds for θ =λ. Depending on parameters, additional equilibria may exist where firms use their expertise. It is easy to construct examples where a partial or no-signalling equilibrium does exist. Figure 6 shows an economy with multiple candidate equilibria. For the candidate equilibrium πP 1 ,θP 1 , condition (19) holds, so it is indeed a partial signalling equilibrium. Instead, for candidate equilibrium πP 2 ,θP 2 , condition (19) fails for some θ >θP 2 , so it is not an equilibrium.10 10. The example uses qH =1,qL =0.4,cH =0.9,cL =0.15,λ=0.6,f (θ)=0.5[sin(13.3(θ −λ)0.4+2.2π)+1]2.8. 11. Specifically, we use the linear density f (θ)=A(θ −λ/2)+B/λ, so the total measure of firms always equals B, with B=1.2. REVIEW OF ECONOMIC STUDIES 24 more expert firms hire more workers.12 Our model thus generates the plausible prediction that more high types forgo signalling through education (or that the amount of retained equity falls) in boom times (see Gee (2018) for descriptive evidence of this effect). This intuitive property is absent when buyers are uninformed: in that case, pure signalling is always the only equilibrium independent of demand. It is also absent in the no-signalling equilibrium where higher demand just translates into higher wages. Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rdaa068/5931860 by University of Zurich user on 05 January 2021 The left panel shows that increasing the relative cost of signalling cH/cL has the same effects as improving expertise on the type of equilibrium we find, holding the other parameters fixed (including A).13 Moreover, we show in Appendix A that, within the class of partial signalling equilibria, the amount of signalling 1−πP decreases with signalling costs. Hence, as signalling gets more expensive, fewer high types signal. Note that the no-information benchmark, somewhat unappealingly, does not have this property: all high types choose e=e∗and e∗does not depend on cH, so high types do not respond to a higher cost of signalling by signalling less. Allowing for heterogeneously informed firms overturns this counterintuitive feature: equilibrium forces do lead workers to respond on the extensive margin. p g Finally, in the right panel, we vary the share of low types on the horizontal axis. To do so in a clean way, we reparametrize the model by assuming that the mass of low-type workers is ˆλ, distributed uniformly in the interval [0,λ], with a density ˆλ λ; correspondingly, the mass of high types is 1−ˆλ, distributed uniformly in the interval [λ,1] with a density 1−ˆλ 1−λ. Changes in ˆλ have the interpretation of changes in the fraction of low types, leaving their relative detectability in the eyes of firms constant. We see that reducing the share of low types this way moves the equilibrium from pure signalling to partial equilibrium and finally to no signalling.14 Indeed, we show formally below that, as the share of low types becomes sufficiently small, a no-signalling equilibrium must always emerge. 5.5.2. Continuity in the symmetric information, no-signalling, and no-information limits. One counterintuitive feature of the uninformed-firms benchmark is that it is discon- tinuous in the buyers’ prior. If all workers have the same productivity, there is no-information asymmetry and no signalling in equilibrium. 12. Mechanically, this is because condition (16) becomes  λ θP f (θ) λ−θ+πP(1−λ)dθ =1, so changing is equivalent to a change in f (θ). 13. The example in the graph uses qH =1,qL =0.4,λ=0.55 in addition to the linear specification of f (θ) from above. 14. The example uses qH =1,qL =0.4,λ=0.55,cH =0.1,cL =0.3. 5.5. Properties of the equilibrium 5.5.1. Equilibrium regions. Figure 7 illustrates what type of equilibrium arises in different regions of the parameter space. As we change parameters, the possible outcomes of the model span the range from pure signalling, via partial signalling, up to the no-signalling allocations in Kurlat (2016). Both panels plot the equilibrium regions as a function of a parameter A on the vertical axis that shifts the distribution of firms F towards more expertise.11 We know from Proposition 4 that the pure signalling equilibrium always exists, and it is indeed the only equilibrium for low enough levels of expertise as captured by the parameter A. As the distribution of expertise improves (in a FOSD sense), holding the other parameters fixed, first a partial signalling and finally a no- signalling equilibrium emerges in addition. Hence, as firms become better informed, less costly signalling is required. Moreover, we show formally in Appendix A that, in the region with a partial signalling equilibrium, the share of high types 1−πP who signal also decreases with a FOSD shift in expertise. Better tools for directly evaluating job applicants, firm shares, asset-backed securities or insurance applicants reduce the need to signal through education, dividends, retained equity tranches, or high deductibles, respectively. In this way, direct information substitutes for traditional signalling. A FOSD increase in F is isomorphic to an increase in demand where each firm hires workers instead of just one. This is because making firms more expert is equivalent to letting the Page: 23 1–50 [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES REVIEW OF ECONOMIC STUDIES 14. The example uses qH =1,qL =0.4,λ=0.55,cH =0.1,cL =0.3. REVIEW OF ECONOMIC STUDIES A second direction to approach the symmetric information limit is making the distribution F approach a mass point at θ =λ, since that limit also implies symmetric information. Again, a no-signalling equilibrium always exists sufficiently close to the limit, so the set of equilibria is lower hemi-continuous in this dimension as well.15 A second form of discontinuity in the uninformed-firms benchmark arises with respect to the cost of signalling. For any cH/cL <1, high types will signal enough to fully separate, whereas when cH/cL =1 the signal does not allow high types to separate and pooling allocations result. The current model, instead, is lower hemi-continuous as cH/cL →1. In the opposite limit, as signalling becomes cheap, the model reduces to the uninformed-firms benchmark. Proposition 6 1. For cH/cL sufficiently close to 1, there is a no-signalling equilibrium. 2. For cH/cL sufficiently close to 0, only the pure signalling equilibrium exists. Part1ofProposition6establishesthatifsignallingissufficientlyexpensive,thereisanequilibrium with no signalling, where all workers pool at e=0. If within this limiting case one takes the limit as F becomes degenerate at 0 (meaning firms have no information), then this reduces to the pooling allocation in Akerlof (1970). Conversely, part 2 establishes that if signalling is sufficiently cheap, then the only equilibrium allocation is the benchmark least-cost separating allocation and firms’ expertise is not used. 15. By contrast, in the degenerate case where F has full mass at some θ <λ, a no-signalling equilibrium never exists. The right-hand side of (18) is zero at θ in this case, so there is always a profitable deviation. Intuitively, when all firms are equally well informed, our model collapses to a standard signalling model and only the pure signalling equilibrium exists. Hence, heterogeneity of information is crucial to obtain the continuity results in this section. REVIEW OF ECONOMIC STUDIES However, as soon as there is even an infinitesimal mass of low types, high types will signal enough to separate. The following result shows that this unappealing property vanishes in our model, as in Daley and Green (2014) where the presence of exogenous information also avoids the discontinuity. Proposition 5 1. Let F be any continuous measure with full support on [0,λ]. For low ˆλ there is a no-signalling equilibrium with limˆλ→0wN =qH. 2. Let F∗be a mass point at θ =λ. For any continuous F sufficiently close to F∗(under the total variation distance), there exists a no-signalling equilibrium, and limF→F∗wN =qH. Proposition 5 1. Let F be any continuous measure with full support on [0,λ]. For low ˆλ there is a no-signalling equilibrium with limˆλ→0wN =qH. λ 0 2. Let F∗be a mass point at θ =λ. For any continuous F sufficiently close to F∗(under the total variation distance), there exists a no-signalling equilibrium, and limF→F∗wN =qH. One way to approach the symmetric information limit is by taking ˆλ→0, since ˆλ=0 implies symmetric information. As ˆλ→0, there is always a no-signalling equilibrium, and wN →qH. Hence, this equilibrium smoothly approaches the symmetric information outcome. Pure signalling is also an equilibrium for any positive ˆλ, so the discontinuity does not go away entirely, 12. Mechanically, this is because condition (16) becomes  λ θP f (θ) λ−θ+πP(1−λ)dθ =1, so changing is equivalent to a change in f (θ). 13. The example in the graph uses qH =1,qL =0.4,λ=0.55 in addition to the linear specification of f (θ) from above. ange in f (θ). 13. The example in the graph uses qH =1,qL =0.4,λ=0.55 in addition to the linear specification of f (θ) from ove. 14. The example uses qH =1,qL =0.4,λ=0.55,cH =0.1,cL =0.3. Page: 24 1–50 RESTUD: The Review of Economic Studies Page: 24 1–50 [19:28 2/11/2020 OP-REST200072.tex] MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES KURLAT & SCHEUER SIGNALLING TO EXPERTS 25 KURLAT & SCHEUER but the set of equilibria is lower hemi-continuous in ˆλ. A second direction to approach the symmetric information limit is making the distribution F approach a mass point at θ =λ, since that limit also implies symmetric information. Again, a no-signalling equilibrium always exists sufficiently close to the limit, so the set of equilibria is lower hemi-continuous in this dimension as well.15 but the set of equilibria is lower hemi-continuous in ˆλ. RESTUD: The Review of Economic Studies 5.6. Welfare The only reason why allocations in the model are not first-best efficient is that signalling is socially wasteful. This does not immediately imply that equilibria with less signalling are Pareto superior: expected wages for different workers are different across equilibria so it is possible that there could be winners and losers from shifting from one equilibrium to another. The following result establishes that partial signalling equilibria can indeed be Pareto ranked against each other (and against the pure signalling equilibrium), but cannot be Pareto ranked against a no-signalling equilibrium if it exists: Proposition 7 1. Suppose there is a partial signalling equilibrium with πP 1 >0. Proposition 7 1. Suppose there is a partial signalling equilibrium with πP 1 >0. a) It Pareto dominates the pure signalling equilibrium in the same economy. P P (a) It Pareto dominates the pure signalling equilibrium in the same economy. (b)If there is another partial signalling equilibrium with πP 1 >πP 2 in the same economy, the first equilibrium Pareto dominates the second. (b)If there is another partial signalling equilibrium with πP 1 >πP 2 in the same economy, the first equilibrium Pareto dominates the second. 2. Suppose there is a no-signalling equilibrium. 2. Suppose there is a no-signalling equilibrium. REVIEW OF ECONOMIC STUDIES 26 0,wP . High-type workers are indifferent because their payoff is wP. The critical step is to show that low-type workers are also better off. They gain from the fact that more firms are hiring in market 0,wP , which (other things being equal) increases their chances of earning wP but lose from the fact that there are more high-type workers looking for work at 0,wP , which lowers their chance of being hired by any given firm. However, using the fact that in both equilibria high types must be hired for sure it is possible to show that the first effect dominates, so low types also prefer the higher-πP equilibrium. 0,wP . High-type workers are indifferent because their payoff is wP. The critical step is to show that low-type workers are also better off. They gain from the fact that more firms are hiring in market 0,wP , which (other things being equal) increases their chances of earning wP but lose from the fact that there are more high-type workers looking for work at 0,wP , which lowers their chance of being hired by any given firm. However, using the fact that in both equilibria high types must be hired for sure it is possible to show that the first effect dominates, so low types also prefer the higher-πP equilibrium. A no-signalling equilibrium (if it exists) cannot be Pareto ranked against partial signalling equilibria. Since the wage is higher and the cutoff firm is lower, workers are better off in the no-signalling equilibrium. However, the best firms are worse off since they have to pay higher wages and their accurate signals mean they benefit little from the improved pool of workers. Intermediate firms with θ ∈ θN,θP are better off in the no-signalling equilibrium while they would make zero profits in the partial signalling equilibrium. The model also makes it possible to ask, assuming there is a technology for firms to choose θ at some cost, whether they have the right incentives to invest in acquiring expertise, such as improving assessment models for job applicants, risk scoring models in insurance markets or pricing models for stocks and financial derivatives. In Appendix B, following the approach in Kurlat (2019), we show that in general the answer is ambiguous: firms may have incentives to either over-invest or under-invest in expertise. REVIEW OF ECONOMIC STUDIES We also provide a simple formula to quantify the ratio of the social and private returns to expertise based on observable properties of the equilibrium. 6. FALSE NEGATIVES We now turn to the case with “false negative” mistakes, where F is continuous with support [λ,1]. Higher-i workers are relatively transparent, since most firms can tell (with certainty) that they are high types, while lower-i high types are relatively obscure, since they can only be identified as high types by the smarter, lower-θ firms. For expositional purposes, assume that the density of firms f (θ) is strictly increasing, meaning that there is a higher density of less informed firms. The general case, which requires working with an “ironed” density, is treated in Appendix D. y g g g y The general case, which requires working with an “ironed” density, is treated in Appendix D. Unlike the false positives case, firms face a non-trivial decision as to what hiring rule to use. There may be markets where a firm θ observes x(i,θ)=0 for all the workers that apply (so if it insisted on hiring only workers with a positive signal it would not be able to hire at all) but it knows that in equilibrium some high-type workers with i∈[λ,θ) do apply, so it may want to hire from the pool of all applicants. We refer to this as non-selective hiring. 2. Suppose there is a no-signalling equilibrium. (a) It Pareto dominates the pure signalling equilibrium in the same economy. P a) It Pareto dominates the pure signalling equilibrium in the same economy. P (b)If there is also a partial signalling equilibrium with πP >0 in the same economy, neither equilibrium Pareto dominates the other. (b)If there is also a partial signalling equilibrium with πP >0 in the same economy, neither equilibrium Pareto dominates the other. In comparing partial signalling equilibria, it is straightforward to show that firms are better off in the higher-πP equilibrium, since wages are the same and there is a better pool of workers at 15. By contrast, in the degenerate case where F has full mass at some θ <λ, a no-signalling equilibrium never exists. The right-hand side of (18) is zero at θ in this case, so there is always a profitable deviation. Intuitively, when all firms are equally well informed, our model collapses to a standard signalling model and only the pure signalling equilibrium exists. Hence, heterogeneity of information is crucial to obtain the continuity results in this section. [19:28 2/11/2020 OP-REST200072.tex] Page: 25 1–50 MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES REVIEW OF ECONOMIC STUDIES 6.1. Description. In equilibrium, only the least transparent high-type workers signal. Letting uL denote the low types’ payoff, there is a cutoff iS such that workers in the interval i∈[λ,iS] signal by choosing: eS = qH −uL cL , (20) (20) while everyone else chooses e=0. Signalling markets with e=eS are straightforward: all the applicants are high types, so less informed firms compete for them and hire them (non-selectively) at a wage w=qH. g qH No-signalling markets, with e=0, are more interesting. Define iH by: f (iH)=1. (21) f (iH)=1. (21) RESTUD: The Review of Economic Studies Page: 26 1–50 [19:28 2/11/2020 OP-REST200072.tex] [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies Page: 26 1–50 Page: 26 1–50 RESTUD: The Review of Economic Studies Page: 26 1–50 MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES KURLAT & SCHEUER SIGNALLING TO EXPERTS 27 KURLAT & SCHEUER Since f (θ) is assumed to be increasing, this means that for all i>iH there are more firms who can detect high-type workers than there are workers. Hence, firms compete for them and hire them (selectively) at wage w=qH. Conversely, for i∈(iS,iH), there are more workers than firms who can identify them as high types. Therefore, some of them have to be hired non-selectively, at wages sufficiently low to attract non-selective firms. At each wage w∈(qL,qH) where there is active hiring, two types of hiring take place: some workers are hired non-selectively, and in addition all the highest remaining i-types are hired selectively and thus drop out of subsequent, lower-wage markets. Let w(0,i) be the highest wage such that all worker types above i have already been hired. The pool of applicants at w(0,i) consists of all the low types plus high types in the interval (iS,i] who have not been hired non-selectively at higher wages. As a result, non-selective firms break even at a wage of:16 w(0,i)= (i−iS)qH +λqL i−iS +λ . (22) (22) Firms with θ =i hire f (i) workers selectively in this market since it involves the cheapest wage at which they can identify high-type workers. Therefore, it must be that the remaining 1−f (i) workers of type i were already hired non-selectively at wages above w(0,i). Since this is true for any i, the probability density for any worker of being hired non-selectively in market (0,w(0,i)) must be f ′(i). for χ(i)=1∀i, where ir(w) is the inverse of w(0,i). 6.1. Description. RESTUD: The Review of Economic Studies Page: 27 1–50 Page: 27 1–50 [19:28 2/11/2020 OP-REST200072.tex] [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES 28 REVIEW OF ECONOMIC STUDIES Figure 8 Signals, wages, and hiring decisions in an interior equilibrium. REVIEW OF ECONOMIC STUDIES REVIEW OF ECONOMIC STUDIES 28 28 REVIEW OF ECONOMIC STUDIES Figure 8 Signals, wages, and hiring decisions in an interior equilibrium. Signals, wages, and hiring decisions in an interior equilibrium. not equal; there is a range of workers i∈ iS,i∗ who are indifferent between signalling and not signalling but choose not to. It is straightforward to show that i∗is increasing in iS. Workers who signal do not apply for jobs in e=0 markets. Higher iS (more signalling) means the pool of applicants for non-selective firms worsens, so in order to maintain zero-profits the wage must fall (equation (22)). In turn, this means that the utility of both high- and low-type workers falls (equations (23) and (25)). It falls more for high types because low types are hired with positive probability in market (0,qL), where the wage is unaffected by higher iS. Hence, other things equal, higher iS makes signalling more attractive, so the indifferent type i∗rises. A fraction 1−f i∗ of workers in the range i∈ iS,i∗ are hired at wages above w 0,i∗ , so the remaining f i∗ i∗−iS workers must be hired at wage w 0,i∗ . For any i∈ iS,i∗ , the measure of firms who are capable of identifying i as being a high type is F(i), so we need f (i∗)(i−iS)≤F(i). By monotonicity of f , this is implied by the market-clearing condition: F i∗ =f i∗ i∗−iS . (27) (27) Equation (27) defines another positive relationship between iS and i∗. If more of the obscure workers decide to signal, then the most informed firms will work their way up to hire slightly less obscure workers. Figure 8 summarizes the equilibrium signals, wages and hiring decisions. 6.1. Description. Hence, the expected utility obtained by worker i is: Firms with θ =i hire f (i) workers selectively in this market since it involves the cheapest wage at which they can identify high-type workers. Therefore, it must be that the remaining 1−f (i) workers of type i were already hired non-selectively at wages above w(0,i). Since this is true for any i, the probability density for any worker of being hired non-selectively in market (0,w(0,i)) must be f ′(i). Hence, the expected utility obtained by worker i is: u(i)=f (i)w(0,i)+ iH i w 0,i′ df i′ . (23) (23) This defines a cutoff worker i∗who is indifferent between signalling (which gives a payoff qH −cHeS) and not signalling: f i∗ w 0,i∗ + iH i∗ w 0,i′ df i′ =qH −cHeS. (24) (24) Market 0,w 0,i∗ is the lowest-wage market at which there is a chance of being hired non- selectively. Low-type workers who have not found a job at or above this wage end up getting hired at w=qL. Therefore, the expected utility of low types is iH Market 0,w 0,i∗ is the lowest-wage market at which there is a chance of being hired non- selectively. Low-type workers who have not found a job at or above this wage end up getting hired at w=qL. Therefore, the expected utility of low types is iH uL =f i∗ qL + iH i∗ w 0,i′ df i′ . (25) (25) Replacing (20), (22), and (25) into (24) and simplifying gives the following indifference condition for the marginal worker i∗: Ŵ(i∗,iS)=f i∗ cH cL − λ i∗−iS +λ −λ  1−cH cL iH i∗ 1 i−iS +λdf (i)=0. (26) (26) Equation (26) defines a positive relationship between i∗(the worker who is indifferent between signalling and not signalling) and iS (the cutoff for actually signalling). In general, i∗and iS are Equation (26) defines a positive relationship between i∗(the worker who is indifferent between signalling and not signalling) and iS (the cutoff for actually signalling). In general, i∗and iS are 16. Accordingly, the non-selective firms’ beliefs are given by g(i;0,w,χ)= I(i<λ)+I(i∈(iS,ir(w)]) λ+ir(w)−iS for χ(i)=1∀i, where ir(w) is the inverse of w(0,i). KURLAT & SCHEUER SIGNALLING TO EXPERTS KURLAT & SCHEUER SIGNALLING TO EXPERTS 29 1−cH cL > F(iH) F(iH)+λ, (28) (28) which is equivalent to Ŵ(iH,iH −F(iH))<0. The following proposition summarizes these results: which is equivalent to Ŵ(iH,iH −F(iH))<0. The following proposition summarizes these results: Proposition 8 Under false negatives, there exists a generically unique equilibrium: Proposition 8 Under false negatives, there exists a generically unique equilibrium: Proposition 8 Under false negatives, there exists a generically unique equilibrium: 1. All high types i∈[iH,1] choose e=0 and are hired at w=qH. 2 F i [0 i ) h ilib i k f h f ll i ibl f 2. For i∈[0,iH), the equilibrium takes one of the following two possible forms: 2. For i∈[0,iH), the equilibrium takes one of the following two possible forms: (a) An interior equilibrium where iS and i∗solve (26) and (27) and: (a) An interior equilibrium where iS and i∗solve (26) and (27) and: i. A measure iS −λ of high types with i∈[λ,i∗) choose e=eS, given by (20) and are hired at w=qH. i. All other high types with i∈[λ,i∗) choose e=0 and are hired at w∈[w(0,i∗),w(0,iH)]. ii. All high types with i∈[i∗,iH) choose e=0 and are hired at w∈[w(0,i),w(0,iH)]. ii. All other high types with i∈[λ,i ) choose e=0 and are hired at w∈[w(0,i ),w(0,iH)]. iii. All high types with i∈[i∗,iH) choose e=0 and are hired at w∈[w(0,i),w(0,iH)]. iii. All high types with i∈[i∗,iH) choose e=0 and are hired at w∈[w(0,i),w(0,iH)]. iv. All low types i∈[0,λ) choose e=0 and are hired at w=qL or w∈[w(0,i∗),w(0,iH)]. (b)A corner equilibrium where Ŵ(iH,iH −F(iH))<0 and: i. A measure F(iH) of high types with i∈[λ,iH) choose e=0 and are hired at w=wP. ii All h hi h i h [ ) h ∗ d hi d . A measure F(iH) of high types with i∈[λ,iH) choose e=0 and are hired at w=wP. i. All other high types with i∈[λ,iH) choose e=e∗and are hired at w=qH. ii. All other high types with i∈[λ,iH) choose e=e and are hired iii. All low types i∈[0,λ) choose e=0 and are hired at w=qL. iii. All low types i∈[0,λ) choose e=0 and are hired at w=qL. The proof is in Appendix D, which also describes all firms’ decisions. 17. In contrast, in the partial or no-signalling equilibria in the “false positives” case there is dispersion in expected wages among low types depending on their chances of being hired at wP or wN versus qL. KURLAT & SCHEUER SIGNALLING TO EXPERTS Moreover, we show that the equilibrium behaves continuously in the symmetric information and expensive signalling limits, and we deal with the general case in which the density of firm types f (θ) is not necessarily monotone. RESTUD: The Review of Economic Studies 6.2. Corner equilibrium. 6.2. Corner equilibrium. Equations (26) and (27) hold for an interior equilibrium where some range of workers are indeed hired by non-selective firms. However, it is possible that all workers below iH prefer to signal rather than being hired at a wage low enough to attract non-selective firms, which would result in a corner equilibrium with i∗=iH. For this corner equilibrium, the market-clearing condition (27) and definition (21) imply iS =iH −F(iH). Also, in this corner equilibrium, there is no non- selective hiring, so uL =qL and eS =e∗. This will be an equilibrium if workers just below iH indeed prefer to signal: qH −cHe∗    > F(iH)qH +λqL F(iH)+λ    utility of signalling wage for non-selective firms to break even so, using (6), Page: 28 1–50 [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES KURLAT & SCHEUER SIGNALLING TO EXPERTS 29 Acknowledgments. We thank Adrien Auclert, Alex Bloedel, Gabriel Carroll, Veronica Guerrieri, Patrick Kehoe, Guido Menzio, Nick Netzer, Venky Venkateswaran, four anonymous referees as well as numerous seminar and conference participants for helpful comments and suggestions. Florian Scheuer acknowledges support through ERC Grant 757721. REVIEW OF ECONOMIC STUDIES We can also ask how the intensive and extensive margins of signalling, measured by eS and iS −λ respectively, depend (locally) on parameters around an interior equilibrium. In Appendix A, we show that an increase in the ratio cH/cL reduces signalling along both margins. For example, an increase in dividend taxes leads to both a smaller fraction of firms paying dividends and a lower dividends per dividend-paying firm. We also show that an increase in demand leads to polarization in signalling: fewer workers choose positive education but those who do choose a higher quantity. 7. CONCLUSION We have developed a general theory to analyse competitive equilibria in economies where buyers possess heterogeneous information about sellers and contracts are multidimensional, specifying both a price and a signal. These information and contracting patterns are the feature of many markets, including labour, asset, and insurance markets, as we have illustrated through a series of examples. Our notion of equilibrium implies that an equilibrium always exists, it may not be unique in the false-positives case but is generically unique in the false-negatives case, and it may not be efficient. Moreover, we uncover a tractable structure to characterize it in both cases, based on the intersection of an indifference and a market-clearing condition. This allows us to provide results on comparative statics. Our model predicts intuitive and continuous equilibrium responses to, for instance, changes in the prior, demand, signalling costs or expertise that cannot be generated in the canonical model with uninformed buyers. g y We expect that our framework can be extended to study other structures of buyers’ direct information, including ones where firms cannot be perfectly ranked by their expertise, such as when both false positive and negative errors occur. In this case, we conjecture the equilibrium to feature a combination of the two pure cases we have analysed: high types are hired in a similar way as in the false-negatives case, except that those in [iS,iH) are partly hired by selective false- positive firms, because those firms have an advantage over non-selective firms by being able to screen out some low types. Our model may also be a useful starting point to study a number of richer environments. First, a market for information may arise, where better informed firms sell their information to less informed ones (e.g. in the form of analyst reports), instead of just trading on it themselves. To prevent the price of information from dropping to zero, some form of capacity constraints would again be required, which would effectively change the distribution of expertise in our model. Second, many of our applications have a dynamic aspect, where the costly signal involves a delay in trading. Our approach could be used to consider settings where some direct information is revealed to buyers gradually at heterogeneous rates, and one could explore how this affects the timing pattern of trades. These issues are left for future research. The replication packages are available at https://dx.doi.org/10.52xx/zenodo.4020466. 6.3. Properties. This model generates dispersion in expected wages among workers who are equally productive and educated, depending on how transparent they are. In particular, the expected wages of high types i∈[i∗,iH), who all select e=0, are increasing in i.17 Similarly, the model can explain, for instance, different prices for asset-backed securities for which both the structure of tranches and the underlying cash flows are similar, but which differ in how many buyers have access to accurate pricing models to evaluate them. Interestingly, this dispersion is driven by break-even conditions of firms that are not making use of expertise. The structure of equilibrium is similar to the pattern of signalling and “countersignalling” (Feltovich et al., 2002): it is the hard-to-identify high types who must use the costly signal in order to differentiate themselves from low types. By contrast, the most obvious high types can be confident that expert buyers are able to tell them apart, thus eliminating the need for signalling. The setup in Feltovich et al. (2002) features three different levels of worker productivity; in our two-type model, countersignalling instead emerges because high types differ in their transparency. Moreover, our model generates the intuitive prediction that expected wages of those high types who “countersignal” increase in their transparency. Page: 29 1–50 [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES 30 RESTUD: The Review of Economic Studies Proposition A.1 1. Signalling decreases with the cost ratio cH/cL. By assumption, the denominator of (A.4) is positive, and equation (A.3) implies that ∂θI ∂πP is negative. 1. Equation (A.3) implies that θI is decreasing in cH/cL, whereas cH/cL does not appear in equation (A.2). Using this in equation (A.4) gives the result. 1. Equation (A.3) implies that θI is decreasing in cH/cL, whereas cH/cL does not appear in equation (A.2). Using this in equation (A.4) gives the result. 2. The distribution F does not appear in equation (A.3). The term inside the integral in equation (A.2) is an increasing function of θ. Therefore, a FOSD increase in F implies that the left-hand side of (A.2) increases, so θP must rise to maintain equality. Using this in equation (A.4) gives the first part of the result. (A.2) implies that θM is increasing in , and does not appear in equation (A.3). Using this in equation (A.4) gives the second part of the result. This follows because neither qH nor qL appear in equation (A.4). 3. This follows because neither qH nor qL appear in equation (A.4). KURLAT & SCHEUER APPENDIX A. COMPARATIVE STATICS Proposition A.1 1. Signalling decreases with the cost ratio cH/cL. 2. SignallingdecreaseswithaFOSDincreaseintheexpertisedistributionF oranincreaseinthedemandforworkers . 2. SignallingdecreaseswithaFOSDincreaseintheexpertisedistributionF oranincreaseinthedemandforworkers . 3. Signalling does not change with productivities qH and qL. 3. Signalling does not change with productivities qH and qL. 3. Signalling does not change with productivities qH and qL. The logic of part A.1 is as follows. The ratio cH/cL governs how much utility high types obtain if they separate by choosing e∗. Since wP is the wage that makes them indifferent, higher cH/cL means a lower wage. This attracts lower-θ firms, so more high types can forgo signalling and still find a job. As for part A.1, a FOSD increase in the distribution of θ means that firms are able to screen out more low types, and therefore hire more high types (and an increase in has the same effect). Therefore, more high types are able to forgo education and still find a job. Finally, productivities have no effect on equilibrium signalling. The wage wP is a weighted average of qH and qL. Therefore, no matter what these productivities are, the indifferent firm θP will be the one whose pool of acceptable workers includes a proportion of exactly cH/cL low types. If, say, the productivity of low types was lower, the wage w adjusts exactly so as to leave firm θP indifferent and the fraction of high types who signal unchanged. Proof. Using the reparametrization of the model where each firm demands workers rather than just one, it is straightforward to show that equations (15) and (16) become θP =λ−πP(1−λ) qH −wP wP −qL (A.1) (A.1) θ π ( ) wP −qL ( . ) λ θP (λ−θ)+πP(1−λ)dF(θ)=1. (A.2) λ θP (λ−θ)+πP(1−λ)dF(θ)=1. λ θP (λ−θ)+πP(1−λ)dF(θ)=1. (A.2) Replacing (6) and (14) into (A.1), the indifference condition reduces to: Replacing (6) and (14) into (A.1), the indifference condition reduces to: θP =λ−πP(1−λ) 1 cL cH −1. (A.3) (A.3) Let θI πP,p and θM πP,p represent the solutions to (A.3) and (A.2), respectively, where p is a parameter. The equilibrium value of πP is given by a solution to the equation θI πP,p −θM πP,p =0. Using the implicit function theorem, the derivative of πP with respect to parameter p is given by: dπP dp = ∂θM ∂p −∂θI ∂p ∂θI ∂πP −∂θM ∂πP . (A.4) (A.4) By assumption, the denominator of (A.4) is positive, and equation (A.3) implies that ∂θI ∂πP is negative. Data Availability Statement The replication package including the MATLAB codes for the simulations underlying Figures 6 and 7 can be found at http://doi.org/10.5281/zenodo.4020466. No new data were generated or analysed in support of this research. Page: 30 1–50 [19:28 2/11/2020 OP-REST200072.tex] Copyedited by: ES MANUSCRIPT CATEGORY: Article Copyedited by: ES RESTUD: The Review of Economic Studies A.1. False positives We compute how the amount of signalling 1−πP in a partial signalling equilibrium depends on parameters. We focus on cases where the locus of the market-clearing condition (16) is steeper than of the indifference condition (15), which corresponds to a heuristic notion of stability of the equilibrium. Proposition A.1 1. Signalling decreases with the cost ratio cH/cL. KURLAT & SCHEUER SIGNALLING TO EXPERTS APPENDIX A. COMPARATIVE STATICS 31 A.2. False negatives We compute how the intensive and extensive margins of signalling depend on parameters around an interior equilibrium. Page: 31 1–50 [19:28 2/11/2020 OP-REST200072.tex] MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES 32 REVIEW OF ECONOMIC STUDIES REVIEW OF ECONOMIC STUDIES Proposition A.2 1. Both the intensive and extensive margins of signalling decrease with the cost ratio cH/cL. 2. The intensive margin of signalling increases but the extensive margin decreases with the demand for workers . 3. The extensive margin of signalling is invariant with respect to productivities qH and qL; the intensive margin eS 2. The intensive margin of signalling increases but the extensive margin decreases with the demand for workers . g g g g 3. The extensive margin of signalling is invariant with respect to productivities qH and qL; the intensive margin eS increases with qH −qL. Higher cH/cL makes separation more costly, so fewer high types signal. This improves the pool of workers in no-signalling markets, so non-selective firms pay higher wages. This raises the utility of low types, so less intense signalling is required to separate from them. An increase in demand means that at every level of expertise there are more selective hires, and therefore fewer non-selective hires, so it is harder for low types and obscure high types to get hired non-selectively. This makes low types worse off; therefore a more intense signal is needed to successfully separate, so fewer high types do so. As in the false-positives case, qH and qL drop out of equations (26) and (27), so the extensive margin is unchanged. However, a greater gap between qH and qL makes it more attractive for low types to mimic high types, so separation requires a more intense signal. Proof. Let i∗I (iS,p) and i∗M (iS,p) represent the solutions to (26) and (27) respectively, where p is a vector of parameters. The equilibrium value of iS is given by a solution to the equation i∗I (iS,p)−i∗M (iS,p)=0. Using the implicit function theorem, the derivatives of i∗and iS with respect to parameter p are given by: diS dp = ∂i∗M ∂p −∂i∗I ∂p ∂i∗I ∂iS −∂i∗M ∂iS (A.5) di∗ dp = ∂i∗I ∂p + ∂i∗I ∂iS ∂iS ∂p . (A.6) diS dp = ∂i∗M ∂p −∂i∗I ∂p ∂i∗I ∂iS −∂i∗M ∂iS (A.5) di∗ dp = ∂i∗I ∂p + ∂i∗I ∂iS ∂iS ∂p . (A.6) (A.5) dp ∂iS −∂iS di∗ dp = ∂i∗I ∂p + ∂i∗I ∂iS ∂iS ∂p . (A.6) di∗ dp = ∂i∗I ∂p + ∂i∗I ∂iS ∂iS ∂p . (A.6) (A.6) The denominator of (A.5) is negative, and equation (26) implies that ∂i∗I ∂iS is positive. RESTUD: The Review of Economic Studies B. EXPERTISE ACQUISITION Following the approach in Kurlat (2019), we ask whether firms have the correct incentives to acquire expertise. Consider an individual firm j and suppose it could invest in becoming better at screening workers. This will affect its profits and also, by affecting the equilibrium, the economy’s total deadweight cost of education. Denote by θj the level of expertise that firm j chooses to acquire. Let θj,F = λ−θj qL +πP(F)(1−λ)qH λ−θj +πP(F)(1−λ) −wP (B.1) (B.1) denote the individual firm’s profits, where we have made explicit that these depend on the firm’s choice θj and the distribution of expertise of all other firms F, which this firm takes as given. Furthermore, let W θj,F denote the equilibrium total payoffs (ignoring their distribution across workers and firms): W θj,F =λqL +(1−λ)qH −(1−λ) 1−πP θj,F cHe∗. (B.2) (B.2) W depends on θj because firm j’s choice of expertise affects equilibrium allocations. W depends on θj because firm j’s choice of expertise affects equilibrium allocations. W depends on θj because firm j’s choice of expertise affects equilibrium allocations. Assume the firm’s cost of acquiring its screening technology is cj θj , where cj(·) is increasing and sufficiently convex Assume the firm s cost of acquiring its screening technology is cj θj , where cj(·) is increasing and sufficiently convex such that θj,F −cj θj is concave in θj. The function cj(·) can be different for different firms, leading to different equilibrium expertise choices. Taking the equilibrium as given, firm j will invest until the marginal cost of better screening equals the marginal benefit: c′ j θj =∂ θj,F /∂θj. A social planner interested in minimizing deadweight costs would instead want the firm to invest up to the point where c′ j θj =∂W θj,F /∂θj. Using the model, we can compute the ratio r θj = ∂W θj,F /∂θj ∂ θj,F /∂θj . (B.3) (B.3) If r θj >1, the marginal social value of better screening is greater than the marginal cost, which would provide a rationale for subsidizing investments in expertise. Conversely, if r θj <1, there would be a case for taxing those investments. j The following proposition provides a formula for r(θj) that relates it to equilibrium objects which, in principle, could be measured, and places a lower bound on it. KURLAT & SCHEUER SIGNALLING TO EXPERTS 33 KURLAT & SCHEUER SIGNALLING TO EXPERTS so evaluating at i∗and taking the total derivative with respect to yields: so evaluating at i∗and taking the total derivative with respect to yields: du(i∗) d = ∂u(i∗) ∂ + ∂u(i∗) ∂i∗ ∂i∗ ∂ (A.9) (A.9) with ∂u(i∗) ∂ =f i∗ w 0,i∗ + iH i∗ w 0,i′ df i′ −∂iH ∂ w(0,iH)f ′(iH)=− w(0,iH)−u i∗  <0 (using (A.8) to replace ∂iH ∂ ), and ∂u(i∗)/∂i∗=f (i∗)∂w(0,i∗)/∂i∗>0. Replacing in (A.9) and using the assumption that du(i∗) d ≥0, this implies that du(i∗) d ≥0, this implies − w(0,iH)−u i∗  +f i∗ ∂w(0,i∗) ∂i∗ ∂i∗ ∂ ≥0⇒∂i∗ ∂ ≥w(0,iH)−u(i∗) f (i∗) ∂w(0,i∗) ∂i∗ >0, which contradicts the first part of the result. which contradicts the first part of the result. 3. The fact that i∗and iS do not depend on qH and qL follows because neither qH nor qL appear in equations (26) and (27). Using (20), (24), and (25): eS = qH −qL cL f i∗ i∗−iS i∗−iS +λ, which is increasing in qH −qL. REVIEW OF ECONOMIC STUDIES Furthermore, the implicit function theorem implies that ∂Ŵ(i∗i ;p) The denominator of (A.5) is negative, and equation (26) implies that ∂i∗I ∂iS is positive. Furthermore, the implicit function theorem implies that ∂Ŵ(i∗i ) ∂i∗I ∂p =− ∂Ŵ(i∗,iS;p) ∂p ∂Ŵ(i∗,iS;p) ∂i∗ (A.7) (A.7) and equation (26) implies that ∂Ŵ(i∗,iS;p) ∂i∗ is positive. 1. Using (26), ∂Ŵ(i∗,iS; cH cL ) ∂cH cL =f i∗ +λ iH i∗ 1 i−iS +λdf (i)≥f i∗ +λ f (iH)−f (i∗)  iH −iS +λ >0 so using (A.7) ∂i∗I ∂cH cL <0. Since ∂i∗M ∂cH cL =0, using this in (A.5) and (A.6) implies that ∂i∗ ∂cH cL <0 and ∂iS ∂cH cL <0. In turn, (25) implies that: so using (A.7) ∂i∗I ∂cH cL <0. Since ∂i∗M ∂cH cL =0, using this in (A.5) and (A.6) implies that ∂i∗ ∂cH cL <0 and ∂iS ∂cH cL <0. In turn, (25) implies that: cL (25) implies that: cL (25) implies that: ∂uL ∂cH cL =−f ′ i∗ ∂i∗ ∂cH cL w 0,i∗ −qL >0 and (20) then implies that ∂eS ∂cH cL <0. and (20) then implies that ∂eS ∂cH cL <0. and (20) then implies that ∂eS ∂cH cL <0. cL 2. Introducing variable demand leaves equations (26) and (27) unchanged except that equation (21) generalizes to f (iH)=1, so ∂i  1 L 2. Introducing variable demand leaves equations (26) and (27) unchanged except that equation (21) generalizes to f (iH)=1, so ∂i  1 ∂iH ∂  =1 =− 1 f ′(iH) ∂iH ∂  =1 =− 1 f ′(iH) (A.8) Therefore Therefore ∂Ŵ(i∗,iS; ) ∂  =1 =−∂iH ∂ λ iH −iS +λf ′(iH)  1−cH cL = λ iH −iS +λ  1−cH cL >0 so using (A.7) ∂i∗I ∂ <0. Since ∂i∗M ∂ =0, using this in (A.5) and (A.6) implies ∂iS ∂ <0 and ∂i∗ ∂ <0. Now assume towards a contradiction that eS falls. This implies that the utility of the marginal high type i∗, given by u(i∗)=qH −cHeS, must rise. Equation (23) generalizes to: u(i)= f (i)w(0,i)+ iH i∗ w 0,i′ df i′ , [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies RESTUD: The Review of Economic Studies Page: 32 1–50 RESTUD: The Review of Economic Studies [19:28 2/11/2020 OP-REST200072.tex] Page: 32 1–50 MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES Proposition B.1 1. The ratio of social to private marginal value of expertise is r θj = cH cL η. 2. The elasticity η is greater than 1. 2. The elasticity η is greater than 1. First, Proposition B.1 establishes, perhaps surprisingly, that r θj does not depend on θj. One might have conjectured that the misalignment of incentives would be different for firms that, e.g. due to different cost functions cj(·), choose different θ in equilibrium. Yet, it turns out that, if the market under- or over-provides incentives to improve direct screening, it does so uniformly for all firms. Second, Proposition B.1 shows that r can be written as the product of the signalling cost ratio and the demand elasticity of πP. The ratio cH/cL enters the formula because, by equation (B.2), it governs the deadweight cost of signalling for a high type that chooses e∗. To understand the role of the elasticity of πP with respect to demand, observe that, again by (B.2), ∂W θj,F /∂θj crucially depends on how the equilibrium πP changes in response to an individual firm’s screening technology θj. If a firm improves its screening technology, it will reject more low type applicants and therefore hire more high types, so the market-clearing condition shifts outwards. Recall from Section 5.5 that demand affects the equilibrium through exactly the same channel: by producing an outward shift in the market-clearing condition. Hence, η precisely summarizes the effect of a firm’s expertise on πP. In particular, we show in the proof of Proposition B.1 that the overall effect on πP depends on the size of the shift to the market-clearing condition and on the difference between the slopes of the indifference and market-clearing conditions. For example, when these slopes are very similar, πP will respond strongly to a firm’s expertise and η will be large. Overall, the result implies that it is desirable to encourage investments in direct screening if the cost of signalling is relatively similar for high and low types (which makes the deadweight cost of signalling high) and if the signalling decisions of high types are highly sensitive to demand (which would make them highly sensitive to improved screening as well). For example, higher dividend taxes make the signalling costs of different types more similar, thereby making an underinvestment in expertise more likely. Moreover, the cost ratio and the demand elasticity of πP are sufficient to determine the magnitude of r. Proposition B.1 1. The ratio of social to private marginal value of expertise is r θj = cH cL η. Conditional on these two statistics, knowledge of other parameters, such as the shape of the cost function c(·), are not required. As usual with sufficient statistics though, η is of course endogenous to the equilibrium. The second part of Proposition B.1 establishes a lower bound of 1 on the elasticity η, which in turn implies a lower bound of cH/cL on r. To understand this, suppose there is an increase in demand of %. If the mix of workers in market (0,wP) remained constant, each firm in θP,λ  would hire % more high types, implying an elasticity of 1. However, precisely because πP increases, the mix of workers available in market (0,wP) improves, so each firm increases its hiring of high types by more than %. Furthermore, higher πP means that marginal firms enter market (0,wP), further increasing demand. The strength of this last effect depends on the density f θP of firms near the cutoff θP. Since this density could be very high (to the point where the slopes of the indifference and market-clearing conditions are the same, leading to an unbounded response of πP to ), there is no upper bound on r. The magnitude of r depends on the relative importance of the various externalities from a firm choosing its screening technology. First, in an interior partial signalling equilibrium, improved screening always helps other firms, since it leads more high types to forgo education and improves the mix of workers available at (0,wP). Second, it is neutral for high-type workers since they get a payoff of wP regardless. Third, the effect on low types with i>θ is also positive. In principle, there are offsetting effects: these workers benefit from having more firms hiring in market (0,wP) and lose from having more high type workers looking for work in (0,wP). However, just like when one compares across equilibria, the market-clearing condition implies that the first effect dominates. Lastly, for low types with i<θ the effect is ambiguous, because better screening increases their chances of being rejected. If this last effect is negative and strong enough, the sum of the externalities could be negative, which would lead to r <1. If instead of being in a partial signalling equilibrium the economy is at a no-signalling equilibrium, it is immediate that improved screening has no marginal social value, since no worker is signalling. B. EXPERTISE ACQUISITION Denote by η≡1 πP ∂πP ∂  =1 (B.4) (B.4) he elasticity of the share of high types who do not signal (in a partial signalling equilibrium) with respect to an increase n demand. RESTUD: The Review of Economic Studies Page: 33 1–50 RESTUD: The Review of Economic Studies Page: 33 1–50 Page: 33 1–50 RESTUD: The Review of Economic Studies [19:28 2/11/2020 OP-REST200072.tex] Page: 33 1–50 MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES REVIEW OF ECONOMIC STUDIES 34 Proposition B.1 1. The ratio of social to private marginal value of expertise is r θj = cH cL η. and using (B.2) [19:28 2/11/2020 OP-REST200072.tex] Proposition B.1 1. The ratio of social to private marginal value of expertise is r θj = cH cL η. (B.9) Replacing (B.9) into equation (B.8) and simplifying gives the result. Replacing (B.9) into equation (B.8) and simplifying gives the result. 2. Rearranging (B.9) and using that f θP ≥0: (B.10) η≥ 1 πP(1−λ)  λ θP (λ−θ)+πP(1−λ) −2dF(θ) (B.10) Now rearrange the market-clearing condition (16) as η≥ 1 πP(1−λ)  λ θP (λ−θ)+πP(1−λ) −2dF(θ) (B.10) learing condition (16) as η≥ 1 πP(1−λ)  λ θP (λ−θ)+πP(1−λ) −2dF(θ) (B.10) ange the market clearing condition (16) as η≥ πP(1−λ)  λ θP (λ−θ)+πP(1−λ) −2dF(θ) Now rearrange the market-clearing condition (16) as λ θP πP(1−λ) −1 (λ−θ)+πP(1−λ) −1dF(θ)= πP(1−λ) −1 Since (λ−θ)+πP(1−λ) −1 is increasing in θ, this implies πP(1−λ) λ θP (λ−θ)+πP(1−λ) −2dF(θ)≤1 Replacing in equation (B.10) gives the result. Replacing in equation (B.10) gives the result. □ Proposition B.1 1. The ratio of social to private marginal value of expertise is r θj = cH cL η. It would still have a positive marginal private value, so r =0. In this region, better screening by one firm has a negative effect on other firms, since it does not improve the pool of workers in market (0,wN) but drives up the wage wN. Proof. 1. Using (B.1) yields 1. Using (B.1) yields 1. Using (B.1) yields ∂ θj,F ∂θj = πP(1−λ) λ−θj +πP(1−λ) 2 (qH −qL) (B.5) (B.5) and using (B.2) and using (B.2) ∂W θj,F ∂θj =(1−λ)cHe∗∂πP ∂θj . (B.6) (B.6) (B.6) RESTUD: The Review of Economic Studies Page: 34 1–50 RESTUD: The Review of Economic Studies Page: 34 1–50 RESTUD: The Review of Economic Studies Page: 34 1–50 [19:28 2/11/2020 OP-REST200072.tex] MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES KURLAT & SCHEUER SIGNALLING TO EXPERTS 35 Around a partial signalling equilibrium, equations (15) and (16) imply ∂πP ∂θj = Sensitivity of market clearing to expertise of one firm    λ−θP +πP(1−λ) λ−θj +πP(1−λ) 2f θP  λ θP (1−λ) (λ−θ)+πP(1−λ) −2dF(θ) λ−θP +πP(1−λ)  f θP    Slope of market clearing −(1−λ) 1 cL cH −1    Slope of indifference . (B.7) Replacing (B.5), (B.6), and (B.7) into (B.3), we obtain r θj = cH/cL πP(1−λ)  λ θP (λ−θ)+πP(1−λ) −2dF(θ)− f(θP) πP(1−λ)+λ−θP 1 cL cH −1 . (B.8) Applying formula (A.4) and definition (B.4) yields η= λ−θP +πP(1−λ)  / πPf θP   λ θP (1−λ) (λ−θ)+πP(1−λ) −2dF(θ) λ−θP +πP(1−λ)  /f θP −(1−λ)/  cL cH −1 . (B.9) Replacing (B.9) into equation (B.8) and simplifying gives the result. P Around a partial signalling equilibrium, equations (15) and (16) imply Around a partial signalling equilibrium, equations (15) and (16) imply Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rda Sensitivity of market clearing to expertise of one firm  ∂πP ∂θj =    λ−θP +πP(1−λ) λ−θj +πP(1−λ) 2f θP  λ θP (1−λ) (λ−θ)+πP(1−λ) −2dF(θ) λ−θP +πP(1−λ)  f θP    Slope of market clearing −(1−λ) 1 cL cH −1    Slope of indifference . (B.7) lacing (B.5), (B.6), and (B.7) into (B.3), we obtain Replacing (B.5), (B.6), and (B.7) into (B.3), we obtain Applying formula (A.4) and definition (B.4) yields η= λ−θP +πP(1−λ)  / πPf θP   λ θP (1−λ) (λ−θ)+πP(1−λ) −2dF(θ) λ−θP +πP(1−λ)  /f θP −(1−λ)/  cL cH −1 . Proof of Proposition 1 1. The proposed {ei,(eθ,wθ,χθ),µ,G} is an equilibrium. Equation (9) implies that low types are indifferent between any e∈ 0,e∗ and high types are indifferent between any e≥e∗, so education decisions (7) solve the workers’ problem. (10) implies that firms can make zero profits by hiring in market (0,qL) (where there are only low types) or (e∗,qH) (where there are only high types), and any other market has either G Iχ;e,w,χ =0 or results in losses. Therefore (8), which places demand only in markets (0,qL) and (e∗,qH) and yields zero profits, is an optimal choice. Furthermore, (8) implies that no firm hires more than one worker. Replacing (8) in (3) implies that demand is: Equation (9) implies that low types are indifferent between any e∈ 0,e∗ and high types are indifferent between any e≥e∗, so education decisions (7) solve the workers’ problem. (10) implies that firms can make zero profits by hiring in market (0,qL) (where there are only low types) or (e∗,qH) (where there are only high types), and any other market has either G Iχ;e,w,χ =0 or results in losses. Therefore (8), which places demand only in markets (0,qL) and (e∗,qH) and yields zero profits, is an optimal choice. Furthermore, (8) implies that no firm hires more than one worker. Replacing (8) in (3) implies that demand is: D(e,w)= ⎧ ⎨ ⎩ λ if e=0,w=qL 1−λ if e=e∗,w=qH 0 otherwise. (C.1) (C.1) Equations (7), (9), (10), and (C.1) imply that Condition 1 holds. Condition 2 is trivially satisfied because (9) is independent of i. Finally, (7) and (9) imply that beliefs (10) satisfy Condition 3 in non-empty markets. Since low types find e∈ 0,e∗ optimal and high types find e≥e∗optimal, (9) implies that beliefs satisfy Condition 4 when they are well defined, and G(Iχ;e,w,χ)=0 only at wages where µ(w;e,i)=0 for all i, so Condition 5 is satisfied as well. RESTUD: The Review of Economic Studies Page: 35 1–50 Page: 35 1–50 [19:28 2/11/2020 OP-REST200072.tex] Page: 35 1–50 RESTUD: The Review of Economic Studies Copyedited by: ES Copyedited by: ES MANUSCRIPT CATEGORY: Article REVIEW OF ECONOMIC STUDIES 36 2. The above equilibrium is unique. a. In any equilibrium, each firm makes zero profits. If there was a firm that made strictly negative profits, it could increase profits by setting χ(i)=0 for all i. On the other hand, suppose there is a firm that makes strictly positive profits in some market (e,w). Recall that F(1)>1, so there must exist a strictly positive measure of firms that do not hire. Any such firm could increase its profits by directing its search to market (e,w), so it cannot be optimizing. a. In any equilibrium, each firm makes zero profits. If there was a firm that made strictly negative profits, it could increase profits by setting χ(i)=0 for all i. On the other hand, suppose there is a firm that makes strictly positive profits in some market (e,w). Recall that F(1)>1, so there must exist a strictly positive measure of firms that do not hire. Any such firm could increase its profits by directing its search to market (e,w), so it cannot be optimizing. b. In any equilibrium, there does not exist a market (e,w) such that I(ei =e)µ(w;e,i)>0 both for some i<λ and some i′ ≥λ. Otherwise, consider a market (e′,w′) with e′ =e+ǫ and w∈ ¯w e,i′ +cHǫ,qH . Suppose type i<λ is in the support of G ·;e′,w′,χ . This requires b. In any equilibrium, there does not exist a market (e,w) such that I(ei =e)µ(w;e,i)>0 both for some i<λ and some i′ ≥λ. Otherwise, consider a market (e′,w′) with e′ =e+ǫ and w∈ ¯w e,i′ +cHǫ,qH . Suppose type i<λ is in the support of G ·;e′,w′,χ . This requires ¯w e′,i −cLe′ ≥¯w(e,i)−cLe. Rearranging gives ¯w e′,i −¯w(e,i)≥cLǫ. Since firms cannot discriminate, it follows that ¯w(e,i) is the same for all i. Also, since µ(w′;e,i) must be weakly decreasing in i by Condition 2, ¯w e′,i is weakly increasing in i. Therefore: ¯w e′,i′ −¯w e,i′ ≥cLǫ, which implies ¯w e′,i′ −¯w e,i′ >cHǫ. This contradicts the premise that type i′ finds e optimal. Hence, no i<λ can be in the support of G ·;e′,w′,χ . If the support of G ·;e′,w′,χ only includes i≥λ, then firms could make profits by hiring in market (e′,w′), which contradicts part (C). Therefore it must be that G Iχ;e′,w′,χ =0. REVIEW OF ECONOMIC STUDIES If they do so by selecting e′ <e∗, then this implies ¯w e′,i −cHe′ >qH −cHe∗, which in turn implies ¯w e′,i −cLe′ >qL and since ¯w e′,i is the same for all i, this implies that low types can obtain a payoff higher than qL, contradicting part (C). If instead e′ ≥e∗, this implies they are hired with positive probability at a wage w>qH and hence strictly negative profits for firms, contradicting part (C). Second, suppose they obtain a payoff uH <qH −cHe∗. This means that for any i≥λ it must be that ¯w(e∗,i)<qH, and therefore ¯w(e∗,i)<qH for i<λ as well. Consider a market with e=e∗and w∈(uH +cHe∗,qH). G Iχ;e∗,w,χ >0 because otherwise µ(w;e∗,i)=0 by Condition 5, so high types can obtain a payoff of at least w−cHe∗>uH by choosing education e∗. But the support of G(·;e∗,w,χ) cannot include low types because choosing e∗implies a payoff of ¯w(e∗,i)−cLe∗<qH −cLe∗<qL, contradicting part (C); and the support of G(·;e∗,w,χ) cannot include only high types because then firms could make profits by hiring in market (e∗,w), contradicting part (C). e. Step (C) implies that all low types select e=0 and get hired for sure in market (0,qL). Step (C) implies that all high types must select e=e∗and get hired for sure in market (e∗,qH). This determines (7) as well as (9) and (10) in these markets. It also requires that there is total demand λ in market (0,qL) and demand 1 λ in (e∗q ) thus (8) must hold For all other markets (9) and (10) then follow from Conditions 1 to 5 e. Step (C) implies that all low types select e=0 and get hired for sure in market (0,qL). Step (C) implies that all high types must select e=e∗and get hired for sure in market (e∗,qH). This determines (7) as well as (9) and (10) in these markets. It also requires that there is total demand λ in market (0,qL) and demand 1−λ in (e∗,qH), thus (8) must hold. For all other markets, (9) and (10) then follow from Conditions 1 to 5. 1−λ in (e∗,qH), thus (8) must hold. For all other markets, (9) and (10) then follow from Conditions 1 to 5. RESTUD: The Review of Economic Studies REVIEW OF ECONOMIC STUDIES This implies that µ w′;e′,i′ =0 by Condition 5, which in turn implies ¯w e′,i′ > ¯w e,i′ +cHǫ, which contradicts the premise that type i′ finds e optimal. c. In any equilibrium, all low types obtain a payoff of qL. Suppose that they obtain a payoff q′ L >qL. This implies that they are hired with positive probability in a market with w>qL. By part (C), the supply in this market only includes low types, which implies negative profits for firms, contradicting part (C). Suppose that they obtain a payoff q′ L <qL and consider a market with e=0 and w∈ q′ L,qL . If G Iχ;e,w,χ >0, then firms can make profits by hiring in this market; otherwise, µ(w;e,i)=0, which means low-type workers can obtain a payoff w>q′ L by choosing e=0. L d. In any equilibrium, all high types obtain payoff qH −cHe∗. Suppose first that they obtain a payoff uH > qH −cHe∗. If they do so by selecting e′ <e∗, then this implies ¯w e′,i −cHe′ >qH −cHe∗, which in turn implies ¯w e′,i −cLe′ >qL and since ¯w e′,i is the same for all i, this implies that low types can obtain a payoff higher than qL, contradicting part (C). If instead e′ ≥e∗, this implies they are hired with positive probability at a wage w>qH and hence strictly negative profits for firms, contradicting part (C). Second, suppose they obtain a payoff uH <qH −cHe∗. This means that for any i≥λ it must be that ¯w(e∗,i)<qH, and therefore ¯w(e∗,i)<qH for i<λ as well. Consider a market with e=e∗and w∈(uH +cHe∗,qH). G Iχ;e∗,w,χ >0 because otherwise µ(w;e∗,i)=0 by Condition 5, so high types can obtain a payoff of at least w−cHe∗>uH by choosing education e∗. But the support of G(·;e∗,w,χ) cannot include low types because choosing e∗implies a payoff of ¯w(e∗,i)−cLe∗<qH −cLe∗<qL, contradicting part (C); and the support of G(·;e∗,w,χ) cannot include only high types because then firms could make profits by hiring in market (e∗,w), contradicting part (C). L d. In any equilibrium, all high types obtain payoff qH −cHe∗. Suppose first that they obtain a payoff uH > qH −cHe∗. 2. Firms’ profits must be weakly increasing in θ. To see this, suppose that θ′ >θ but firm θ makes strictly higher profits than θ′, and consider the market and hiring rule (eθ,wθ,χθ) chosen by firm θ. By hiring in market (eθ,wθ) and setting χθ′(i)=I(i≥θ′), firm θ′ could make profits at least as high as firm θ since it accepts all the high types but rejects more low types than firm θ possibly can. SIGNALLING TO EXPERTS Since not being hired in market (˜e, ˜w) implies getting a wage qL, this implies that the payoff from choosing e= ˜e is bounded above by: i 7. In any equilibrium, all low types get education e=0. To see this, assume to the contrary that some i<λ chooses e= ˜e>0. By step (C), we have that ¯w(˜e,i)≥qL +cL˜e>qL. Together with step (C), this implies that in every market (w,˜e) with w>qL where type i has some chance of being hired, there are also high-type applicants, because otherwise firms would make losses by paying more than qL. Step (C) implies that there can be only one such market; label it (˜e, ˜w). Letting uH be the utility obtained by high types in equilibrium, this implies ˜w=uH +cH ˜e. Let θ be the lowest firm type that hires in market (˜e, ˜w) and πH be the measure of high types that choose e= ˜e. Using the fact that all high types that choose e= ˜e are hired in market (˜e, ˜w), the probability that type i<λ is hired in market (˜e, ˜w) is bounded above by  i θ 1 πH dF(θ). Since not being hired in market (˜e, ˜w) implies getting a wage qL, this implies that the payoff from choosing e= ˜e is bounded above by: i ¯ui(˜e)=qL + i θ 1 πH dF(θ)( ˜w−qL)−cL˜e, which is lower than qL for i sufficiently close to θ. Let i be the lowest worker type such that there is a δ1 >0 such that all workers i∈ i,i+δ1  choose e= ˜e. We know that i>θ. Assume that some firm ˜θ ∈ θ,i  prefers to hire in some market (e′′,w′′)̸=(˜e, ˜w). This implies firms θ ∈  ˜θ,i  also Assume that some firm ˜θ ∈ θ,i  prefers to hire in some market (e′′,w′′)̸=(˜e, ˜w). This implies firms θ ∈  ˜θ,i  also prefer (e′′,w′′) over (˜e, ˜w), since they hire from the same pool of workers as firm ˜θ in market (˜e, ˜w) but from a more selected pool in other markets. But then the fact that worker i= ˜θ does not choose ˜e implies that worker i does not want to choose ˜e either, since he obtains the same payoff as worker i= ˜θ upon choosing ˜e but weakly higher in every other market. This contradicts the assumption that worker i chooses ˜e. SIGNALLING TO EXPERTS To see this, consider two wage levels w′ >w and suppose that high types are hired with positive probability in both of them if they choose e. Let θ′ be the highest-type firm that hires at wage w′. Conditions 1 and 3 imply that the expected productivity of workers that firm θ′ will find in markets e,w′ and (e,w) is the same, and therefore, it cannot be optimal for firm θ′ to hire at wage w′. Therefore it must be that all high types are hired at the same wage, which implies that µ(·;e,i) is a step function for every i. 6. For any i≥λ and any e, µ(·;e,i) has a point mass at a single wage. To see this, consider two wage levels w′ >w and suppose that high types are hired with positive probability in both of them if they choose e. Let θ′ be the highest-type firm that hires at wage w′. Conditions 1 and 3 imply that the expected productivity of workers that firm θ′ will find in markets e,w′ and (e,w) is the same, and therefore, it cannot be optimal for firm θ′ to hire at wage w′. Therefore it must be that all high types are hired at the same wage, which implies that µ(·;e,i) is a step function for every i. 7. In any equilibrium, all low types get education e=0. To see this, assume to the contrary that some i<λ chooses e= ˜e>0. By step (C), we have that ¯w(˜e,i)≥qL +cL˜e>qL. Together with step (C), this implies that in every market (w,˜e) with w>qL where type i has some chance of being hired, there are also high-type applicants, because otherwise firms would make losses by paying more than qL. Step (C) implies that there can be only one such market; label it (˜e, ˜w). Letting uH be the utility obtained by high types in equilibrium, this implies ˜w=uH +cH ˜e. Let θ be the lowest firm type that hires in market (˜e, ˜w) and πH be the measure of high types that choose e= ˜e. Using the fact that all high types that choose e= ˜e are hired in market (˜e, ˜w), the probability that type i<λ is hired in market (˜e, ˜w) is bounded above by  i θ 1 πH dF(θ). Proof of Proposition 3 We first show that, in any equilibrium, all low types choose e=0 and get hired at least at wage w=qL. Some fraction π ∈[0,1] of the high types choose e=0 and find a job for sure at wage w=wP (if π <1) or w≥wP (if π =1). The rest of the high types choose e=e∗and get hired with certainty at wage w=qH. We prove this claim based on the following sequence of steps: 1. By the same argument as in the proof of Proposition 1, all firms make non-negative profits. 2. Firms’ profits must be weakly increasing in θ. To see this, suppose that θ′ >θ but firm θ makes strictly higher profits than θ′, and consider the market and hiring rule (eθ,wθ,χθ) chosen by firm θ. By hiring in market (eθ,wθ) and setting χθ′(i)=I(i≥θ′), firm θ′ could make profits at least as high as firm θ since it accepts all the high types but rejects more low types than firm θ possibly can. 2. Firms’ profits must be weakly increasing in θ. To see this, suppose that θ′ >θ but firm θ makes strictly higher profits than θ′, and consider the market and hiring rule (eθ,wθ,χθ) chosen by firm θ. By hiring in market (eθ,wθ) and setting χθ′(i)=I(i≥θ′), firm θ′ could make profits at least as high as firm θ since it accepts all the high types but rejects more low types than firm θ possibly can. Page: 36 1–50 [19:28 2/11/2020 OP-REST200072.tex] MANUSCRIPT CATEGORY: Article Copyedited by: ES 37 SIGNALLING TO EXPERTS SIGNALLING TO EXPERTS 3. There exists some ¯θ such that all firms θ ≤¯θ make zero profits, and F( ¯θ)>0. To see this, recall that at least a measure F(1)−1>0 of firms do not hire, which implies zero profits. The claim then follows from the monotonicity of profits in θ. 3. There exists some ¯θ such that all firms θ ≤¯θ make zero profits, and F( ¯θ)>0. To see this, recall that at least a measure F(1)−1>0 of firms do not hire, which implies zero profits. The claim then follows from the monotonicity of profits in θ. Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rdaa068/5931860 by University of Zurich user on 05 January 2021 4. In any equilibrium, low types obtain a payoff of at least qL. Suppose that they obtain a payoff q′ L <qL and consider a market with e=0 and w∈ q′ L,qL . If G Iχ;0,w,χ >0, then firms θ < ¯θ can make profits by hiring in this market; otherwise, µ(w;0,i)=0 by Condition 1, which means low-type workers can obtain a payoff w>q′ L by choosing e=0. 4. In any equilibrium, low types obtain a payoff of at least qL. Suppose that they obtain a payoff q′ L <qL and consider a market with e=0 and w∈ q′ L,qL . If G Iχ;0,w,χ >0, then firms θ < ¯θ can make profits by hiring in this market; otherwise, µ(w;0,i)=0 by Condition 1, which means low-type workers can obtain a payoff w>q′ L by choosing e=0. 5. In any equilibrium, high types obtain a payoff of at least wP =qH −cHe∗. Suppose they obtain a payoff uH < qH −cHe∗. This means that for any i≥λ it must be that ¯w(e∗,i)<qH, and therefore ¯w(e∗,i)<qH for i<λ as well. Consider a market with e=e∗and w∈(uH +cHe∗,qH). G Iχθ ;e∗,w,χθ >0 for all θ because otherwise µ(w;e∗,λ)=0 by Condition 5, so high types can obtain a payoff of at least w−cHe∗>uH by choosing education e∗. But the support of G(·;e∗,w,χθ) cannot include low types for any θ because choosing e∗implies a payoff of ¯w(e∗,i)−cLe∗<qH −cLe∗=qL; and the support of G(·;e∗,w,χθ) cannot include only high types for θ < ¯θ because then firms θ < ¯θ could make profits by hiring in market (e∗,w). 6. For any i≥λ and any e, µ(·;e,i) has a point mass at a single wage. RESTUD: The Review of Economic Studies [19:28 2/11/2020 OP-REST200072.tex] REVIEW OF ECONOMIC STUDIES For any w≥w, beliefs can only place weight on high types since by part (C), no low types choose e′. This implies that if w<qH, any firm, including those with θ < ¯θ, could make profits by hiring in market e′,w , which contradicts part (C). Therefore we must have w=qH. Note that this implies that there can only be a single e′ ∈(0,e∗] such that ei =e′ for some i≥λ since otherwise the high types would only select the lowest such e. Let π be the fraction of high types who choose e=0 and 1−π the fraction who choose e=e′. Since they must be indifferent, it follows that high types who choose e=0 get a wage of w′ =qH −cHe′. Since all low types choose e=0, firms will find it profitable to hire in market e=0,w=w′ iff 8. In any equilibrium, the high types select either e=0 or e=e∗. If some types selected e>e∗, then by step (C) this would require paying them w>qH and therefore involve negative profits for firms. On the other hand, suppose some high type i′ sets e′ ∈(0,e∗) and let w be the highest wage such that µ w;e′,i′ =0. For any w≥w, beliefs can only place weight on high types since by part (C), no low types choose e′. This implies that if w<qH, any firm, including those with θ < ¯θ, could make profits by hiring in market e′,w , which contradicts part (C). Therefore we must have w=qH. Note that this implies that there can only be a single e′ ∈(0,e∗] such that ei =e′ for some i≥λ since otherwise the high types would only select the lowest such e. Let π be the fraction of high types who choose e=0 and 1−π the fraction who choose e=e′. Since they must be indifferent, it follows that high types who choose e=0 get a wage of w′ =qH −cHe′. Since all low types choose e=0, firms will find it profitable to hire in market e=0,w=w′ iff π (1−λ)qH +(λ−θ)qL π (1−λ)+(λ−θ) >qH −cHe′. This defines the cutoff firm θ′ such that firms with θ <θ′ make zero profits. Furthermore, this implies that all workers with i<θ′ do not get hired in market 0,w′ and therefore obtain a payoff of qL. Let ⊆ 0,θ′ be the set of firms who hire workers in market (e′,qH). REVIEW OF ECONOMIC STUDIES higher profits in market (0,uH +ǫ) than in market (˜e, ˜w), a contradiction. Instead, if G  Iχ ˆθ ;0,uH +ǫ,χ ˆθ  = 0, this requires µ(uH +ǫ;0,i)=0 for all i≥λ, which implies that e=0 is a better choice than ˜e for high types, again a contradiction. Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rdaa068/5931860 by University of Zurich user on 05 January 2021 b. Assume instead that ˆθ ≤λ. This implies that all firms that hire in market (˜e, ˜w) accept workers i∈  ˆθ,λ  . Since high-type workers are hired for sure in market (˜e, ˜w), this implies that workers i∈  ˆθ,λ  are hired for sure as well, and therefore obtain utility uH −(cL −cH)˜e. Now consider a market with e′ = ˜e+ǫ and w′ ∈( ˜w+cHǫ, ˜w+cLǫ). Suppose type i′ ∈  ˆθ,λ  is in the support of G ·;e′,w′,χθ . This requires: ¯w e′,i′ −cLe′ ≥uH −(cL −cH)˜e ⇒¯w e′,i′ − uH +cH ˜e  ≥cLǫ. ¯w e′,i′ −cLe′ ≥uH −(cL −cH)˜e ⇒¯w e′,i′ − uH +cH ˜e  ≥cLǫ. ¯w e′,i′ −cLe′ ≥uH −(cL −cH)˜e ⇒¯w e′,i′ − uH +cH ˜e  ≥cLǫ. Since ¯w e′,i must be increasing in i by Condition 2, for any high-type worker i′′: Since ¯w e′,i must be increasing in i by Condition 2, for any high-type worker i′′: ¯w e′,i′′ − uH +cH ˜e  ≥cLǫ ⇒¯w e′,i′′ − uH +cH ˜e  >cHǫ, ¯w e′,i′′ − uH +cH ˜e  ≥cLǫ ⇒¯w e′,i′′ − uH +cH ˜e  >cHǫ, which contradicts the premise that i′′ finds ˜e optimal. Hence, no i′ ∈  ˆθ,λ  can be in the support of G ·;e′,w′,χθ . If the support of G ·;e′,w′,χθ only includes i≥λ, then for small enough ǫ, firm θ would find it more profitable to hire in market (e′,w′) than in market (˜e, ˜w). Therefore it must be that G Iχθ ;e′,w′,χθ =0. This implies that µ w′;e′,i′′ =0, which in turn implies that high types prefer e′ to ˜e, a contradiction. 8. In any equilibrium, the high types select either e=0 or e=e∗. If some types selected e>e∗, then by step (C) this would require paying them w>qH and therefore involve negative profits for firms. On the other hand, suppose some high type i′ sets e′ ∈(0,e∗) and let w be the highest wage such that µ w;e′,i′ =0. SIGNALLING TO EXPERTS Therefore, it must be that all firms in the interval θ,i  hire in market (˜e, ˜w). Since there are no workers with i<i in market (˜e, ˜w), then upon hiring in market (˜e, ˜w), any firm θ ≤i hires from the entire pool of applicants, without rejecting any. Since this hiring rule is available to all firms, part (C) implies that all θ ≤i firms must make zero profits by hiring in market (˜e, ˜w). For this to be true, it must mean that they cannot make profits in any other market, including any markets with e=0. But any firm with θ >i will be able to reject some workers in the interval i,i+δ  , which implies it can make strictly positive profits by hiring in market (˜e, ˜w). Therefore, all firms in the interval (i,i+δ1] hire in market (˜e, ˜w). This in turn implies that if worker i is willing to choose ˜e, then worker i+δ1 strictly prefers ˜e, since, compared to worker i, he has a higher chance of being hired in market (˜e, ˜w) and the same chance of being hired in any other market. By continuity, this implies that there is a number δ2 >δ1 such that all workers in i,i+δ2  choose e= ˜e. Repeating the same reasoning, this implies that there is a strictly increasing sequence {δn} such that all workers in i,δn  choose ˜e. Therefore all workers i∈ i,λ  choose e= ˜e. Let ˆθ be the highest firm type that hires in market (˜e, ˜w). a. Assume ˆθ =λ . Consider market (0,uH +ǫ). If G  Iχ ˆθ ;0,uH +ǫ,χ ˆθ  >0, then they can only include high types because firm ˆθ only accepts high types. For sufficiently small ǫ, this implies that firm ˆθ could make a. Assume ˆθ =λ . Consider market (0,uH +ǫ). If G  Iχ ˆθ ;0,uH +ǫ,χ ˆθ  >0, then they can only include high types because firm ˆθ only accepts high types. For sufficiently small ǫ, this implies that firm ˆθ could make Page: 37 1–50 [19:28 2/11/2020 OP-REST200072.tex] MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES 38 REVIEW OF ECONOMIC STUDIES RESTUD: The Review of Economic Studies KURLAT & SCHEUER SIGNALLING TO EXPERTS 39 KURLAT & SCHEUER SIGNALLING TO EXPERTS 39 Partial signalling equilibrium. 1. Necessity of condition (19). Let ˜w(e,i) be the wage that would make worker i indifferent between their equilibrium payoff and choosing education e, given by: ˜w(e,i)=  u(i)+cLe if i<λ u(i)+cHe if i≥λ, (C.2) (C.2) where u(i) is given by (17). Suppose firm θ considers hiring in market (e,w). For it to believe that it will find χθ-acceptable low types, i.e. workers with i∈[θ,λ), it must be that: ˜w(e,θ)≤˜w(e,i)= ¯w(e,i)≤¯w(e,λ)≤˜w(e,λ). (C.3) (C.3) REVIEW OF ECONOMIC STUDIES Since all high types who choose e′ get a job at w=qH it follows that F()=1−π. Suppose worker i′ <θ′ chooses e=e′. His chance of finding a job at wage qH will be given by: 1−µ qH;e′,i′ = F ∩ 0,i′ 1−π = F ∩ 0,i′ F() . Since F is continuous, then for i′ sufficiently close to θ′ , µ qH;e′,i′ will be arbitrarily close to 0, and therefore (since e′ <e∗), (1−µ qH;e′,i′ )qH −cLe′ >qL. Thus, there is a low type who would prefer e=e′ to e=0, which contradicts step (C). To complete the proof, let uH be the equilibrium payoff of high types. To complete the proof, let uH be the equilibrium payoff of high types. 1. If uH >wP, then it must be that all high types choose e=0 and get hired at a wage w=uH. Firms will find it profitable to hire in this market if (1 λ) +(λ θ) 1. If uH >wP, then it must be that all high types choose e=0 and get hired at a wage w=uH. Firms will find it profitable to hire in this market if (1−λ)qH +(λ−θ)qL (1−λ)+λ−θ >uH This defines a cutoff ¯θ, so (12) holds. Furthermore, since all high types must be hired at this wage, (11) must hold. This defines a cutoff ¯θ, so (12) holds. Furthermore, since all high types must be hired at this wage, (11) must hold 2. If uH =wP, then high types are indifferent between choosing e=0 and getting hired at wage wP and choosing e=e∗and getting hired at a wage qH. Let π be the fraction that choose e=0. Firms will find it profitable to hire in market 0,wP iff 2. If uH =wP, then high types are indifferent between choosing e=0 and getting hired at wage wP and choosing e=e∗and getting hired at a wage qH. Let π be the fraction that choose e=0. Firms will find it profitable to hire in market 0,wP iff π (1−λ)qH +(λ−θ)qL π (1−λ)+(λ−θ) >wP This defines the cutoff ¯θ, so (15) holds. Furthermore, since all high types who choose e=0 must be hired at w, (16) must hold too. [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies Page: 38 1–50 Copyedited by: ES Copyedited by: ES MANUSCRIPT CATEGORY: Article ˜w(e,θ)≤˜w(e,i)= ¯w(e,i)≤¯w(e,λ)≤˜w(e,λ). The first inequality follows from the fact that u(i) and therefore ˜w(e,i) is increasing in i. The second step follows from Condition 4: if beliefs place weight on type i, then i must be indifferent between e and his equilibrium choice. The third follows from Condition 2, which implies that ¯w is monotonic in i. The last inequality follows from the fact that otherwise worker λ could exceed his equilibrium payoff by choosing e. By Condition 4, the only markets where firm θ can place beliefs on χθ-acceptable low types are those with education levels that worker i=θ is willing to choose for weakly lower wages than high types. over, for firm θ not to have well-defined beliefs about market (e,w Moreover, for firm θ not to have well-defined beliefs about market (e,w) it must be that: w≤˜w(e,θ), (C.4) (C.4) since otherwise Condition 5 requires µ(w;e,θ)=0, so some χθ-acceptable worker could exceed his equilibrium payoff by choosing e. Together, conditions (C.3) and (C.4) imply that for any market (e,w) such that ˜w(e,λ)< ˜w(e,θ) and w> ˜w(e,λ), firm θ’s beliefs G(·;e,w,χθ) can only place weight on high types. Denote by (eD θ ,wD θ ) the lowest-wage market where firm θ’s beliefs are guaranteed to only include high types, which satisfies wD θ = ˜w eD θ ,λ = ˜w eD θ ,θ . Using (6), (14), (17), and (C.2) and rearranging, the profits that firm θ can obtain by hiring in market (eD θ ,wD θ ) are θ D(θ)=qH −wD θ = cH cL θ θP 1 πP(1−λ)+λ−θ dF(θ)(qH −qL). By (B.1), profits in market (eD θ ,wD θ ) exceed those that firm θ obtains in equilibrium if condition (19) is violated, which implies it cannot be an equilibrium. By (B.1), profits in market (eD θ ,wD θ ) exceed those that firm θ obtains in equilibrium if condition (19) is violated, which implies it cannot be an equilibrium. Sufficiency of condition (19). We construct the equilibrium objects {ei,(eθ,wθ,χθ),µ,G}. 2. Sufficiency of condition (19). We construct the equilibrium objects {ei,(eθ,wθ,χθ),µ,G}. ˜w(e,θ)≤˜w(e,i)= ¯w(e,i)≤¯w(e,λ)≤˜w(e,λ). (a) Worker decisions: ei =  0 if i<λ+πP(1−λ) e∗if i≥λ+πP(1−λ) (C.5) (b) Firm decisions: ei =  0 if i<λ+πP(1−λ) e∗if i≥λ+πP(1−λ) (C.5) (eθ,wθ,χθ)= ⎧ ⎪⎪⎨ ⎪⎪⎩ (0,wP,χ(i)=I(i≥θ)) for θ ≥θP (0,qL,χ(i)=1∀i) for a measure λ−ϕP of firms θ <θP (e∗,qH,χ(i)=1∀i) for a measure (1−λ) 1−πP of firms θ <θP (0,0,χ(i)=1∀i) otherwise (C.6) where ϕP = λ θP λ−θ πP(1−λ)+λ−θ dF(θ) (c) Probabilities: µ(w;e,i)= ⎧ ⎪⎨ ⎪⎩ 1 if e=0,w≥wP 1−  min{i,λ} θP 1 λ−θ+πP(1−λ)dF(θ) if e=0,wP >w≥qL I(w≥min{ ˜w(e,i), ˜w(e,λ)}) otherwise (C.7) (eθ,wθ,χθ)= ⎧ ⎪⎪⎨ ⎪⎪⎩ (0,wP,χ(i)=I(i≥θ)) for θ ≥θP (0,qL,χ(i)=1∀i) for a measure λ−ϕP of firms θ <θP (e∗,qH,χ(i)=1∀i) for a measure (1−λ) 1−πP of firms θ <θP (0,0,χ(i)=1∀i) otherwise (C.6) (C.6) where we used (C.2). RESTUD: The Review of Economic Studies Page: 39 1–50 RESTUD: The Review of Economic Studies Page: 39 1–50 [19:28 2/11/2020 OP-REST200072.tex] Page: 39 1–50 MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES REVIEW OF ECONOMIC STUDIES 40 (d) Beliefs: for selection rule χ(i)=I(i≥θ), (d) Beliefs: for selection rule χ(i)=I(i≥θ), (d) Beliefs: for selection rule χ(i)=I(i≥θ), g(i;e,w,χ)= ⎧ ⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎩ I(i∈[θ,λ))+πPI(i≥λ) πP(1−λ)+λ−θ if e=0,w≥wP I(i∈[θ,λ])µ(w;e,i)  λ θ µ(w;e,i)di if e=0,wP >w≥qL I(i=θ) if e∈ 0,eD θ ,w≥˜w(e,θ) I(i≥λ) 1−λ if e≥eD θ ,w≥˜w(e,λ) 0 for any other (e,w) (C.8) and for selection rule χ(i)=1∀i, g(i;e,w,χ)= ⎧ ⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎩ I(i<λ)+πPI(i≥λ) πP(1−λ)+λ if e=0,w≥wP µ(w;e,i)  1 0 µ(w;e,i)di if e=0,wP >w≥qL I  i<θP θP if e∈(0,e∗),w≥˜w(e,0) I(i≥λ) 1−λ if e≥e∗,w≥˜w(e,λ) 0 for any other (e,w) (C.9) We now verify that {ei,(eθ,wθ,χθ),µ,G} satisfies all the equilibrium conditions from Definition 1. (C.7) implies that low types i∈[0,λ) are indifferent between any e∈ 0,eD i )  and high types are indifferent between any e≥0, so the education decisions (C.5) solve the workers’ problem. The beliefs (C.8) and (C.9) together with the fact that condition (19) holds implies that firms θ ≥θP maximize profits by hiring selectively in market (0,wP). All other firms make zero profits by hiring non-selectively either in market (0,qL) or (e∗,qH), and any other market has either G Iχθ ;e,w,χθ =0 or results in losses. Therefore the demands (C.6) are an optimal choice. Furthermore, replacing (C.6) in (3) implies that demand in market (e,w) for a set of selection rules X0 θ′ ={χ (i)=I(i≥θ): θ ∈ 0,θ′ is: D(e,w,X0 θ′ )= ⎧ ⎨ ⎩ λ−ϕP if e=0,w=qL max  F θ′ −F θP ,0  if e=0,w=wP (1−πP)(1−λ) if e=e∗,w=qH Together with (C.8) and (C.9), this implies that Condition 1 holds. Condition 2 is satisfied because, by (C.7), µ(·;e,i) is weakly decreasing in i. Finally, (C.5) and (C.7) imply that beliefs (C.8) and (C.9) satisfy Condition 3 in non-empty markets. Since low types i find e∈ 0,eD i )  optimal and high types find any e≥0 optimal, beliefs satisfy Condition 4 when they are well defined, and G(Iχ;e,w,χ)=0 only at wages where µ(w;e,i)=0 for all i such that χ(i)=1, so Condition 5 is satisfied as well. RESTUD: The Review of Economic Studies Proof of Proposition 6 1. Using (14) and (6): wP =  1−cH cL qH + cH cL qL, and therefore lim cH cL →1wP =qL. Using (12) we have wN >wP, so there is a candidate corner equilibrium. h / 1 di i (18) h ld h did ilib i i i d d ilib i and therefore lim cH cL →1wP =qL. Using (12) we have wN >wP, so there is a candidate corner equilibrium. and therefore lim cH cL →1wP =qL. Using (12) we have wN >wP, so there is a candidate corner equilibrium. Furthermore, as cH/cL →1, condition (18) holds so the candidate equilibrium is indeed an equilibrium. and therefore lim cH cL →1w qL. Using (12) we have w >w , so there is a candidate corner equilibrium. Furthermore, as cH/cL →1, condition (18) holds so the candidate equilibrium is indeed an equilibrium. L Furthermore, as cH/cL →1, condition (18) holds so the candidate equilibrium is indeed an equilibrium. 2. Taking the limit, lim cH cL →0wP =qH. Using (15), this implies θP →λ, so condition (16) cannot hold for any πP >0. KURLAT & SCHEUER SIGNALLING TO EXPERTS KURLAT & SCHEUER SIGNALLING TO EXPERTS 41 and for selection rule χ(i)=1∀i, Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rdaa068/5931860 by University of Zurich user on 05 January 2021 g(i;e,w,χ)= ⎧ ⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎩ 1∀i if e=0,w≥wN µ(w;e,i)  1 0 µ(w;e,i)di if e=0,wN >w≥qL I  i<θN  θN if e∈ 0,eN ,w≥˜w(e,0) I(i≥λ) 1−λ if e≥eN,w≥˜w(e,λ) 0 for any other (e,w) (C.14) with eN =(wN −qL)/(cL −cH) with eN =(wN −qL)/(cL −cH) with eN =(wN −qL)/(cL −cH) Proof of Proposition 5 1. Using the reparametrization of the model in terms of ˆλ, equation (12) generalizes to wN = λ−θN ˆλ λqL +  1−ˆλ  qH λ−θN ˆλ λ +  1−ˆλ  . (C.15) (C.15) For ˆλ low enough, wN >wP so there is a candidate corner equilibrium. Condition (18) generalizes to (λ−θ) ˆλ λ λ−θN ˆλ λ +  1−ˆλ   (λ−θ) ˆλ λ +  1−ˆλ  > cH cL −cH  1−ˆλ  ⎛ ⎝ 1 θ 1 (λ−t) ˆλ λ +  1−ˆλ dF(t) ⎞ ⎠, which cannot hold for sufficiently low ˆλ, so the candidate equilibrium is indeed an equilibrium. Furthermore, taking the limit in (C.15) we obtain limˆλ→0wN =qH. which cannot hold for sufficiently low ˆλ, so the candidate equilibrium is indeed an equilibrium. Furthermore, taking the limit in (C.15) we obtain limˆλ→0wN =qH. 2. Equation (11) implies that limF→F∗θN =λ, which implies, using (12), that limF→F∗wN =qH for F sufficiently close to F∗, so a candidate equilibrium with the desired properties exists. Furthermore, as θN →λ, condition (18) cannot hold so the candidate equilibrium is indeed an equilibrium. Pure signalling equilibrium. The above analysis applies for the special case with πP =0. Pure signalling equilibrium. The above analysis applies for the special case with πP =0. No-signalling equilibrium. Necessity and sufficiency of condition (18) are proved by the same steps as for the partial signalling equilibrium. For completeness, we state the equilibrium objects {ei,(eθ,wθ,χθ),µ,G}. (a) Worker decisions: ei =0∀i (C.10) (a) Worker decisions: ei =0∀i (C.10) (b) Firm decisions: (eθ,wθ,χθ)= ⎧ ⎨ ⎩ (0,wN,χ(i)=I(i≥θ)) for θ ≥θN (0,qL,χ(i)=1∀i) for a measure 1−F(λ)+F(θN) of firms θ <θN (0,0,χ(i)=1∀i) otherwise (C.11) (c) Probabilities: µ(w;e,i)= ⎧ ⎨ ⎩ 1 if e=0,w≥wN 1−  min{i,λ} θN 1 1−θ dF(θ) if e=0,wN >w≥qL I(w≥min{ ˜w(e,i), ˜w(e,λ)}) otherwise (C.12) (d) Beliefs: for selection rule χ(i)=I(i≥θ), RESTUD: The Review of Economic Studies Page: 40 1–50 RESTUD: The Review of Economic Studies [19:28 2/11/2020 OP-REST200072.tex] Page: 40 1–50 Copyedited by: ES MANUSCRIPT CATEGORY: Article Copyedited by: ES RESTUD: The Review of Economic Studies Proof of Proposition 7 1. It is sufficient to prove claim (b) because claim (a) is a special case with πP 2 =θP 2 =0. By equation (B.1), firm profits are increasing in πP, and since wP is the same across equilibria, firms are better off in the higher-πP equilibrium. High-type workers obtain a payoff of wP in both equilibria, so they are indifferent. Using (17), workers with i≤θP 2 get a payoff of qL in both equilibria, so they are also indifferent. Workers with i∈(θP 2 ,θP 1 ] get qL in the first equilibrium and more than qL in the second, so they are better off in the second. For workers with i∈ θP 1 ,λ , their payoff is: 1. It is sufficient to prove claim (b) because claim (a) is a special case with πP 2 =θP 2 =0. By equation (B.1), firm profits are increasing in πP, and since wP is the same across equilibria, firms are better off in the higher-πP equilibrium. High-type workers obtain a payoff of wP in both equilibria, so they are indifferent. Using (17), workers with i≤θP 2 get a payoff of qL in both equilibria, so they are also indifferent. Workers with i∈(θP 2 ,θP 1 ] get qL in the first equilibrium and more than qL in the second, so they are better off in the second. For workers with i∈ θP 1 ,λ , their payoff is: u(i)=qL + i θP 1 πP(1−λ)+λ−θ dF(θ) wP −qL =wP − λ i 1 πP(1−λ)+λ−θ dF(θ) wP −qL u(i)=qL + i θP 1 πP(1−λ)+λ−θ dF(θ) wP −qL =wP − λ i 1 πP(1−λ)+λ−θ dF(θ) wP −qL where we used (16). This is increasing in πP, so they are also better off in the second equilibrium. [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies Page: 41 1–50 [19:28 2/11/2020 OP-REST200072.tex] MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES REVIEW OF ECONOMIC STUDIES 42 2. (a) In the first equilibrium, all firms make zero profits, so they are better off in the second equilibrium. Low-type workers get a payoff of qL in the first equilibrium, but those with i>θN get more in the second equilibrium. High-type workers get a payoff of wP in the first equilibrium but wN in the second, so they are also better off. Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rdaa068/5931860 by University of Zurich user on 05 January 2021 (b) By equation (B.1), for θ sufficiently close to λ, firm θ’s profits approach qH −w, so wP <wN implies they are higher in the first equilibrium. High-type workers get a payoff of wP in the first equilibrium but wN in the second, so they are better off in the second. (b) By equation (B.1), for θ sufficiently close to λ, firm θ’s profits approach qH −w, so wP <wN implies they are higher in the first equilibrium. High-type workers get a payoff of wP in the first equilibrium but wN in the second, so they are better off in the second. D. FALSE NEGATIVES Uniqueness in case f (θ) is strictly increasing Proposition D.1 If condition (28) holds, the system of equations (26), (27) has no solution. Otherwise, it has a unique solution. Proof. Solving (27) for iS and replacing in (26), a solution requires: Proof. Solving (27) for iS and replacing in (26), a solution requires: i∗ ≡f i∗ " cH cL −λ  F(i∗) f (i∗) +λ −1# −  1−cH cL iH i∗ λ i−i∗+ F(i∗) f (i∗) +λ df (i)=0 (D.1) (D.1) Taking the derivative and rearranging: Taking the derivative and rearranging: ∂ ∂i∗=f ′ i∗ cH cL $ 1−λ  F(i∗) f (i∗) +λ −1% +λ " f i∗  F(i∗) f (i∗) +λ −2 1−F(i∗)f ′(i∗) f (i∗)2 −  1−cH cL iH i∗  i−i∗+ F(i∗) f (i∗) +λ −2 df (i)  F(i∗)f ′(i∗) f (i∗)2 # ≥f ′ i∗ cH cL $ 1−λ  F(i∗) f (i∗) +λ −1% +λ " f i∗  F(i∗) f (i∗) +λ −2 1−F(i∗)f ′(i∗) f (i∗)2 −  1−cH cL iH i∗  i−i∗+ F(i∗) f (i∗) +λ −1 df (i)  F(i∗)f ′(i∗) f (i∗)2  F(i∗) f (i∗) +λ −1# where the inequality follows because i>i∗. If i∗satisfies (D.1), then: where the inequality follows because i>i∗. If i∗sati ∂ ∂i∗≥f ′ i∗ cH cL $ 1−λ F(i∗) f (i∗) +λ −1% +λ " f i∗  F(i∗) f (i∗) +λ −2 1−F(i∗)f ′(i∗) f (i∗)2 −f i∗ " cH cL −λ  F(i∗) f (i∗) +λ −1# F(i∗)f ′(i∗) f (i∗)2 F(i∗) f (i∗) +λ −1# =λf i∗  F(i∗) f (i∗) +λ −2 >0 so the function (i∗) is increasing at any i∗such that (i∗)=0. Condition (28) is equivalent to (iH)<0. Furthermore, so the function (i∗) is increasing at any i∗such that (i∗)=0. Condition (28) is equivalent to (iH)<0. Furthermore, (λ)=−  1−cH cL " f (λ)+ iH i∗ λ i df (i) # <0 Therefore, if (28) holds, there can be no i∗∈[λ,iH] that satisfies (i∗)=0 because (λ)<0 and (iH)<0 and must be increasing at any solution. Instead, if condition (28) does not hold, (iH)≥0, so by continuity and using the fact that is increasing at any solution, there is exactly one i∗∈[λ,iH] that satisfies (i∗)=0. □ is increasing at any solution, there is exactly one i∗∈[λ,iH] that satisfies (i∗)=0. □ 3. lim cH cL →1iS =λ. s.t. ω∈[0,1], θ1,θ2 ∈[λ,1]and ωθ1+(1−ω)θ2 =θ. s.t. ω∈[0,1], θ1,θ2 ∈[λ,1]and ωθ1+(1−ω)θ2 =θ. The corresponding density ¯f (θ), which is weakly increasing, is the “ironed” version of the original density f (θ). We now show how the analysis in Section 6 extends to this general case, replacing F with ¯F. Let iH be defined as iH ≡min i∈[λ,1]  i: ¯f (i)≥1  . iH ≡min i∈[λ,1]  i: ¯f (i)≥1  . This generalizes the definition of iH in (21), allowing both for the possibility of ironing and the case where f (i)>1 for all i (in which case trivially iH =λ). Let the reservation wage for type i∈[i∗,iH) be given by (i′ i ) +λ This generalizes the definition of iH in (21), allowing both for the possibility of ironing and the case where f (i)>1 for all i (in which case trivially iH =λ). Let the reservation wage for type i∈[i∗,iH) be given by (i′ i )q +λq w(0,i)=max i′ (i′−iS)qH +λqL i′−iS +λ s.t. ¯f i′ = ¯f (i). (D.4) (D.4) Hence, when ¯f is strictly increasing, this coincides with (22), but in a flat region (due to ironing), w(0,i) equals the value for the top of the ironing range. In other words, in intervals [i0,i1] where the ironed density ¯f is constant, there will be “bunching:” all remaining workers who are not hired non-selectively at higher wages are hired at the same wage ¯w(0,i1) by firms θ ∈[i0,i1]. Based on the same steps as underlying (26) but using (D.4) instead of (22), we obtain Ŵ(i,iS)= ¯f (i)  cH cL − λ ib(i)−iS +λ −λ  1−cH cL iH i 1 i′−iS +λd¯f (i′) (D.5) (D.5) where ib(i)=max  i′ : ¯f (i′)= ¯f (i)  . Let i∗and iS solve where ib(i)=max  i′ : ¯f (i′)= ¯f (i)  . Let i∗and iS solve i∗= min i∈[λ,1]{i:Ŵ(i,iS)≥0} (D.6) ¯F i∗ = ¯f i∗ i∗−iS . (D.7) i∗= min i∈[λ,1]{i:Ŵ(i,iS)≥0} ¯F i∗ = ¯f i∗ i∗−iS . di i (26) f (D.6) (D.6) (D.7) (D.6) (D.7) (D.7) and Equation (D.6) generalizes the indifference condition (26) to account for the fact that, with bunching, the reservation wage function (D.4) and hence Ŵ(i,iS) can be discontinuous in i. Note that, by (D.6), whenever i∗falls into a bunching region, it corresponds to the lower end of it. KURLAT & SCHEUER SIGNALLING TO EXPERTS 43 KURLAT & SCHEUER SIGNALLING TO EXPERTS 43 43 Proof. Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rda 1. Letting the fraction of low types be ˆλ, equations (21), (22) and (26) generalize, respectively, to: f iH = 1−ˆλ 1−λ, w(0,i)= 1−ˆλ 1−λ (i−iS)qH + ˆλqL 1−ˆλ 1−λ (i−iS)+ ˆλ ,and (D.2) Ŵ i∗,iS =cH cL f i∗ −ˆλ  1−cH cL iH i∗ ⎡ ⎣ 1 1−ˆλ 1−λ (i−iS)+ ˆλ ⎤ ⎦df i′ −f i∗ ˆλ 1 1−ˆλ 1−λ (i∗−iS)+ ˆλ =0 (D.3) f iH = 1−ˆλ 1−λ, w(0,i)= 1−ˆλ 1−λ (i−iS)qH + ˆλqL 1−ˆλ 1−λ (i−iS)+ ˆλ ,and (D.2) (D.2) (D.2) Ŵ i∗,iS =cH cL f i∗ −ˆλ  1−cH cL iH i∗ ⎡ ⎣ 1 1−ˆλ 1−λ (i−iS)+ ˆλ ⎤ ⎦df i′ −f i∗ ˆλ 1 1−ˆλ 1−λ (i∗−iS)+ ˆλ =0 (D.3) (D.3) while the market-clearing condition is unchanged. The first statement follows directly from equation (D.2), the second from equation (D.3) and the last from equation (D.7). while the market-clearing condition is unchanged. The first 2. Since the measure of firms is assumed to be greater than 1, for F sufficiently close to F∗, then f (i)>1 for all i, which implies iH =λ. 3. Equation (26) implies lim cH cL →1i∗−iS =0. Using this and equation (27), we have lim cH cL →1F(i∗)=0, which implies lim cH cL →1i∗=λ and therefore lim cH cL →1iS =λ. □ 3. Equation (26) implies lim cH cL →1i∗−iS =0. Using this and equation (27), we have lim cH cL →1F(i∗)=0, which implies lim cH cL →1i∗=λ and therefore lim cH cL →1iS =λ. □ □ By part 1, as the fraction of low types goes to zero, nobody signals and everyone is paid qH. Part 2 says that as firms become fully informed, again nobody signals and all high types are paid qH. Finally, part 3 shows that if signalling is sufficiently expensive, no workers signal in equilibrium. In all cases the equilibrium allocations are continuous in the limit. General case For the case where f (i) is not monotone, the argument in Section 6 needs to be modified. Consider two workers, i and i′ with i∗<i<i′ <iH and assume f (i)>f i′ . The argument above, unmodified, implies that any worker will be able to sell a fraction 1−f i′ of his labour to non-selective firms at wages above w 0,i′ . This means that only f i′ of i-workers will be available for hire in market 0,w(0,i) , which is less than the f (i) workers that firms with θ =i want to hire. Realizing this, firms would bid up the wage, displacing non-selective firms. To characterize exactly what will happen, it is useful to define ¯F(θ) as the convex hull of F(θ), i.e. the highest convex function on [λ,1] such that ¯F(θ)≤F(θ): ¯F(θ) ≡ min ω,θ1,θ2 {ωF(θ1)+(1−ω)F(θ2)} ¯F(θ) ≡ min ω,θ1,θ2 {ωF(θ1)+(1−ω)F(θ2)} ¯F(θ) ≡ min ω,θ1,θ2 {ωF(θ1)+(1−ω)F(θ2)} Continuity in the limit Proposition D.2 1. limˆλ→0w(0,i)=qH, limˆλ→0i∗=λ, and limˆλ→0iS =λ, for all i∈[i∗,iH 2. Let F∗be a mass point at θ =λ. limF→F∗iH =λ. Proposition D.2 1. limˆλ→0w(0,i)=qH, limˆλ→0i∗=λ, and limˆλ→0iS =λ, for all i∈[i∗,iH). 2 L t F∗b i t t θ λ li i λ 2. Let F∗be a mass point at θ =λ. limF→F∗iH =λ. 3. lim cH cL →1iS =λ. Page: 42 1–50 [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES RESTUD: The Review of Economic Studies Page: 43 1–50 Uniqueness We prove uniqueness based on the following sequence of steps: Uniqueness We prove uniqueness based on the following sequence of steps: 1. By the same arguments as in the proof of Proposition 3: 1. By the same arguments as in the proof of Proposition 3: (a) all firms make non-negative profits (b) profits are decreasing in θ ¯ (c) all firms θ ≥¯θ make zero profits in equilibrium, with ¯θ ∈[λ,1) and F(1)−F( ¯θ)>0 (d) low types obtain a payoff of at least qL P (e) high types obtain a payoff of at least wP =qH −cHe∗. 2. Because all low types are indistinguishable for all firms, all low types must obtain the same utility. Denote this by uL. 3. Utility for workers is weakly increasing in i. This follows immediately from Condition 2. 4. In any equilibrium, all low types choose e=0. Suppose some low types choose e′ >0. By step (D), we have ¯w e′,i ≥qL +cLe′ for all i<λ. Consider all markets with e=e′ and w>qL where low types are hired with positive probability. For low types to be hired, in any such market there must be firms that hire non-selectively, setting χ(i)=1 for all i. By step (D), there must be high-type applicants in all these markets. Let w′ be the highest wage where anyone choosing e′ is hired with positive probability. Suppose first that some high types i′ ≥λ are hired in market (e′,w′) by selective firms θ ≤i′ setting selection rule χθ(i)=I(i≥θ). The equilibrium payoff of these high types must be u′ H ≤w′−cHe′. Consider a market (0, ˜w) with ˜w∈(w′−cHe′,w′). Then G(Iχθ ;0, ˜w,χθ)>0 since otherwise µ( ˜w;0,i′)=0 by Condition 5, so type i′ could obtain a payoff of at least ˜w>u′ H by choosing e=0. The support of G(·;0, ˜w,χθ) can only include high types by construction of χθ. But this would imply that firm θ could increase its profits by hiring high types in market (0, ˜w) instead of market (e′,w′) at wage ˜w<w′. Hence, everyone in market (e′,w′) must be hired by non-selective firms. Since this is feasible for any firm and by step (D), all firms must make zero profits in market (e′,w′). This implies w′ <qH. Because all firms hire non-selectively in market (e′,w′), µ(w′;e′,i)=µ′ is the same for all i. Suppose first that µ′ >0. Consider a market (e′,w′−ǫ). Then for sufficiently small ǫ >0, the applicant pool is the same in markets (e′,w′−ǫ) and (e′,w′). s.t. ω∈[0,1], θ1,θ2 ∈[λ,1]and ωθ1+(1−ω)θ2 =θ. These definitions allow us to state the following general existence and uniqueness result, of which Proposition 8 in Section 6 is a special case. RESTUD: The Review of Economic Studies Page: 43 1–50 RESTUD: The Review of Economic Studies Page: 43 1–50 [19:28 2/11/2020 OP-REST200072.tex] Page: 43 1–50 RESTUD: The Review of Economic Studies Page: 43 1–50 MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES (i) A measure F(iH) of high types with i∈[λ,iH) choose e=0. (ii) All other high types with i∈[λ,iH) choose e=e∗. The corner equilibrium is of the same form as described in Section 6. If the equilibrium is interior, the proposition encompasses two cases. Either there is no bunching at i∗, in which case our previous analysis goes through: the indifference condition Ŵ(i∗,iS)=0 implies that type i∗is just indifferent between signalling or not, and all high types below i∗who do not signal are hired at least at wage wS =w(0,i∗). The other case allows for i∗to be in a bunching region. Because there is a discontinuity in u(i) at i∗in this case, i∗is given by the smallest i that still prefers choosing e=0 over signalling (so u(i∗)>qH −cHeS and hence Ŵ(i∗,iS)>0). All high types i<i∗are indifferent between signalling or not. The wages at which workers are hired and all firms’ decisions are specified in the proof below. When there is bunching at the bottom (i.e. on the interval [λ,i∗]), the market-clearing condition (D.7) implies iS =λ, so there is no signalling whatsoever in equilibrium. This occurs when there is a high density of precisely informed buyers relative to less informed ones. We now provide a proof of Proposition D.3, establishing first the uniqueness and then the existence of the stated equilibrium. RESTUD: The Review of Economic Studies REVIEW OF ECONOMIC STUDIES 44 oposition D.3 There exists a generically unique equilibrium: Proposition D.3 There exists a generically unique equilibrium: 1. All low types i∈[0,λ) choose e=0. 1. All low types i∈[0,λ) choose e=0. 3. For i∈[λ,iH), the equilibrium takes one of the following two possible forms: (a) An interior equilibrium where iS and i∗solve (D.6) and (D.7) and: (a) An interior equilibrium where iS and i∗solve (D.6) and (D.7) and: (i) A measure iS −λ of high types with i∈[λ,i∗) choose e=eS. (ii) All other high types with i∈[λ,i∗) choose e=0. (i) A measure iS −λ of high types with i∈[λ,i∗) choose e=eS. (ii) All other high types with i∈[λ,i∗) choose e=0. (b) A corner equilibrium where Ŵ(iH,iH −F(iH))<0 and: (b) A corner equilibrium where Ŵ(iH,iH −F(iH))<0 and: (i) A measure F(iH) of high types with i∈[λ,iH) choose e=0. (ii) All other high types with i∈[λ,iH) choose e=e∗. 8. Define ¯wS ≡qH −cHeS =  1−cH cL qH + cH cL uL. (D.8) (D.8) There exists a cutoff i∗such that: for i<i∗, high types’ utility is u(i)= ¯wS and for i≥i∗, utility is u(i)≥¯wS and e=0. Steps (D) and (D) imply that high types who choose e>0 must obtain utility equal to ¯wS. Therefore the only possible way to obtain higher utility is to choose e=0. The result then follows from step (D). There exists a cutoff i∗such that: for i<i∗, high types’ utility is u(i)= ¯wS and for i≥i∗, utility is u(i)≥¯wS and e=0. Steps (D) and (D) imply that high types who choose e>0 must obtain utility equal to ¯wS. Therefore the only possible way to obtain higher utility is to choose e=0. The result then follows from step (D). 9. For workers i≥i∗(who choose e=0) the minimum wage in their support w(0,i) is weakly increasing in i. This follows from the fact that w(0,i) solves µ(w;0,i)=0, and µ(w;0,i) is weakly increasing in w and weakly decreasing in i by Condition 2. 9. For workers i≥i∗(who choose e=0) the minimum wage in their support w(0,i) is weakly increasing in i. This follows from the fact that w(0,i) solves µ(w;0,i)=0, and µ(w;0,i) is weakly increasing in w and weakly decreasing in i by Condition 2. 10. If some type i≥0 who chooses e=0 is hired by a selective firm, this can only occur at the minimum wage in worker i′s support w(0,i). To see this, consider a market (0,w) where a high type i≥λ is hired by a selective firm θ <i setting χθ(i)=I(i≥θ), and suppose µ(w;0,i)>0. This implies that there are i-type applicants in some market (0,w−ǫ). As a result, firm θ could increase its profits by shifting demand to market (0,w−ǫ) using the same selection rule. 11. There does not exist a market (0,w) with w>qL where all firms hire non-selectively. Suppose there were such a market and let (0,w) be the highest-wage market where all firms hire non-selectively. All firms must make zero profits in (0,w) and µ(w;0,i)= ¯µ for all i. Suppose ¯µ>0. Consider a market (0,w−ǫ). For sufficiently small ǫ >0, the pool of applicants is the same in markets (0,w−ǫ) and (0,w). Then all firms could make positive profits by hiring in market (0,w−ǫ), contradicting (i). Hence we must have ¯µ=0. Uniqueness We prove uniqueness based on the following sequence of steps: This implies that the equilibrium payoff of the low types is u′ L =w′−cLe′ and the equilibrium payoff of those high types who choose e′ is u′ H =w′−cHe′. Consider a market (e′′,w′′) such that e′′ =e′+ǫ and w′′ ∈(w′+cHǫ,w′+cLǫ). Suppose χ(i)=1 for all i. Then G(Iχ;e′′,w′′,χ)>0 since otherwise µ(w′′;e′′,i)=0 for all i, so all high types who choose e′ could obtain payoff w′′−cHe′′ >w′−cHe′ =u′ H, a contradiction. The support of G(·;e′′,w′′,χ) cannot include low types since w′′− cLe′′ <w′−cLe′ =u′ L. ThesupportofG(·;e′′,w′′,χ)cannotincludeonlyhightypessincethenanyfirm θ > ¯θ could make strictly positive profits in market (e′′,w′′) for ǫ ∈(0,(qH −w′)/cL). This delivers the final contradiction. 5. Any high type who chooses e>0 is hired with probability 1 at w=qH. Suppose otherwise, then there exists a market (e,w) with w<qH such that there are high-type applicants. Since there are no low types in market (e,w) by step (D), any firm θ > ¯θ could then make positive profits by hiring non-selectively in market (e,w), contradicting step (D). 5. Any high type who chooses e>0 is hired with probability 1 at w=qH. Suppose otherwise, then there exists a market (e,w) with w<qH such that there are high-type applicants. Since there are no low types in market (e,w) by step (D), any firm θ > ¯θ could then make positive profits by hiring non-selectively in market (e,w), contradicting step (D). 5. Any high type who chooses e>0 is hired with probability 1 at w=qH. Suppose otherwise, then there exists a market (e,w) with w<qH such that there are high-type applicants. Since there are no low types in market (e,w) by step (D), any firm θ > ¯θ could then make positive profits by hiring non-selectively in market (e,w), contradicting step (D). 6. No firm hires high types selectively at any e>0. Suppose there was a high type i>λ who is hired in market (e,qH) with e>0 by a firm θ <i that sets selection rule χθ(i)=I(i≥θ). Consider market (0,w′) with w′ ∈(qH −cHe,qH). Then G(Iχθ ;e,qH,χθ)>0 since otherwise µ(qH,0,i)=0 by Condition 5, so type i could obtain a payoff w′ > qH −cHe by choosing e=0. Since by construction the support of G(·;e,qH,χθ) only includes high types and since w′ <qH, firm θ can increase its profits by hiring high types in market (0,w′) rather than (e,qH), a contradiction. Uniqueness We prove uniqueness based on the following sequence of steps: Hence, all high types selecting e>0 are hired by firms using selection rule χ(i)=1 for all i. 6. No firm hires high types selectively at any e>0. Suppose there was a high type i>λ who is hired in market (e,qH) with e>0 by a firm θ <i that sets selection rule χθ(i)=I(i≥θ). Consider market (0,w′) with w′ ∈(qH −cHe,qH). Then G(Iχθ ;e,qH,χθ)>0 since otherwise µ(qH,0,i)=0 by Condition 5, so type i could obtain a payoff w′ > qH −cHe by choosing e=0. Since by construction the support of G(·;e,qH,χθ) only includes high types and since w′ <qH, firm θ can increase its profits by hiring high types in market (0,w′) rather than (e,qH), a contradiction. Hence, all high types selecting e>0 are hired by firms using selection rule χ(i)=1 for all i. 6. No firm hires high types selectively at any e>0. Suppose there was a high type i>λ who is hired in market (e,qH) with e>0 by a firm θ <i that sets selection rule χθ(i)=I(i≥θ). Consider market (0,w′) with w′ ∈(qH −cHe,qH). Then G(Iχθ ;e,qH,χθ)>0 since otherwise µ(qH,0,i)=0 by Condition 5, so type i could obtain a payoff w′ > qH −cHe by choosing e=0. Since by construction the support of G(·;e,qH,χθ) only includes high types and since w′ <qH, firm θ can increase its profits by hiring high types in market (0,w′) rather than (e,qH), a contradiction. Hence, all high types selecting e>0 are hired by firms using selection rule χ(i)=1 for all i. 7. If any high types choose some education eS >0, it must satisfy qH −cLeS =uL. Suppose first that some high types choose e∈(0,eS). By step (D), they are hired at wage qH and by step (D) they are hired by non-selective firms. However, this implies that the low types, by choosing e, could obtain qH −cLe>uL, a contradiction. Suppose next that some high types choose e>eS. Consider some market (eS,qH −ǫ) and selection rule χ(i)=1 for all i, which is feasible for all firms. For sufficiently small ǫ, G(Iχ;eS,qH −ǫ,χ)>0 since otherwise µ(qH −ǫ;eS,i)=0 and those high types choosing e could do better by choosing education eS. By Condition 4, the support cannot include low types because qH −cLe<uL. Hence, firms θ ≥¯θ could make strictly positive profits in market (eS,qH −ǫ), contradicting step (D). 7. If any high types choose some education eS >0, it must satisfy qH −cLeS =uL. Uniqueness We prove uniqueness based on the following sequence of steps: Suppose first that some high types choose e∈(0,eS). By step (D), they are hired at wage qH and by step (D) they are hired by non-selective firms. However, this implies that the low types, by choosing e, could obtain qH −cLe>uL, a contradiction. Suppose next that some high types choose e>eS. Consider some market (eS,qH −ǫ) and selection rule χ(i)=1 for all i, which is feasible for all firms. For sufficiently small ǫ, G(Iχ;eS,qH −ǫ,χ)>0 since otherwise µ(qH −ǫ;eS,i)=0 and those high types choosing e could do better by choosing education eS. By Condition 4, the support cannot include low types because qH −cLe<uL. Hence, firms θ ≥¯θ could make strictly positive profits in market (eS,qH −ǫ), contradicting step (D). 8. Define Uniqueness We prove uniqueness based on the following sequence of steps: Since profits are zero in market (e′,w′), all firms could make positive profits by hiring in Page: 44 1–50 [19:28 2/11/2020 OP-REST200072.tex] MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES 45 KURLAT & SCHEUER SIGNALLING TO EXPERTS market (e′,w′−ǫ), contradicting step (D). Hence we must have µ′ =0. This implies that the equilibrium payoff of the low types is u′ L =w′−cLe′ and the equilibrium payoff of those high types who choose e′ is u′ H =w′−cHe′. Consider a market (e′′,w′′) such that e′′ =e′+ǫ and w′′ ∈(w′+cHǫ,w′+cLǫ). Suppose χ(i)=1 for all i. Then G(Iχ;e′′,w′′,χ)>0 since otherwise µ(w′′;e′′,i)=0 for all i, so all high types who choose e′ could obtain payoff w′′−cHe′′ >w′−cHe′ =u′ H, a contradiction. The support of G(·;e′′,w′′,χ) cannot include low types since w′′− cLe′′ <w′−cLe′ =u′ L. ThesupportofG(·;e′′,w′′,χ)cannotincludeonlyhightypessincethenanyfirm θ > ¯θ could make strictly positive profits in market (e′′,w′′) for ǫ ∈(0,(qH −w′)/cL). This delivers the final contradiction. market (e′,w′−ǫ), contradicting step (D). Hence we must have µ′ =0. This implies that the equilibrium payoff of the low types is u′ L =w′−cLe′ and the equilibrium payoff of those high types who choose e′ is u′ H =w′−cHe′. Consider a market (e′′,w′′) such that e′′ =e′+ǫ and w′′ ∈(w′+cHǫ,w′+cLǫ). Suppose χ(i)=1 for all i. Then G(Iχ;e′′,w′′,χ)>0 since otherwise µ(w′′;e′′,i)=0 for all i, so all high types who choose e′ could obtain payoff w′′−cHe′′ >w′−cHe′ =u′ H, a contradiction. The support of G(·;e′′,w′′,χ) cannot include low types since w′′− cLe′′ <w′−cLe′ =u′ L. ThesupportofG(·;e′′,w′′,χ)cannotincludeonlyhightypessincethenanyfirm θ > ¯θ could make strictly positive profits in market (e′′,w′′) for ǫ ∈(0,(qH −w′)/cL). This delivers the final contradiction. market (e′,w′−ǫ), contradicting step (D). Hence we must have µ′ =0. This implies that the equilibrium payoff of the low types is u′ L =w′−cLe′ and the equilibrium payoff of those high types who choose e′ is u′ H =w′−cHe′. Consider a market (e′′,w′′) such that e′′ =e′+ǫ and w′′ ∈(w′+cHǫ,w′+cLǫ). Suppose χ(i)=1 for all i. Then G(Iχ;e′′,w′′,χ)>0 since otherwise µ(w′′;e′′,i)=0 for all i, so all high types who choose e′ could obtain payoff w′′−cHe′′ >w′−cHe′ =u′ H, a contradiction. The support of G(·;e′′,w′′,χ) cannot include low types since w′′− cLe′′ <w′−cLe′ =u′ L. ThesupportofG(·;e′′,w′′,χ)cannotincludeonlyhightypessincethenanyfirm θ > ¯θ could make strictly positive profits in market (e′′,w′′) for ǫ ∈(0,(qH −w′)/cL). This delivers the final contradiction. market (e′,w′−ǫ), contradicting step (D). Hence we must have µ′ =0. REVIEW OF ECONOMIC STUDIES 46 from the fact that ¯f (θ)≥1 for all θ ≥iH. Moreover, generically the second inequality is strict. By Condition 1, this implies µ(w;0,i)<0 for some worker i in this interval, a contradiction. Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rdaa068/5931860 by University of Zurich user on 05 January 2021 13. Consider first the case where the equilibrium is interior with i∗<iH and let iS −λ denote the measure of high type workers who choose e=eS. For all other i∈[λ,iH], the lower bound on their wage distribution wS must satisfy: i 13. Consider first the case where the equilibrium is interior with i∗<iH and let iS −λ denote the measure of high type workers who choose e=eS. For all other i∈[λ,iH], the lower bound on their wage distribution wS must satisfy: iH ¯f (i∗)wS + iH i∗w(0,i′)d¯f (i′)= ¯wS (D.9) (D.9) i with the cutoff i∗defined in step (D) given by (D.6). i with the cutoff i∗defined in step (D) given by (D.6). Suppose first that there exist workers in [λ,iH] with lower bounds on wages lower than those defined by (D.9), and let ˜i be the highest worker such that for some ǫ >0, the lower bound is higher for all i∈  ˜i−ǫ,˜i  . (a) If ˜i∈(i∗,iH] is in a region where ¯f (i) is strictly increasing, let ˜w(0,i) be the lower bounds on the wages of i∈(˜i−ǫ,˜i). Define markets M  ˜i  ={(e,w):e=0,w= ˜w(0,i),i∈(˜i−ǫ,˜i)}. Firms θ ≤˜i−ǫ can find high types in markets with wages below ˜w(0,i−ǫ), so they don’t want to hire selectively in any market (e,w)∈M  ˜i  . Therefore total selective hiring in markets (e,w)∈M  ˜i  will be  ˜i ˜i−ǫ d ¯F(i). By construction of ˜i, all workers ˜i+ε for ε>0 have lower bounds on wages w  0,˜i+ε  given by (D.4). By step (D), a fraction ¯f  ˜i+ε  of them are hired by selective firms, and step (D) implies that the selective hiring occurs at the lower bound of their wage w(0,˜i+ε). (Since ˜i≤iH, we have ¯f (˜i+ε)≤1.) Taken together, this implies that a share 1−¯f  ˜i+ε  of workers ˜i+ε must be hired by non-selective firms at or above w(0,˜i+ε). Continuity of ¯f then implies that a fraction 1−¯f  ˜i  of workers of type ˜i will be hired by non-selective firms at wages at or above w(0,˜i). 8. Define This implies that there can only be a single such market (0,w) where all firms hire non-selectively, and that all workers must obtain utility of at least w in equilibrium. Let ¯i denote the highest i∈[λ,1] that applies to market (0,w). By zero profits and w>qL, we must have ¯i>λ. To ensure that no firm wants to hire selectively in market (0,w), all firms θ ≤¯i must at least make profits qH −w in equilibrium, i.e. they must hire high types in some market (e′,w′)̸=(0,w) with w′ ≤w. However, because all workers obtain utility of at least w, there cannot be any supply of workers in market (e′,w′). 12. All types i>iH, who select e=0 by step (D), must be hired with probability 1 at w=qH. They cannot be hired with positive probability above qH because no firm would hire at such a wage. Suppose some ˜i>iH is not hired with probability 1 at w=qH. This implies that all firms θ ∈  iH,˜i  maximize profits by hiring selectively at the lower bound of the support of the wages of worker i=θ, which is below qH by step (D). The total number of workers these firms would hire is F  ˜i  −F(iH)≥¯F  ˜i  −¯F(iH)≥˜i−iH. The first inequality follows from the fact that F(θ)≥¯F(θ) for all θ by construction of ¯F, and F(iH)= ¯F(iH) by definition of iH. The second follows 12. All types i>iH, who select e=0 by step (D), must be hired with probability 1 at w=qH. They cannot be hired with positive probability above qH because no firm would hire at such a wage. Suppose some ˜i>iH is not hired with probability 1 at w=qH. This implies that all firms θ ∈  iH,˜i  maximize profits by hiring selectively at the lower bound of the support of the wages of worker i=θ, which is below qH by step (D). The total number of workers these firms would hire is F  ˜i  −F(iH)≥¯F  ˜i  −¯F(iH)≥˜i−iH. The first inequality follows from the fact that F(θ)≥¯F(θ) for all θ by construction of ¯F, and F(iH)= ¯F(iH) by definition of iH. The second follows RESTUD: The Review of Economic Studies [19:28 2/11/2020 OP-REST200072.tex] Page: 45 1–50 MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES REVIEW OF ECONOMIC STUDIES Suppose first that ˜w(0,i) is strictly increasing in (˜i−ǫ,˜i). For each i∈(˜i−ǫ,˜i), all workers i′ >i have lower bounds on wages ˜w(0,i), so the supply of workers in market (0, ˜w(0,i)) includes i−iS high types and λ low types. Therefore (D.4) and the fact that ˜w(0,i)>w(0,i) imply that no firms want to hire non-selectively in any market (0, ˜w(0,i)). Alternatively, suppose ˜w(0,i) is flat in (˜i−ǫ,˜i) at level w(0,˜i). Since a fraction 1−¯f  ˜i  of workers of type ˜i will be hired by non-selective firms at wages at or above w(0,˜i), the same must then be true for all i∈(˜i−ǫ,˜i]. In both cases, the total measure of workers in (˜i−ǫ,˜i] not hired at wages at or above w(0,˜i) by non-selective firms is ¯f  ˜i  ǫ. Since ¯f (i) is strictly increasing, ¯f  ˜i  ǫ >  ˜i ˜i−ǫ d ¯F(i), which implies that µ( ˜w(0,i);0,i)>0 for some workers i∈(˜i−ǫ,˜i), and the lower bound on wages must be lower than ˜w(0,i). step (D), a fraction ¯f  ˜i+ε  of them are hired by selective firms, and step (D) implies that the selective hiring occurs at the lower bound of their wage w(0,˜i+ε). (Since ˜i≤iH, we have ¯f (˜i+ε)≤1.) Taken together, this implies that a share 1−¯f  ˜i+ε  of workers ˜i+ε must be hired by non-selective firms at or above w(0,˜i+ε). Continuity of ¯f then implies that a fraction 1−¯f  ˜i  of workers of type ˜i will be hired by non-selective firms at wages at or above w(0,˜i). Suppose first that ˜w(0,i) is strictly increasing in (˜i−ǫ,˜i). For each i∈(˜i−ǫ,˜i), all workers i′ >i have lower bounds on wages ˜w(0,i), so the supply of workers in market (0, ˜w(0,i)) includes i−iS high types and λ low types. Therefore (D.4) and the fact that ˜w(0,i)>w(0,i) imply that no firms want to hire non-selectively in any market (0, ˜w(0,i)). Alternatively, suppose ˜w(0,i) is flat in (˜i−ǫ,˜i) at level w(0,˜i). Since a fraction 1−¯f  ˜i  of workers of type ˜i will be hired by non-selective firms at wages at or above w(0,˜i), the same must then be true for all i∈(˜i−ǫ,˜i]. In both cases, the total measure of workers in (˜i−ǫ,˜i] not hired at wages at or above w(0,˜i) by non-selective firms is ¯f  ˜i  ǫ. REVIEW OF ECONOMIC STUDIES Since ¯f (i) is strictly increasing, ¯f  ˜i  ǫ >  ˜i ˜i−ǫ d ¯F(i), which implies that µ( ˜w(0,i);0,i)>0 for some workers i∈(˜i−ǫ,˜i), and the lower bound on wages must be lower than ˜w(0,i). (b) If ˜i∈(i∗,iH] is in a region where ¯f (i) is constant or if ˜i<i∗then this implies that the lower bound on the wages of worker ˜i is higher than that of some worker i′ >˜i, which would violate step (D). ˜ (b) If ˜i∈(i∗,iH] is in a region where ¯f (i) is constant or if ˜i<i∗then this implies that the lower bound on th wages of worker ˜i is higher than that of some worker i′ >˜i, which would violate step (D). (c) If ˜i=i∗, this would imply that some workers i∈[λ,i∗] have a lower bound on their wage ˜w(0,i)>wS. This can only occur without violating step (D) when i∗corresponds to the lower end of a bunching region and wS <w(0,i∗). Let i′ be the lowest i∈[λ,i∗] such that ˜w(0,i)>wS for all i>i′. We must have i′ >λ since otherwise no one signals by (D.9). No firm θ <i′ wants to hire any type i>i′ since they maximize profits by hiring in market (0,wS). Hence, total selective hires in (i′,i∗) are given by F(i∗)−F(i′)≤¯F(i∗)−¯F(i′)<i∗−i′. The first inequality follows from the fact that, since i∗is the lower end of a bunching region, we have F(i∗)= ¯F(i∗). The second follows from the definition of iH and the fact that i∗<iH. Since there is no non-selective hiring (if there was, step (D) would apply), this implies that µ( ˜w(0,i),0,i)>0 for some workers i∈(i′,i∗), and therefore the lower bound on wages must be lower than ˜w(0,i). (c) If ˜i=i∗, this would imply that some workers i∈[λ,i∗] have a lower bound on their wage ˜w(0,i)>wS. This can only occur without violating step (D) when i∗corresponds to the lower end of a bunching region and wS <w(0,i∗). Let i′ be the lowest i∈[λ,i∗] such that ˜w(0,i)>wS for all i>i′. We must have i′ >λ since otherwise no one signals by (D.9). No firm θ <i′ wants to hire any type i>i′ since they maximize profits by hiring in market (0,wS). Hence, total selective hires in (i′,i∗) are given by F(i∗)−F(i′)≤¯F(i∗)−¯F(i′)<i∗−i′. The first inequality follows from the fact that, since i∗is the lower end of a bunching region, we have F(i∗)= ¯F(i∗). RESTUD: The Review of Economic Studies KURLAT & SCHEUER Since this is feasible for all firms, it contradicts (D). Downloaded from https://academic.oup.com/restud/advance-article/doi/10.1093/restud/rdaa068/5931860 by University of Zurich user on 05 January 2021 (b) If ˜i∈ i∗,iH  is in a region [i0,i1] where ¯f (i) is constant, then a fraction 1−¯f (i1) of all workers i<i1 are     (b) If ˜i∈ i∗,iH  is in a region [i0,i1] where ¯f (i) is constant, then a fraction 1−¯f (i1) of all workers i<i1 are hired by non-selective firms at wages at least w(0,i1). The measure of firms in  i0,˜i  is F  ˜i  −F(i0)≥ ¯F(˜i)−¯F(i0)= ¯f (i1)  ˜i−i0  , with strict inequality in the generic case where the original density f is not exactly constant and equal to ¯f (i1). For all these firms, it is profit maximizing to hire selectively at the lower bound on wages of worker i=θ, which implies that µ(w;0,i)<0 for some i in this interval, and which therefore cannot be part of an equilibrium. ˜ ˜ (b) If ˜i∈ i∗,iH  is in a region [i0,i1] where ¯f (i) is constan (c) If ˜i<i∗then equation (D.6) implies that the utility of all workers i≤˜i is below ¯wS. By (D.9) and (D.8), they would be better off choosing e=eS. (c) If ˜i<i∗then equation (D.6) implies that the utility of all workers i≤˜i is below ¯wS. By (D.9) and (D.8), they would be better off choosing e=eS. 14. In any interior equilibrium, the cutoff i∗defined in step (D) must also satisfy (D.7). To see this, observe first that not all workers i∈[λ,i∗] can possibly signal. If this were the case, consider the beliefs of a firm θ ∈[λ,i∗) in a market (0,wS +ε). Then G(Iχθ ;0,wS +ε,χθ)>0 since otherwise µ(wS +ε;0,i)=0 for i∈[λ,i∗) by Condition 5, so these workers could get payoff in excess of ¯wS by choosing e=0 instead of signalling. Moreover, the support of G(·;0,wS +ε,χθ) cannot include high types since otherwise some firms could increase their profits by shifting demand to market (0,wS +ε) for ε sufficiently small. Hence, for all firms θ ∈[λ,i∗), it is profit maximizing to hire selectively in market (0,wS), so total selective hires will be F(i∗)= ¯F(i∗), where the equality follows from the fact that, by (D.6), if i∗falls in a bunching region, it corresponds to the lower end of it. SIGNALLING TO EXPERTS 47 KURLAT & SCHEUER As a result, (i)≡Ŵ(i,i−¯F(i)/¯f (i)) is continuous in i except when i is the lower end of a bunching interval, in which case (i) discontinuously jumps up at that point as i increases. Recall that the solution to (D.6) and (D.7) is i∗=mini∈[λ,iH]{i| (i)≥0}. Together with the result from Proposition D.1 that ′(i)>0 when i is not in a bunching region and =0, this implies the following: 17. Finally, we show that there is a unique solution to equations (D.6) and (D.7). The argument in the proof of Proposition (D.1) applies, except that, with bunching, the function (i∗) is no longer continuous. From (D.5) we see that Ŵ is still continuous in iS but, as i increases, jumps up at the lower end of each bunching interval. This is because when i enters a bunching region, ib(i) jumps to the upper end of that region. As a result, (i)≡Ŵ(i,i−¯F(i)/¯f (i)) is continuous in i except when i is the lower end of a bunching interval, in which case (i) discontinuously jumps up at that point as i increases. Recall that the solution to (D.6) and (D.7) is i∗=mini∈[λ,iH]{i| (i)≥0}. Together with the result from Proposition D.1 that ′(i)>0 when i is not in a bunching region and =0, this implies the following: (a) If (iH)<0, then (i)<0 for all i∈[λ,iH], so there cannot be any solution to (D.6) and (D.7) and the corner equilibrium is the unique equilibrium. (b)If (iH)≥0, then either (i)>0 for all i∈[λ,iH], in which case i∗=λ, or there exists a unique solution i∗∈(λ,iH]. Hence, if there is an interior equilibrium, it is also unique. (a) If (iH)<0, then (i)<0 for all i∈[λ,iH], so there cannot be any solution to (D.6) and (D.7) and the corner equilibrium is the unique equilibrium. (a) If (iH)<0, then (i)<0 for all i∈[λ,iH], so there cannot be any solution to (D.6) and (D.7) and the corner equilibrium is the unique equilibrium. (b)If (iH)≥0, then either (i)>0 for all i∈[λ,iH], in which case i∗=λ, or there exists a unique solution (b)If (iH)≥0, then either (i)>0 for all i∈[λ,iH], in which case i∗=λ, or there exists a unique solution i∗∈(λ,iH]. Hence, if there is an interior equilibrium, it is also unique. Existence We have established the existence of a solution to equations (D.6) and (D.7). We now provide the equilibrium decisions, probabilities and beliefs, and verify the conditions in Definition 1. REVIEW OF ECONOMIC STUDIES The second follows from the definition of iH and the fact that i∗<iH. Since there is no non-selective hiring (if there was, step (D) would apply), this implies that µ( ˜w(0,i),0,i)>0 for some workers i∈(i′,i∗), and therefore the lower bound on wages must be lower than ˜w(0,i). Suppose next that there exist workers with lower bounds on wages lower than those defined by (D.9), and let ˜i be the highest worker such that for some ǫ >0, lower bounds on wages are lower than those defined by (D.9) for all i∈  ˜i−ǫ,˜i  . Suppose next that there exist workers with lower bounds on wages lower than those defined by (D.9), and let ˜i be the highest worker such that for some ǫ >0, lower bounds on wages are lower than those defined by (D.9) for all i∈  ˜i−ǫ,˜i  . (a) If ˜i∈ i∗,iH  is in a region where ¯f (i) is strictly increasing, take some i′ ∈  ˜i−ǫ,˜i  with a lower bound on wages w′ < (i′−iS)qH +λqL  / i′−iS +λ  and consider the market (0,w′). The supply of workers in this market includes all low types (a measure λ) and at least the high types i∈ λ,i′ who do not signal (a measure at least i′−iS). Therefore, a firm that hired non-selectively in market (0,w′) would make profits of at least (a) If ˜i∈ i∗,iH  is in a region where ¯f (i) is strictly increasing, take some i′ ∈  ˜i−ǫ,˜i  with a lower bound on wages w′ < (i′−iS)qH +λqL  / i′−iS +λ  and consider the market (0,w′). The supply of workers in this market includes all low types (a measure λ) and at least the high types i∈ λ,i′ who do not signal (a measure at least i′−iS). Therefore, a firm that hired non-selectively in market (0,w′) would make profits of at least (i′−iS)qH +λqL i′−iS +λ −w′ >0. [19:28 2/11/2020 OP-REST200072.tex] Page: 46 1–50 MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES RESTUD: The Review of Economic Studies KURLAT & SCHEUER On the other hand, the measure of workers who are not hired by non-selective firms at higher wages is ¯f (i∗)(i∗−iS). Hence, if ¯F(i∗)> ¯f (i∗)(i∗−iS), thenµ wS;0,i <0 forsomei inthisinterval,whichisacontradiction.If ¯F(i∗)< ¯f (i∗)(i∗−iS),thenµ wS;0,i >0 for some i in this interval, so wS cannot be the lower bound on wages. 15. For the case of a corner equilibrium, note first that Ŵ(iH,iH −F(iH))<0 implies w(0,iH)<wP, where we used ¯f (iH)=1 (abstracting from the trivial case iH =λ, which is fully characterized by step (D)). Together with steps (D) and (D), this means that there cannot be any non-selective hiring. Hence, uL =qL and eS =e∗. Moreover, since there are only F(iH) firms with θ <iH that can hire selectively at e=0, this immediately implies that at least a measure iS −λ=iH −λ−F(iH) of workers must signal. All other workers in [λ,iH] must have a lower bound on wages wP. The bound cannot be lower than wP by step (D). Suppose for some workers the bound is higher and let i′ be the lowest i∈[λ,iH) such that w(0,i)>wP for all i>i′. We must have i′ >λ since otherwise no-one would signal. No firm θ <i′ wants to hire any type i>i′ since they maximize profits by hiring in market (0,wP). Hence, total selective hires in (i′,iH) are given by F(iH)−F(i′)≤¯F(iH)−¯F(i′)<iH −i′. The first inequality follows from F(iH)= ¯F(iH) and the second from the definition of iH. Since there is no non-selective hiring, this implies that µ(w(0,i);0,i)>0 for some workers i∈(i′,iH), and therefore wage w(0,i) cannot be the lower bound on their wages. 16. By the same argument as in step (D), in the corner equilibrium not every worker with i<iH can signal. Moreover, again by the same argument as in step (D), it is not possible that the measure iS −λ of workers who signal exceeds iH −F(iH)−λ. Hence, together with the previous step, we must have iS =iH −F(iH). 17. Finally, we show that there is a unique solution to equations (D.6) and (D.7). The argument in the proof of Proposition (D.1) applies, except that, with bunching, the function (i∗) is no longer continuous. From (D.5) we see that Ŵ is still continuous in iS but, as i increases, jumps up at the lower end of each bunching interval. This is because when i enters a bunching region, ib(i) jumps to the upper end of that region. REVIEW OF ECONOMIC STUDIES (a) Education decisions: ei =  0 if i<λor i≥iS eS otherwise (b) Probabilities: µ(w;e,i)= ⎧ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩ I(w≥qH) if e=0, i≥iH ¯f (ir(w)) if ⎧ ⎪⎨ ⎪⎩ e=0, i∈[i∗,iH), w∈[w(0,i),w(0,iH)) e=0, i∈[λ,i∗), w∈[wS,w(0,iH)) e=0, i∈[0,λ), w∈[qL,w(0,iH)) 0 if ⎧ ⎪⎨ ⎪⎩ e=0, i∈ i∗,iH  , w<w(0,i) e=0, i∈[λ,i∗), w<wS e=0, i∈[0,λ), w<qL 1 if e=0, i≤iH, w≥w(0,iH) I(w≥˜w(e,i)) if e>0, i≥λ I(w≥min{ ˜w(e,i), ˜w(e,λ)}) if e>0, i<λ where ir(w)= min i∈[i∗,iH]  i:w(0,i)≥w  , ˜w(e,i) is given by (C.2) and u(i)= ⎧ ⎪⎪⎪⎨ ⎪⎪⎪⎩ qH if i>iH ¯f (i)w(0,i)+  iH i w(0,i′)d¯f (i′) if i∈ i∗,iH  ¯f (i∗)wS +  iH i∗w(0,i)d¯f (i) if i∈[λ,i∗) ¯f (i∗)qL +  iH i∗w(0,i)d¯f (i) if i<λ (c) Demand decisions: (eθ,wθ,χθ)= ⎧ ⎪⎪⎪⎪⎨ (0,wS,χ(i)=I(i≥θ)) if θ ∈[λ,i∗) (0,w(0,θ),χ(i)=I(i≥θ)) if θ ∈[i∗,iH) (0,qH,χ(i)=I(i≥θ)) if θ ∈[iH,θ∗) q (a) Education decisions: ei =  0 if i<λor i≥iS eS otherwise (b) Probabilities: µ(w;e,i)= ⎧ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩ I(w≥qH) if e=0, i≥iH ¯f (ir(w)) if ⎧ ⎪⎨ ⎪⎩ e=0, i∈[i∗,iH), w∈[w(0,i),w(0,iH)) e=0, i∈[λ,i∗), w∈[wS,w(0,iH)) e=0, i∈[0,λ), w∈[qL,w(0,iH)) 0 if ⎧ ⎪⎨ ⎪⎩ e=0, i∈ i∗,iH  , w<w(0,i) e=0, i∈[λ,i∗), w<wS e=0, i∈[0,λ), w<qL 1 if e=0, i≤iH, w≥w(0,iH) I(w≥˜w(e,i)) if e>0, i≥λ I(w≥min{ ˜w(e,i), ˜w(e,λ)}) if e>0, i<λ where ir(w)= min i∈[i∗,iH]  i:w(0,i)≥w  , ˜w(e,i) is given by (C.2) and u(i)= ⎧ ⎪⎪⎪⎨ ⎪⎪⎪⎩ qH if i>iH ¯f (i)w(0,i)+  iH i w(0,i′)d¯f (i′) if i∈ i∗,iH  ¯f (i∗)wS +  iH i∗w(0,i)d¯f (i) if i∈[λ,i∗) ¯f (i∗)qL +  iH i∗w(0,i)d¯f (i) if i<λ (c) Demand decisions: ( ) ⎧ ⎪⎪⎪⎪⎨ (0,wS,χ(i)=I(i≥θ)) if θ ∈[λ,i∗) (0,w(0,θ),χ(i)=I(i≥θ)) if θ ∈[i∗,iH) (0 (i) I(i≥θ)) if θ [i θ∗) (b) Probabilities: where ir(w)= min i∈[i∗,iH]  i:w(0,i)≥w  , ˜ u(i)= ⎧ ⎪⎪⎪⎨ ⎪⎪⎪⎩ qH if i>iH ¯f (i)w(0,i)+  iH i w(0,i′)d¯f (i′) if i∈ i∗,iH  ¯f (i∗)wS +  iH i∗w(0,i)d¯f (i) if i∈[λ,i∗) ¯f (i∗)qL +  iH i∗w(0,i)d¯f (i) if i<λ (c) Demand decisions: (eθ,wθ,χθ)= ⎧ ⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎩ (0,wS,χ(i)=I(i≥θ)) if θ ∈[λ,i∗) (0,w(0,θ),χ(i)=I(i≥θ)) if θ ∈[i∗,iH) (0,qH,χ(i)=I(i≥θ)) if θ ∈[iH,θ∗) (0,qL,χ(i)=1∀i) for a measure λ(1−¯f (i∗)) of firms θ ≥θ∗ (eS,qH,χ(i)=1∀i) for a measure iS −λ of firms θ ≥θ∗ (eθ,wθ,χθ)= ⎧ ⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎩ (0,wS,χ(i)=I(i≥θ)) if θ ∈[λ,i∗) (0,w(0,θ),χ(i)=I(i≥θ)) if θ ∈[i∗,iH) (0,qH,χ(i)=I(i≥θ)) if θ ∈[iH,θ∗) (0,qL,χ(i)=1∀i) for a measure λ(1−¯f (i∗)) of firms θ ≥θ∗ (eS,qH,χ(i)=1∀i) for a measure iS −λ of firms θ ≥θ∗ where θ∗is such that F(θ∗)−F(iH)=1−iH. REVIEW OF ECONOMIC STUDIES REVIEW OF ECONOMIC STUDIES 48 Interior equilibrium. Interior equilibrium. terior equilibrium. KURLAT & SCHEUER Page: 47 1–50 [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES KURLAT & SCHEUER SIGNALLING TO EXPERTS 49 and for selection rule χ(i)=1∀i, g(i;e,w,χ)= ⎧ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩ I(i<λ)+I(i≥iS) 1−iS+λ if e=0, w≥qH I(i<λ)+I(i∈[iS,iH)) λ+iH−iS if e=0, w∈[w(0,iH),qH) I(i<λ)+I(i∈[iS,ir(w)]) λ+ir(w)−iS if e=0, w∈[wS,w(0,iH)) 1 λI(i<λ) if e=0, w∈[qL,wS) 1 λI(i<λ) if e∈(0,eS), w≥˜w(e,0) I(i∈[λ,iS]) iS−λ if e≥eS, w≥˜w(e,λ) ∅ otherwise To see that the proposed {ei,(eθ,wθ,χθ),µ,G} is an equilibrium, note first that the probabilities defined in (b) imply that low types are indifferent between any e∈[0,eS] and high types are indifferent between any e, so the education decisions defined in (a) solve the workers’ problem. The beliefs defined in (d) imply that it is profit maximizing for firms θ ≤i∗to hire selectively in market (0,w=wS) and for firms θ ∈(i∗,iH) to hire in market (0,w(0,θ)). Firms θ ≥iH make zero profits by hiring selectively in market (0,qH). Moreover, firms θ ≥iH make zero profits by hiring non-selectively in markets (0,qL), (eS,qH) or (0,w(0,i)),i∈[i∗,iH). Any other market has either G Iχθ ;e,w,χθ =0 or results in losses. Therefore the demands defined in (c) are an optimal choice. Finally, using the above-specified demand and beliefs, Condition 1 holds. It is straightforward to verify that µ(w;e,i) given in (b) is weakly decreasing in i, so Condition 2 is also satisfied. Beliefs satisfy Condition 3 in nonempty markets. In zero supply markets, beliefs are also constructed to satisfy Condition 4 when they are well defined, and G(·;e,w,χθ)=0 only at wages where µ(w;e,i)=0 for all i such that χ(i)=1, so Condition 5 is satisfied as well. REVIEW OF ECONOMIC STUDIES The non-selective demand in markets (0,w(0,i)) with i∈[i∗,iH) and ¯f ′(i)>0 remains to be specified. For a small interval of types [i0,i0 + ] the change in the probability of being hired non-selectively is: ¯f (i0 + )−¯f (i0)≈¯f ′(i0) ¯f (i0 + )−¯f (i0)≈¯f ′(i0) Using that in a no-bunching region ir (w)= iS(qH−w)+λ(w−qL) qH−w , this implies that total non-selective hires over an interval of wages [w,w+ǫ] are f (i0 + )−f (i0)≈f ′(i0) Using that in a no-bunching region ir (w)= iS(qH−w)+λ(w−qL) qH−w , this implies that total non-selective hires over an interval of wages [w,w+ǫ] are ǫ λ(qH −qL) (qH −w)2 ir (w)−(iS −λ) ¯f ′ ir (w) . Hence, the total measure of demand from firms θ ≥θ∗using the non-selective hiring rule χ(i)=1∀i placed on any set of markets (E0,W0)=  (e,w):e=0,w∈(w0,w1)⊂[w(0,i∗),w(0,iH)]  must be D(E0,W0,χ)= w1 w0 λ(qH −qL) (qH −w)2 ir (w)−(iS −λ) ¯f ′ ir (w) dw. All firms θ ≥θ∗are indifferent between hiring in any of these markets and remaining inactive, for instance by setting (eθ,wθ,χθ)=(0,0,χ(i)=1∀1). (d) Beliefs: for selection rule χ(i)=I(i≥θ), (d) Beliefs: for selection rule χ(i)=I(i≥θ), if e=0, w≥qH if e=0, w∈[w(0,iH),qH),θ <iH if e=0, w∈[wS,w(0,iH)), θ <iH if e>0,w−cHe∈ ˜w(e,λ), ˜w(e,iH)),θ <iH if e>0, w−cHe≥˜w(e,iH) otherwise Page: 48 1–50 [19:28 2/11/2020 OP-REST200072.tex] [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES Corner equilibrium. We state the equilibrium objects {ei,(eθ,wθ,χθ),µ,G}. Verifying that this is an equilibrium is analogous to the interior equilibrium case. (a) Education decisions: (eθ,wθ,χθ)= ⎧ ⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎩ (0,wP,χ(i)=I(i≥θ)) for θ <iH (0,qH,χ(i)=I(i≥θ)) for θ ∈[iH,θ∗) (0,qL,χ(i)=1∀i) for a measure λ of firms θ ≥θ∗ (e∗,qH,χ(i)=1∀i) for a measure iH −F(iH)−λ of firms θ ≥θ∗ (0,0,χ(i)=1∀i) otherwise where θ∗is such that F(θ∗)−F(iH)=1−iH where θ∗is such that F(θ∗)−F(iH)=1−iH (c) Probabilities: µ(w;e,i)= ⎧ ⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎩ I(w≥qH) if e=0, i>iH I w≥wP if e=0, i∈[λ,iH] I(w≥qL) if e=0, i<λ I(w≥˜w(e,i)) if e>0, i≥λ I(w≥min{ ˜w(e,i), ˜w(e,λ)}) if e>0, i<λ where ˜w(e,i) is given by (C.2) and u(i)= ⎧ ⎨ ⎩ qH if i>iH wP if i∈[λ,iH] qL if i<λ (c) Probabilities: (d) Beliefs: for selection rule χ(i)=I(i≥θ), g(i;e,w,χ)= ⎧ ⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎩ I(i≥max{θ,iH−F(iH)}) 1−max{θ,iH−F(iH)} if e=0, w≥qH I(i∈[max{iH−F(iH),θ},iH]) min{F(iH),iH−θ} if e=0, w∈[wP,qH), θ <iH I(i∈[θ,iH)) iH−θ if e>0,w∈ ˜w(e,λ), ˜w(e,iH)),θ <iH I(i≥θ) 1−θ if e>0, w≥˜w(e,iH) 0 otherwise RESTUD: The Review of Economic Studies Page: 49 1–50 [19:28 2/11/2020 OP-REST200072.tex] [19:28 2/11/2020 OP-REST200072.tex] MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES 50 REVIEW OF ECONOMIC STUDIES 50 and for selection rule χ(i)=1∀i, and for selection rule χ(i)=1∀i, and for selection rule χ(i)=1∀i, g(i;e,w,χ)= ⎧ ⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎩ I(i<λ)+I(i≥iH−F(iH)) λ+1−iH+F(iH) if e=0, w≥qH I(i<λ)+I(i∈[iH−F(iH),iH)) λ+F(iH) if e=0, w∈[wP,qH) 1 λI(i<λ) if e=0,w∈ qL,wP 1 λI(i<λ) if e∈(0,e∗), w≥˜w(e,0) I(i∈[λ,iH−F(iH)]) iH−F(iH)−λ if e≥e∗, w≥˜w(e,λ) 0 otherwise REFERENCES REFERENCES AKERLOF, G. 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(2001), “Housing-Price Cycles and Prepayment Rates of U.S. Mortgage Pools”, Journal of Real Estate Finance and Economics, 23, 161–184. Page: 50 1–50 Page: 50 1–50 [19:28 2/11/2020 OP-REST200072.tex] RESTUD: The Review of Economic Studies MANUSCRIPT CATEGORY: Article Copyedited by: ES Copyedited by: ES RESTUD: The Review of Economic Studies [19:28 2/11/2020 OP-REST200072.tex] KURLAT & SCHEUER 51 SIGNALLING TO EXPERTS SIGNALLING TO EXPERTS MILGROM, P. R. and ROBERTS, J. (1986), “Price and Advertising Signals of Product Quality”, Journal of Political Economy, 94, 796–821. y MIYAZAKI, H. (1977), “The Rat Race and Internal Labor Markets”, The Bell Journal of Economics, 8, 394–418. N, P. (1974), “Advertising as Information”, Journal of Political Eco NETZER, N. and SCHEUER, F. (2014) “A Game Theoretic Foundation of Competitive Equilibria with Adverse Selection”, International Economic Review, 55, 399–422. ROSENTHAL, R. W. and WEISS, A. 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https://thescipub.com/pdf/ajbbsp.2009.1.6.pdf
English
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The Effect of Higher Sludge Recycling Rate on Anaerobic Treatment of Palm Oil Mill Effluent in a Semi-Commercial Closed Digester for Renewable Energy
American journal of biochemistry & biotechnology/American journal of biochemistry and biotechnology
2,009
cc-by
3,947
Corresponding Author: Alawi Sulaiman, Faculty of Engineering, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia, Faculty of Chemical Engineering, University Technology MARA, 40450 Shah Alam, Selangor, Malaysia 1 The Effect of Higher Sludge Recycling Rate on Anaerobic Treatment of Palm Oil Mill Effluent in a Semi-Commercial Closed Digester for Renewable Energy 1,2Alawi Sulaiman, 3Zainuri Busu, 3Meisam Tabatabaei, 4Shahrakbah Yacob, 3Suraini Abd-Aziz, 1,3Mohd Ali Hassan and 5Yoshihito Shirai 1Faculty of Engineering, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 2Faculty of Chemical Engineering, University Technology MARA, 40450 Shah Alam, Selangor, Malaysia 3Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 4Applied Agricultural Resources Sdn. Bhd., ½ km off Jalan Sg Buloh-Subang, 47000 Sg. Buloh, Selangor, Malaysia g g y 5Graduate School of Life Sciences and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0196, Japan Abstract: Problem statement: A 500 m3 semi-commercial closed anaerobic digester was constructed for Palm Oil Mill Effluent (POME) treatment and methane gas capture for renewable energy. During the start-up operation period, the Volatile Fatty Acids (VFA) accumulation could not be controlled and caused instability on the system. Approach: A settling tank was installed and sludge was recycled as to provide a balanced microorganisms population for the treatment of POME and methane gas production. The effect of sludge recycling rate was studied by applying Organic Loading Rates (OLR) (between 1.0 and 10.0 kgCOD m−3 day−1) at different sludge recycling rates (6, 12 and 18 m3 day−1). Results: At sludge recycling rate of 18 m3 day−1, the maximum OLR was 10.0 kgCOD m−3 day−1 with biogas and methane productivity of 1.5 and 0.9 m3 m−3 day−1, respectively. By increasing the sludge recycling rate the VFA concentration was controlled below its inhibitory limit (1000 mg L−1) and the COD removal efficiency recorded was above 95% which indicated good treatment performance for the digester. Two methanogens species (Methanosarcina sp. and Methanosaeta concilii) had been identified from sludge samples obtained from the digester and recycled stream. Conclusion: By increasing the sludge recycling rate upon higher application of OLR, the treatment process was kept stable with high COD removal efficiency. The biogas and methane productivity were initially improved but reduced once OLR and recycling rate were increased to 10.0 kg COD m3 day−1 and 18 m3 day−1 respectively. Key words: Palm oil mill effluent, methane, biogas, clean development mechanism Key words: Palm oil mill effluent, methane, biogas, clean development mechanism American Journal of Biochemistry and Biotechnology 5 (1): 1-6, 2009 ISSN 1553-3468 © 2009 Science Publications American Journal of Biochemistry and Biotechnology 5 (1): 1-6, 2009 ISSN 1553-3468 © 2009 Science Publications American Journal of Biochemistry and Biotechnology 5 (1): 1-6, 2009 ISSN 1553-3468 © 2009 Science Publications The Effect of Higher Sludge Recycling Rate on Anaerobic Treatment of Palm Oil Mill Effluent in a Semi-Commercial Closed Digester for Renewable Energy 1,2Alawi Sulaiman, 3Zainuri Busu, 3Meisam Tabatabaei, 4Shahrakbah Yacob, 3Suraini Abd-Aziz, 1,3Mohd Ali Hassan and 5Yoshihito Shirai 1Faculty of Engineering, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 2Faculty of Chemical Engineering, University Technology MARA, 40450 Shah Alam, Selangor, Malaysia 3Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 4Applied Agricultural Resources Sdn. Bhd., ½ km off Jalan Sg Buloh-Subang, 47000 Sg. Buloh, Selangor, Malaysia The Effect of Higher Sludge Recycling Rate on Anaerobic Treatment of Palm Oil Mill Effluent in a Semi-Commercial Closed Digester for Renewable Energy 1,2Alawi Sulaiman, 3Zainuri Busu, 3Meisam Tabatabaei, 4Shahrakbah Yacob, 3Suraini Abd-Aziz, 1,3Mohd Ali Hassan and 5Yoshihito Shirai 1Faculty of Engineering, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 2Faculty of Chemical Engineering, University Technology MARA, 40450 Shah Alam, Selangor, Malaysia 3Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 4Applied Agricultural Resources Sdn. Bhd., ½ km off Jalan Sg Buloh-Subang, 47000 Sg. Buloh, Selangor, Malaysia INTRODUCTION effluent discharge compared to other oil extraction process[1]. Unfortunately in Malaysia, the most popular treatment method for POME which is utilized by more than 85% of the mills is the open ponds system[2]. This is due to its low capital and operating costs. However with the implementation of CDM project under the Kyoto Protocol, open ponds treatment system is becoming less attractive because the valuable gas (methane) is wasted to the atmosphere and the system could not be certified for Carbon Emission Reduction (CER) trading. Globally there is a great concern for reducing the emission of Green Houses Gases (GHG) through Clean Development Mechanism (CDM) projects for renewable energy. In Malaysia one of the attractive projects for CDM and renewable energy is the methane gas capture from anaerobic treatment of Palm Oil Mill Effluent (POME) because it contains high amount of organic substances which are mainly lignocellulosic materials. In the world, POME contributes the largest 1 Am. J. Biochem. & Biotech., 5 (1): 1-6, 2009 Am. J. Biochem. & Biotech., 5 (1): 1-6, 2009 In an anaerobic digestion process, the organic materials are converted into biogas which mainly composed of methane and carbon dioxide by the action of a consortium of microorganisms through series of metabolic stages namely hydrolysis, acidogenesis, acetogenesis and methanogenesis[3-5]. By closing the digester in oppose to the conventional system, the methane gas could be captured and used for electricity generation. In addition, high Chemical Oxygen Demand (COD) removal efficiency could be achieved in shorter retention time. So far there were many reports on the anaerobic treatment of POME and methane generation, however none has been tested at a large pilot scale specifically for CDM study[6-10]. Therefore in 2005, a 500 m3 semi-commercial anaerobic digester was constructed and the digester has been in operation since then[1]. The objective of this study is to investigate the digester performances on COD removal efficiency and methane gas productivity at different OLR and sludge recycling rates applied. settled sludge was then returned to the digester at different recycling rates (6, 12 and 18 m3 day−1). Minimal mixing was applied intermittently for every 6 h (for 30 min) by using the mixing pump as to provide good contact between substrate and microorganisms. INTRODUCTION The COD, pH, Volatile Fatty Acids (VFA), alkalinity, biogas mass flow and POME mass flow were measured by using the standard methods for the examination of water and wastewater (American Public Health Association)[11]. The methane concentration was measured using a portable methane gas analyzer (XP-314A, Shin-Cosmos Electric Co. Ltd. Japan). The analyzer was calibrated at the manufacturer’s site in Japan. The probe MSMX860, complementary to the 16S rRNA of some methanogens including Methanosarcina spp., Methanococcoides spp., Methanolobus spp., Methanohalophilus spp. and Methanosaeta spp. was used to directly analyze the methanogenic population[12]. To determine the sludge bacteria, the 16S rRNA probe EUB338 for the bacteria domain was used[13]. Oligonucleotides and their fluorescent derivatives (5 -labelled with either FITC or rhodamine) were purchased from First Base (Malaysia) Sdn. Bhd. Cells were fixed and hybridized using the protocol reported by Amman[14] with some modifications[15]. Fluorescence was observed using an epifluorescence microscope (BX50; Olympus, Tokyo, Japan) and photomicrographs were taken using a chilled 3-CCD color camera (640×483 pixels, C5810; Hamamatsu Photonics, Shizuoka, Japan). RESULTS Figure 2 shows the VFA concentration in the digester, methane yield achieved and productivity of biogas and methane at different OLR and sludge recycling rate applied. The VFA level was initially low in the digester but increased once OLR was increased. By increasing the sludge recycling rate the VFA level was controlled below 1000 mg L−1 and OLR was able to be applied up to 10.0 kgCOD m−3 day−1. Productivity of biogas and methane also increase with OLR but reduced towards the end of study. Initially the methane yield was high but slowly reduced once higher OLR and sludge recycling rate was applied. Table 1 shows the sludge recycling rate applied, COD levels in the raw POME, raw POME feeding rate, Hydraulic Retention Time (HRT), Organic Loading Rate (OLR), digester’s pH and COD removal efficiency recorded in this study. A total of 111 days of operation was sustained in this study at different COD concentration (66,400-118,100 mg L−1), OLR (1.0-10.0 kgCOD m−3 day−1) and sludge recycling rate (6.0-18.0 m3 day−1). The variation of OLR applied resulted in different POME feeding rate and HRT applied as well. The last two columns show the digester’s stability in terms of pH and COD removal efficiency achieved. The COD removal efficiency recorded the same performance for different OLR and sludge recycling rate applied which indicated lower recycling rate was sufficient for lower OLR application and higher recycling rate was required once OLR was increased in the system. The results of methanogens population analysis in the digester and the recycled sludge by using Fluorescent In Situ Hybridization (FISH) technique is shown in Fig. 3A and B respectively. These diagrams confirm the presence of filamentous Methanosaeta concilii and the clover-leaved Methanosarcina sp. in both sludge samples. MATERIALS AND METHODS Figure 1 shows the diagram of the digester system used previously by Yacob et al.[1] except for the sludge settling tank which was installed for this study. The POME used was obtained daily from the mixing pond of the Serting Hilir Palm Oil Mill by using a pump. The POME was stored temporarily in the holding tank prior to feeding. The sludge from the treated effluent was collected in the settling tank and let to settle. The 12 12 1 11 7 10 2 Ana erob ic d igester H olding tank 3 4 5 6 Treated effluent 8 9 Biogas storage 3 3 Fig. 1: Schematic diagram of the 500 m3 semi-commercial closed anaerobic digester; (1): Fresh POME inlet; (2): Feeding pump; (3): Sampling ports; (4): Gas collection chamber; (5): Biogas safety relief system; (6): Settling tank; (7): Sludge recycling pump; (8): pH probe; (9): Temperature probe; (10): Mixing pump; (11): POME mass flow meter; (12): Biogas mass flow meter 12 1 11 7 10 2 Ana erob ic d igester H olding tank 3 4 5 6 Treated effluent 8 9 Biogas storage 3 3 Biogas storage Fig. 1: Schematic diagram of the 500 m3 semi-commercial closed anaerobic digester; (1): Fresh POME inlet; (2): Feeding pump; (3): Sampling ports; (4): Gas collection chamber; (5): Biogas safety relief system; (6): Settling tank; (7): Sludge recycling pump; (8): pH probe; (9): Temperature probe; (10): Mixing pump; (11): POME mass flow meter; (12): Biogas mass flow meter 2 Am. J. Biochem. & Biotech., 5 (1): 1-6, 2009 RESULTS Table 1: Feeding profiles, stability and COD removal performance of the digester Feeding profiles Digester stability and performance Sludge ---------------------------------------------------------------------------------------------- ----------------------------------------- Operation recycling COD of raw Feeding rate Hydraulic retention Organic loading COD removal period days rate m3 day−1 POME mg L−1 m3 day−1 time days rate kgCOD m−3 day−1 pH efficiency (%) 1-9 6.0 66,400 7.7 64.9 1.0 6.8 97.3 10-28 6.0 75,900 15.5 32.2 2.0 6.8 97.6 29-38 6.0 80,700 21.8 22.9 3.0 6.9 96.6 39-58 12.0 86,300 27.4 18.2 4.0 6.9 96.7 59-78 12.0 98,900 33.5 14.9 5.0 7.0 98.1 79-84 12.0 103,000 38.3 13.0 6.0 7.1 97.7 85-95 18.0 111,600 37.8 13.2 7.0 7.0 97.7 96-105 18.0 118,100 39.3 12.7 8.0 7.0 97.6 106-111 18.0 111,100 46.6 10.7 10.0 7.0 97.5 Fig. 2: Productivities of biogas and methane, methane yield and VFA concentration profiles Table 1: Feeding profiles, stability and COD removal performance of the digester Feeding profiles Digester stability and performance Sludge ---------------------------------------------------------------------------------------------- ----------------------------------------- Operation recycling COD of raw Feeding rate Hydraulic retention Organic loading COD removal period days rate m3 day−1 POME mg L−1 m3 day−1 time days rate kgCOD m−3 day−1 pH efficiency (%) 1-9 6.0 66,400 7.7 64.9 1.0 6.8 97.3 10-28 6.0 75,900 15.5 32.2 2.0 6.8 97.6 29-38 6.0 80,700 21.8 22.9 3.0 6.9 96.6 39-58 12.0 86,300 27.4 18.2 4.0 6.9 96.7 59-78 12.0 98,900 33.5 14.9 5.0 7.0 98.1 79-84 12.0 103,000 38.3 13.0 6.0 7.1 97.7 85-95 18.0 111,600 37.8 13.2 7.0 7.0 97.7 96-105 18.0 118,100 39.3 12.7 8.0 7.0 97.6 106-111 18.0 111,100 46.6 10.7 10.0 7.0 97.5 Fig. 2: Productivities of biogas and methane, methane yield and VFA concentration profiles Table 1: Feeding profiles, stability and COD removal performance of the digester Feeding profiles Digester stability and performance Fig. 2: Productivities of biogas and methane, methane yield and VFA concentration profiles 3 Am. J. Biochem. & Biotech., 5 (1): 1-6, 2009 Fig. 3: Fluorescent In Situ Hybridization (FISH) picture for the samples taken from the digester (A): 1000X magnification and recycled sludge (B): 200X magnification showing the distribution of methanogens (Methanosaeta concilii and Methanosarcina sp.) productivity once the project has been approved for CDM. From Table 1, the COD removal efficiency maintained removal efficiency of greater than 95% despite higher OLR applied to the system. RESULTS This signify the importance of applying higher sludge recycling rate in order to maintain high COD removal once OLR was increased. During 111 days of continuous operation, the OLR was increased in step-wise (from 1.0-10.0 kgCOD m−3 day−1). This resulted in higher POME feeding rate and shorter Hydraulic Retention Time (HRT). In the previous study at OLR of between 5.0 and 6.0 kgCOD m−3 day−1 the digester became unstable due to VFA accumulation inside the digester and the treatment could not be sustained[1]. However in this study higher OLR was achieved (up to 10.0 kgCODm−3 day−1) and the system remained stable (neutral pH with COD removal efficiency greater than 95%) by increasing the sludge recycling rate from the settling tank at different rates. From days 1-40 the sludge recycling rate was fixed at 6 m3 day−1 and increased to 12 m3day−1 (from days 39-78) and finally to 18 m3 day−1 (from days 91-110) as shown in Table 1. During these different periods, the OLR were also increased steadily from 1.0-10.0 kgCOD m−3 day−1 with an increment of 1.0 kgCOD m−3 day−1 except for the last increment. Volatile Fatty Acids (VFA) accumulation: Figure 2 shows the VFA concentration in the digester, productivity of biogas and methane and methane yield achieved in this study. From this study at lower OLR applied (from 1.0- 4.0 kgCOD m−3 day−1), the measured VFA concentration inside the digester was low which indicates good VFA utilization by the methanogens. However after 40 days of operation, the VFA concentration was recorded higher (nearly 900 mg L−1). In order to avoid the process to turn acidic and inhibit the methanogenesis process, higher sludge recycling rate was applied at 12 m3 day−1 by increasing the pump’s timer. The VFA concentration in the digester reduced steadily with time as shown in Fig. 2. In earlier study the sludge recycling strategy was suggested in order to supplement alkalinity and maintain the system’s pH[10]. By recycling the sludge, the active microorganisms could be returned to the digester and provide a balanced population of microorganisms that responsible for converting organics materials to VFA and methane gas. The system was stable until OLR of 6.0 kgCOD m−3 day−1 where the VFA concentration in the digester showed an increasing trend. In order to avoid the process to turn acidic, the sludge recycling rate was increased to 18 m3 day−1. RESULTS As a result, the VFA concentration was maintained below 1000 mg L−1. Fig. 3: Fluorescent In Situ Hybridization (FISH) picture for the samples taken from the digester (A): 1000X magnification and recycled sludge (B): 200X magnification showing the distribution of methanogens (Methanosaeta concilii and Methanosarcina sp.) Fig. 3: Fluorescent In Situ Hybridization (FISH) picture for the samples taken from the digester (A): 1000X magnification and recycled sludge (B): 200X magnification showing the distribution of methanogens (Methanosaeta concilii and Methanosarcina sp.) In the recycled sludge sample, the population of Methanosarcina sp. was lower but there is a considerable amount of Methanosaeta concilii observed. DISCUSSION COD removal efficiency and OLR applied: In many studies the COD removal efficiency recorded was high although high fluctuation of COD concentration were applied[6-10]. Similarly in this study, the COD concentrations fluctuated between 66,000 and 118,000 mg L−1 as shown in Table 1. The anaerobic treatment system was stable which was due the suitable anaerobic treatment system design for POME. The actual organic level in POME varies daily and the ability of the system to run continuously is important as to ensure minimal down-time and increase biogas 4 Am. J. Biochem. & Biotech., 5 (1): 1-6, 2009 Am. J. Biochem. & Biotech., 5 (1): 1-6, 2009 Productivity of biogas and methane and methane yield: The productivity of biogas and methane and methane yield are shown in Fig. 2. The productivity shows the biogas volume at standard temperature and pressure measured in a day over the volume of reactor. In this study, the gas productivity increased with OLR applied where the maximum biogas and methane productivity were recorded at 1.5 and 0.9 m3 m−3 day−1, respectively at OLR of 8.0 kgCOD m−3 day−1. However, after 100 days of operation when OLR and sludge recycling rate were increased to 10.0 kgCOD m−3 day−1 and 18 m3 day−1 respectively, biogas and methane productivity reduced to 1.3 and 0.4 m3 m−3 day−1, respectively. This reflects the occurrence of anaerobic inhibition in the system. Theoretically, higher biogas productivity should be achieved with higher OLR applied but this was not observed towards the end of the study which may be due to the organic washout. Towards the end, the sludge recycling rate was increased to 18 m3 day−1 and some of the organics were washed out from the digester and could not be converted into biogas even though higher amount of microorganisms available. In addition, higher feeding volume was also introduced to the digester which may have resulted in shock loading to the microorganism as well and might have contributed to the reduced biogas generation[1]. For the methane yield the trend was different where it was highest at lowest OLR applied and slowly reduced once it was increased as shown in Fig. 2. At lower OLR the methane yield was around 0.15 kgCH4 kgCODremoved−1 but reduced to 0.10 kgCH4 kgCODremoved−1 and finally to 0.06 kgCH4 kgCODremoved−1 when OLR was increased to 5.0 and 10.0 kgCOD m−3 day−1, respectively. CONCLUSION This study demonstrated the feasibility of methane gas capturing project for CDM from anaerobic treatment of POME. The maximum OLR achieved was 10.0 kgCOD m−3 day−1 and gas productivity were 1.5 and 0.9 m3 m−3 day−1, respectively for biogas and methane. By increasing the sludge recycling rate upon higher application of POME loading rate, the treatment process was stable with VFA concentration recorded below its inhibitory limit (1000 mg L−1) and the COD removal efficiency recorded was higher than 95%. This indicated good treatment performance of the digester. However, the methane productivity was recorded lower at high OLR which was due to organic washout and methanogens shock loading. The total methane emission reduction for a 54 tons h−1 palm oil mill was more than 2 millions m3. DISCUSSION Although the sludge recycling rate were increased to 12.0 and 18.0 m3 day−1 it was not sufficient to increase the methane yield performance. population of both important mesophilic acetolastic methanogens which responsible for methanogenesis process[16]. Methane emission reduction: Based on this study the methane emission reduction could be estimated by using the volumetric ratio of methane produced and POME utilized. From this study the calculated ratio is approximately 12.0. In 2007, the total volume of POME generated for a 54 tones h−1 Serting Hilir Palm Oil Mill was 192,372 m3. This accounted for methane emission reduction of 2,308,464 m3 of methane. Considering that there are hundreds of palm oil mills in Malaysia and CER could be traded at the rate of USD10/ton of CO2 equivalent, there is a huge potential for CDM business in Malaysia. ACKNOWLEDGEMENT The authors would like to acknowledge various support from University Putra Malaysia, FELDA Palm Industries Sdn. Bhd., Kyushu Institute of Technology, Japan Society for Promotion of Science Asian Core Program, University Teknologi MARA and Serting Hilir Palm Oil Mill. Fluorescent In Situ Hybridization (FISH): The analysis of methanogens population in the digester and the recycled sludge by using the Fluorescent In Situ Hybridization (FISH) technique confirmed the presence of filamentous Methanosaeta concilii and the clover- leaved Methanosarcina sp. in both recycled and digester sludge samples as shown in Fig. 3. The population of Methanosarcina sp. was lower in the recycled sludge sample (Fig. 3B) as compared to the digester sludge sample (Fig. 3A). However there is a considerable amount of Methanosaeta concilii in the recycled sludge which highlights the importance of recycling the sludge to the digester to maintain high REFERENCES 1. Yacob, S., Y. Shirai, M.A. Hassan, M. Wakisaka and S. Subash, 2006. Start-up operation of semi- commercial closed anaerobic digester for palm oil mill effluent treatment. Proc. Biochem., 41: 962-964. DOI: 10.1016/J.Procbio.2005.10.021 1. Yacob, S., Y. Shirai, M.A. Hassan, M. Wakisaka and S. Subash, 2006. Start-up operation of semi- commercial closed anaerobic digester for palm oil mill effluent treatment. Proc. Biochem., 41: 962-964. DOI: 10.1016/J.Procbio.2005.10.021 5 Am. J. Biochem. & Biotech., 5 (1): 1-6, 2009 10. Husaini, S.T. and A. Djajadiningrat, 1996. Palm oil mill effluent treatment by anaerobic baffled reactors: Recycle effects and biokinetic parameters. Water Sci. Technol., 34: 59-66. DOI: 10.1016/S0273-1223(96)00821-9 2. Najafpour, G.D., A.A.L. Zinatizadeh, A.R. Mohamed, M.H. Isa and H. Nasrollahzadeh, 2006. High-rate anaerobic digestion of palm oil mill effluent in an up flow anaerobic sludge-fixed film bioreactor. Proc. Biochem., 41: 370-379. DOI: 10.1016/j.procbio.2005.06.031 11. Greenberg, A.E., L.S. Clesceri and A.D. Eaton, 1992. Standard Methods for the Examination of Water and Wastewater. 18th Edn., American Public Health Association, Washington DC., USA., ISBN: 08-755- 32071, pp: 269-273, 429-436, 505-506, 537-538. 3. Chynoweth, D.P., J.M. Owens and R. Legrand, 2001. Renewable methane from anaerobic digestion of biomass. Renew. Energ., 22: 1-8. DOI: 10.1016/S0960-1481(00)00019-7 12. Crocetti, G., M. Murto and L. Björnsson, 2006. An update and optimisation of oligonucleotide probes targeting methanogenic Archaea for use in fluorescence In Situ Hybridisation (FISH). J. Microbiol. Methods, 65: 194-201.DOI: 10.1016/j.mimet.2005.07.007 4. Bouallagui, H., Y. Touhami, R. Ben Cheikh and M. Hamdi, 2005. Bioreactor performance in anaerobic digestion of fruit and vegetable wastes. Proc. Biochem., DOI: 40: 989-995. DOI: 10.1016/j.procbio.2004.03.007 5. Hassan, M.A., S. Yacob, Y. Shirai and Y.T. Hung, 2004. Treatment of Palm Oil Wastewaters. In: Handbook of Industrial and Hazardous Wastes Treatment, Wang, L.K., Y. Hung, H.H. Lo and C. Yapijakis (Eds.). Marcel Dekker, New York, ISBN: 0824741145, pp: 719-736. 13. Amann, R.I., L. Krumholz and D.A. Stahl, 1990. Fluorescent oligonucleotide probing of whole cells for determinative phylogenetic and environmental studies in microbiology. J. Bacteriol., 172: 762-770. http://jb.asm.org/cgi/content/abstract/172/2/762 14. Amann, R., 1995. In Situ Identification of Micro- Organisms by Whole Cell Hybridization with rRNA- Targeted Nucleic Acid Probes, 3.3.6. In: Molecular Microbial Ecology Manual. Akkermans, A.D.L., J.D. Elsas and F.J. Bruijn (Eds.). Kluwer Academic Publisher, Dordrecht¸ ISBN: 978-1-4020-2176-3, pp: 1-15. 6. Faisal, M. and U. Hajime, 2001. Kinetic analysis of palm oil mill wastewater treatment by a modified anaerobic baffled reactor. Biochem. Eng., 9: 25-31. http://cat.inist.fr/?aModele=afficheN&cpsidt=1414 6279 7. Fakhrul-Razi, A. REFERENCES and M.J.M.M. Noor, 1999. Treatment of palm oil mill effluent with the membrane anaerobic system. Water Sci. Technol., 39: 159-163. DOI: 10.1016/S0273-1223(99)00268-1 15. Sakai, K., M. Mori, A. Fujii, Y. Iwami, E. Chukeatirote and Y. Shirai, 2004. Fluorescent in situ hybridization analysis of open lactic acid fermentation of kitchen refuse using rrna-targeted oligonucleotide probes. J. Biosci. Bioeng., 98: 48-56. DOI: 10.1016/s1389-1723(04)70241-8 8. Borja, R. and C.J. Banks, 1994. Anaerobic digestion of palm oil mill effluent using an up-flow anaerobic sludge blanket reactor. Biomass Bioener., 6: 381-389. DOI: 10.1016/0961- 9534(94)E0028-Q 16. Westernmann, P., K. Birgitte, T. Ahring and R.A. Mah, 1989. Threshold acetate concentrations for acetate catabolism by aceticlastic methanogenic bacteria. Applied Environ. Microbiol., 55: 514-515. http://aem.asm.org/cgi/content/abstract/55/2/514 9. Cail, R.G. and J.P. Barford, 1985. Mesophilic semi-continuous anaerobic digestion of palm oil mill effluent. Biomass, 7: 287-295. DOI: 10.1016/0144-4565(85)90067-8 6 6
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HIV vaccines: Lessons from basic research, clinical trials, and public policy
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HIGHLIGHTS • Decades of HIV research have paved the way for new HIV vaccines, although there remain technical challenges to produce an effective vaccine. • Public-private partnerships have been integral in modern vaccine development, particularly for HIV. • Several factors will impact future vaccine rollout, including healthcare data systems, vaccine sharing plans, and disparities in care accessibility. Transmission of the virus may occur through several modes, such as sexual contact, injection with a contaminated syringe, or spread from mother to offspring. An individual’s vulnerability—the capacity to control their level of risk—depends heavily on the social environment in which the individual encounters the virus [3]. Vulnerability to HIV is disproportionately higher in certain demographics, that, in turn, face a disproportionately more devastating impact. For example, male-to-male sexual contact accounts for the most new cases per year in the U.S. (68% of new cases in 2020), and statistically, one in six bisexual and gay men will be diagnosed with HIV in their lifetime [4]. Forty years since its discovery in 1983, HIV/AIDS still lacks a cure, despite advances in biomedicine that have dramatically improved global understanding of the virus’s biology. Decades of research and improvements to clinical trial infrastructure have promoted the development of novel vaccines for HIV, but these vaccines have shown limited efficacy. The landmark clinical trial “RV144” in Thailand (2003) showed modest efficacy of an HIV vaccine for the first time ever, sparking the formation of public-private partnerships and a wave of other HIV vaccine clinical trials. Around two decades later, the COVID-19 pandemic provided an impetus to move vaccine technology forward and exposed areas for improvement in vaccine and clinical-trial infrastructure. Looking at historical examples from both HIV and COVID-19 research, vaccine trials, and global statistics, it is clear that a successful vaccination campaign will require generous funding, collaboration, clinical trial infrastructure, sharing of data and resources amongst researchers, and policy based on scientific evidence and ethics. Such a successful campaign will also require overcoming several hurdles, such as delivering vaccines to individuals in countries with underdeveloped health care systems and both demographic and geographic disparities in access to care. The perspectives in this article are apropos given the recent development of HIV vaccines that have entered clinical trials. Black people are also affected disproportionately, as they account for the most new infections per year in the U.S. 1Broad Institute of MIT and Harvard, Cambridge, MA 2Whitehead Institute for Biomedical Research, Cambridge, MA 3MIT Department of Biology, Cambridge, MA ∗Email: saiz@broadinstitute.org The authors declare no conflict of interest. © 2023 The Author(s) HIV vaccines: Lessons from basic research, clinical trials, and public policy Andrew C. Saiz1,3,∗and Kelsey L. Farenhem2,3 Edited by Hannah LeBlanc and Steven Cheng Andrew C. Saiz1,3,∗and Kelsey L. Farenhem2,3 Edited by Hannah LeBlanc and Steven Cheng T he fight against the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) epidemic has been arduous —- a sustained effort for decades to combat one of the deadliest pandemics of the modern era. Over forty years since its discovery, and despite medical advances and prevention efforts, HIV continues to spread with millions of new cases per year. At the end of 2019, an estimated 1.19 million people aged 13 and older had HIV in the United States alone, 13% of whom did not know they had HIV [1]. The global impact is staggering, with approximately 84.2 million infections and a global death toll of 40.1 million people since the start of the epidemic [2]. T HIGHLIGHTS amongst racial and ethnic categories, representing 43% of all new cases in 2020 [1, 2], despite making up only 13.6% of the population. Hispanic/Latino and white people accounted for 27% and 26% of new cases, respectively [1]. The impact of HIV/AIDS goes beyond the extreme risk to health and life, with a drastic impact on social and societal structures as well as economic growth, particularly for vulnerable populations [3]. https://doi.org/10.38105/spr.0cg04tczoe ARTs are incredibly effective at preventing further replication of HIV, as they stop the cell-to-cell spread of the virus within an individual. However, HIV is able to insert its genetic material into the host genome, a behavior that classifies HIV as a retrovirus. Once this integration occurs, the virus’s genetic material is irremovable from the cell. Without consistent dosage of ARTs, this infected cell will spread the virus throughout the body, allowing the disease to progress. Therefore, a person living with HIV cannot stop taking ARTs without a high risk of viral recurrence in a matter of weeks [12, 13]. The development of a therapeutic vaccine may make treatment more accessible for those living with HIV, and a preventative vaccine may reduce or eliminate the need for these therapies, which are often inaccessible to those who need them most. An efficacious HIV vaccine, therefore, could be beneficial for both protecting the uninfected population against infection (preventative) and increasing the quality of life of the HIV-positive population (therapeutic). Therapeutic vaccines may follow, but as of now preventative vaccines are the primary focus of research and development. A preventative HIV vaccine would be a groundbreaking therapy but will require policies to support the entire process from development to distribution and post-vaccination monitoring. HIV can hide from the immune system: Given that HIV is a retrovirus that integrates its genome into the host genome, one might ask, "How does the body detect and kill infected cells?" For many viruses, an infected cell will be producing viral proteins that can be detected by antibodies and immune cells that destroy the infected cell. This detection prevents the virus from replicating further, but HIV presents an additional challenge. After the viral genome has been integrated into the host, it does not always produce more virus. Instead, HIV can go into a latent phase, where the infected cell appears completely normal [14]. The immune system, therefore, fails to detect these infected cells, and they can divide for many years in the latent stage. Over time, this creates a large number of cells containing HIV’s genetic material, known as an HIV reservoir. Why is there no vaccine to prevent HIV infection? https://doi.org/10.38105/spr.0cg04tczoe https://doi.org/10.38105/spr.0cg04tczoe Article Figure 1: Mutations in HIV allow the virus to evade the immune system. After a successful immune response, triggered by a vaccine or past infection, antibodies can bind to the virus and prevent it from infecting cells. When the virus mutates, the shape of its surface proteins change, and antibodies can no longer bind. This allows the virus to enter cells and reproduce. severe side-effects, and HIV rapidly acquired resistance to this therapy [5, 6]. severe side-effects, and HIV rapidly acquired resistance to this therapy [5, 6]. Another major advance in HIV treatment came in 1996 with the approval of indinavir, a protease inhibitor drug that ushered in the era of highly-active antiretroviral therapy (HAART), which differed from ART in that it contained a combination of multiple drugs [7]. In 2012, the World Health Organization (WHO) began recommending pre-exposure prophylaxis (PrEP) with the approval of tenofovir disoproxil fumarate, an oral drug that effectively prevented HIV infection in clinical trials when taken prior to exposure, further lessening the impact of HIV [4, 8]. PrEP reduces the risk of getting HIV from sex by about 99% when taken as prescribed [9]. Figure 1: Mutations in HIV allow the virus to evade the immune system. After a successful immune response, triggered by a vaccine or past infection, antibodies can bind to the virus and prevent it from infecting cells. When the virus mutates, the shape of its surface proteins change, and antibodies can no longer bind. This allows the virus to enter cells and reproduce. Although oral antiretroviral drugs have drastically improved patient outcomes and quality of life, a therapeutic HIV vaccine would dramatically improve treatment regimens. A therapeutic vaccine is one given to those already infected with a virus in order to further prevent its spread throughout the body, thereby reducing the need for long-term treatment. People living with HIV currently must take antiretroviral drugs indefinitely, which requires lifelong adherence and can be accompanied by side effects, such as nausea, fatigue, trouble sleeping, or high cholesterol. A major goal of therapeutic HIV vaccines is to eliminate the need and the cost for lifelong care such as ART therapy, slow down the progression of the virus, and maintain undetectable levels of virus in patients who already have HIV [10, 11]. curative. Saiz and Farenhem Major milestones in HIV care Decades of research, investment, and clinical trials have informed scientific innovations that have slowed the spread of HIV and improved patient outcomes. A landmark development occurred in 1987, when the U.S. Food and Drug Administration approved the use of azidothymidine (AZT), the first antiretroviral therapy (ART) for the treatment of HIV/AIDS. However, AZT extended life only on the order of months with MIT Science Policy Review | August 31, 2023 | vol. 4 | pg. 31 The modern vaccine development landscape The modern vaccine development landscape The endeavor to produce an HIV vaccine is a multi-billion-dollar, worldwide effort. Without clear and well-defined communication of scientific advances and distribution of funding, such an effort could easily result in wasted resources and loss of lives. The need for organization became clear several decades ago, so vast networks of collaborators were formed from both the public and private spheres to combine their efforts for a more effective push toward vaccine production. These partnerships, which often include scientists, healthcare professionals, and community-focused groups, remain a crucial aspect of the development of HIV-related therapeutics, preventative measures, and vaccines. In cases where a suitable antigen can be formulated for viruses, a successful immune response (and therefore immunity to future infection) necessitates multiple factors. One such response pursued by vaccine researchers is the activation of killer T-cells that home toward infected cells and eliminate them to thwart the infection. Another defensive response is the production of antibodies, where over a few weeks, the vaccine induces the immune system to produce both short- and long-term antibodies that can target the virus [17]. A successful vaccine would induce a rapid and highly specific immune response upon exposure to the virus, preventing significant spread of infection throughout the body. Formation of public-private collaboration networks: Even before the landmark RV144 trial, partnerships in HIV vaccine research between the private and public spheres began to emerge in the late 1990s. Such partnerships aimed to understand the biology behind HIV infection and to leverage that knowledge for development of a vaccine. The first to emerge was the International AIDS Vaccine Initiative (IAVI) in 1996. The cooperation allowed for streamlined design of vaccines, alleviated some of the challenges associated with sharing of data, reduced redundancies in funding and research, and focused all parties on a common goal. However, this entire premise relies on the ability of the vaccine to closely mimic the virus that the individual may later be exposed to—and herein lies the issue, particularly with HIV. With such large genetic diversity and a high evolution rate among many other mechanisms of evading the immune system, it has been very difficult to create a vaccine that provides robust protection against future exposure to HIV. https://doi.org/10.38105/spr.0cg04tczoe There is no cure for HIV: Generally speaking, the goal of any virus is to infect a target cell, called a host cell, and use the cell’s resources to create as many copies of the virus as possible. Newly produced viral particles, called virions, then escape the host cell to infect other cells, and the process repeats. To enter a host cell, a virus must first bind to a receptor on the cell’s surface. This is a highly specific interaction, meaning the proteins on the surface of a virus will interact only with the target cell type. In the case of HIV, the protein on the surface of the virus is known as the envelope protein (Env). This protein directs HIV to infect healthy immune cells known as helper T-cells, among a few other types of immune cells [9]. As a result of the infection, HIV patients experience symptoms resulting from an impaired immune system; without treatment, this impairment becomes severe and culminates in AIDS. Even with ART, these HIV reservoirs still exist, particularly within lymph nodes. One troubling study found that beginning ARTs as early as 10 days after infection was not sufficient to prevent the formation of these latent virus reservoirs [14]. These findings mean that preventing initial infection is absolutely critical, and the hope is that a vaccine would do exactly that. Though several life-saving therapies for HIV have been developed and improved over the decades, none of them are HIV evolves to evade vaccines: Another factor that enables MIT Science Policy Review | August 31, 2023 | vol. 4 | pg. 32 https://doi.org/10.38105/spr.0cg04tczoe https://doi.org/10.38105/spr.0cg04tczoe Article Article HIV’s ability to evade the immune system is its high rate of evolution. Each time the virus replicates within an individual, it can experience multiple mutation events [15, 16]. These mutations change the appearance of the proteins that are typically targeted by antibodies; such changes prevent the immune system from recognizing the virus (Figure 1). Mutations create extensive diversity in the composition and appearance of HIV proteins, both within an individual and amongst different people living with HIV. The RV144 clinical trial, which began in 2003 in Thailand and had 16,402 participants at high risk for infection, demonstrated 31.2% protection against infection after 42 months [19]. The study involved the administration of two different vaccines, each targeting multiple HIV proteins and each with multiple rounds of injections over 6 months. By using a vaccine adapted from the RV144 trial, the HVTN702 trial conducted in South Africa from October 2016 to January 2020 sought to improve upon RV144 in preventing HIV infection. Promising immune responses were observed in Phase 1-2a clinical trials, so the trial was advanced to phase 2b-3 in which 5,404 adults were randomly administered the candidate vaccine or placebo. By January 2020, however, the vaccine had failed to prevent HIV infection, so the trial was discontinued [20]. Broadly speaking, vaccines mimic components of the disease-causing virus to trigger an immune response in the body. This "mimic" is the vaccine antigen, the component of the virus that’s expressed or delivered by the vaccine so that the body can develop an immunity to it. The design and selection of such an antigen is therefore critical for a vaccine’s efficacy. Not only must the antigen be located in a region of the protein that is easily targeted by the immune system, but also in a region that is mostly conserved, one where mutations would be unlikely to occur. https://doi.org/10.38105/spr.0cg04tczoe https://doi.org/10.38105/spr.0cg04tczoe Article Article companies, the NIH, and academic institutions such as Fred Hutchinson Cancer Center—which houses the HVTN’s headquarters—has been developed for over 20 years and was vital for the rapid response to COVID-19. For example, the HVTN already had validated immunology assays and quality assurance systems that were quickly adapted for SARS-CoV-2 [24]. for HIV vaccine research and development. That same year, NIAID launched the Center for HIV/AIDS Vaccine Immunology (CHAVI), and in 2006 the Bill Melinda Gates Foundation launched the Collaboration for AIDS Vaccine Discovery (CAVD). The CHAVI program was a multi-institutional partnership founded at Duke University and responsible for discovering key aspects of HIV immunology that have informed the design of the latest vaccine candidates [21]. The CAVD is focused on developing novel vaccine candidates utilizing cutting-edge immunology labs, animal models, and data management and sharing tools to bring discoveries from research to clinical testing. The collaborative network has undoubtedly accelerated HIV vaccine development and enabled studies that would not have been possible had the many collaborators worked independently. As proven during the COVID-19 pandemic, the collaborative networks built for HIV response and vaccine development benefit both scientific development and implementation of medical interventions. These joint efforts remain critical in the development and testing of current vaccine candidates and will promote a successful HIV vaccine rollout if and when it becomes available. Current HIV vaccine clinical trials: The technologies and strategies used for vaccine development are constantly evolving. Of particular impact is the increased use of public-private partnerships, as well as novel vaccine technologies such as messenger RNA (mRNA) vaccines. Although the technology underlying mRNA vaccines had been in development for over a decade before the COVID-19 pandemic, the unprecedented funding and manpower dedicated to a rapid response led to the creation of the first FDA-approved mRNA vaccines. These partnerships have sped up progress towards an HIV vaccine and impacted vaccine progress for other diseases as well. The clearest example of this occurred during the COVID-19 pandemic, where many of the partnerships and collaborations that had initially been established for HIV detection, treatment, and vaccine development were pivoted to address COVID-19 in the early stages of the pandemic. The U.S. President’s Emergency Plan for AIDS Relief (PEPFAR) accelerated other aspects of HIV prevention and treatment. https://doi.org/10.38105/spr.0cg04tczoe PEPFAR was founded in 2003 with the goal of “leveraging years of HIV/AIDS research, coordinated humanitarian effort, bipartisan support from Congress, and engagement from community and faith-based organizations as well as the private sector to create an unprecedented response to a global health crisis [23].” PEPFAR’s $1 billion in annual funding supports over 70,000 health clinics, including 3,000 laboratories and 290,000 healthcare workers. Critically, through these networks, PEPFAR also supports supply chains for healthcare commodities, as well as systems for data collection and use in disease response. The Moderna mRNA vaccine against SARS-CoV-2, for example, was developed in partnership with NIAID, but this collaboration was initially created for HIV and emerging infectious diseases [25]. Though this joint effort was leveraged during the COVID-19 pandemic, it has proven to be fruitful for developments in HIV vaccine research. A phase 1 clinical trial of two Moderna mRNA vaccines for HIV began in 2022 and will be completed later in 2023 (ClinicalTrials.gov identifier: NCT05217641). This study is sponsored by IAVI and NIAID; it includes many private and public research collaborators, including the University of Texas at San Antonio, George Washington University, the Fred Hutchinson Cancer Center, and Emory University, further indicating the extent of these collaborations. PEPFAR shifted to aiding in the COVID-19 pandemic in 2020 and was able to help in several critical ways: PEPFAR labs provided COVID-19 testing and were able to track novel SARS-CoV-2 mutations in order to find variants of concern, assisted in identifying worldwide COVID-19 hot spots and provide intervening measures to prevent transmission, and leveraged their community-contact networks to provide the vaccine to those most in need once it was available [24]. It remains to be seen whether the rapid timelines employed for COVID-19 vaccine development will similarly expedite vaccines against other diseases. Operation Warp Speed (OWS), a massive collaborative COVID-19 response initiative, enabled Moderna to go from enrolling their first clinical trial volunteers to applying for Emergency Use Authorization—which makes the vaccine available for public use—in under five months [26]. Such a timeline for HIV vaccine development has yet to be seen; for comparison, the current phase 1 trial is estimated to run for over a year and a half. In addition to PEPFAR, the COVID-19 vaccine trial would not have been possible without the HIV Vaccine Trials Network (HVTN), a cooperative partnership between the National Institutes of Health (NIH) and academia founded in 1999. Saiz and Farenhem The modern vaccine development landscape The central problem plaguing the design of an efficacious HIV vaccine is the lack of a specific epitope, or small region of the virus, that can be used as an antigen to elicit a protective immune response when administered as a vaccine. Together with Scripps Research, IAVI used newly-developed vaccine design techniques in pursuit of an efficacious vaccine [21]. The partnership still stands, and in 2020, IAVI, Scripps Research, and the National Institute of Allergy and Infectious Disease (NIAID) announced a collaboration with the Serum Institute of India and USAID to advance the development of vaccines. As a prime example of collaboration-based scientific research, they are optimizing combinations of broadly neutralizing antibodies (bnAbs), types of immune molecules that bind to and attenuate multiple strains of HIV, as a potential passive immunization tool. BnAbs would be a novel technology for HIV protection [21, 22]. A look at past HIV vaccine trials: The first HIV vaccine was tested in clinical trials in 1987; since then, multiple worldwide clinical trials have evaluated various HIV vaccines. Nearly all of these vaccines were designed to mimic a portion of the Env protein, and several later vaccines also targeted co-receptors that are closely associated with Env [18]. Several of these trials generated the expected immune responses and created non-neutralizing antibodies, or antibodies that can bind to viral proteins and even initiate T-cell responses. Unfortunately these antibodies were unable to neutralize the virus and therefore could not prevent its spread throughout the body. Only one trial, whose vaccine is identified as RV144, had any measurable protection against future HIV infection. IAVI incited more collaboration, and the Global HIV Vaccine Enterprise launched in 2005 to provide resources MIT Science Policy Review | August 31, 2023 | vol. 4 | pg. 33 Saiz and Farenhem https://doi.org/10.38105/spr.0cg04tczoe https://doi.org/10.38105/spr.0cg04tczoe Article Article clinical setting, and if successful in phase 1, may be studied further in phase 2 and 3 clinical trials. clinical setting, and if successful in phase 1, may be studied further in phase 2 and 3 clinical trials. management systems prior to an HIV vaccine rollout would ensure that if vaccinated, individuals could be properly monitored and statistics could be accurately tracked. Standardized data collection procedures and a unified technology platform would benefit both clinicians and vaccine recipients, the former, by alleviating technology incompatibility issues across regions, and the latter, by organizing clinical data in a secure, reliable platform that contains all necessary information for quality health care. Despite the novelty of an HIV mRNA vaccine, mRNA-based technology could provide several advantages to HIV prevention. Previously, development of HIV vaccine candidates required the design, stabilization, and purification of various viral proteins. Using this strategy, a single antigen can take years to develop and test. With mRNA vaccines, the components of the vaccine are mostly the same, and the sequence of the mRNA itself is what varies depending on the target. Given the appropriate technology necessary to synthesize the components of these vaccines, they are easier to design and produce than traditional vaccines, which often require the growth of entire virions or viral proteins in complex biological systems, such as chicken eggs. mRNA vaccines, which can be produced in vitro and do not require extensive purification steps, can be developed more quickly and inexpensively than other vaccine technologies. This lowers the cost barrier to vaccine accessibility, though it should be noted that mRNA vaccines require extremely cold storage that may not be accessible in some places. Using mRNA, it is also much easier to create vaccines that are designed against multiple HIV epitopes, theoretically targeting several highly-mutating HIV variants [27]. Complete demographic information and statistics can help provide a detailed understanding of existing vaccine disparities, which are otherwise overlooked without proper IISs. IISs are subject to regulation by hundreds of federal, state, and local policies, which may hamper their effectiveness, whereas individual IISs managed at the state or local level could perhaps provide more tailored service to a particular community. During COVID-19, IIS databases were federally managed, but data acquisition was overseen by state and local entities, which made it challenging to track nationwide vaccinations. https://doi.org/10.38105/spr.0cg04tczoe A conscious HIV vaccination campaign might include standardized IISs, policies authorizing data transfers between state IISs, and policies to encourage IIS innovation, for example, smartphone app integration, text-message reminders, and conversion of paper-documented records to electronic. Outlook on policies affecting a future vaccine Vaccine-related data systems: While the timeline for an efficacious HIV vaccine is unknown, many other factors will affect a future vaccine’s rollout and overall impact. The effectiveness of an HIV vaccine program would be improved by policies intended to streamline healthcare data management. An example is immunization information systems (IISs), databases that health care providers use to manage patient vaccine information, records, and medical information. The primary purpose of these systems is to track vaccination rates in real time, which is necessary for the timely and appropriate allocation of resources during a large-scale vaccine rollout [28]. IISs have room for improvement, as some operate on different technology platforms that are not interoperable with other IISs [29, 30]. In order to provide the best care to the largest number of people, IISs may benefit from accurate and complete information, technology updates, and additional funding [28]. The $8.3 billion stimulus package supplied by the March 2020 Coronavirus Preparedness and Response Supplemental Appropriations Act (CPRSAA)(2) provided funds to develop, manufacture, and procure vaccines and medical supplies, provide humanitarian assistance and support for health systems in countries affected by COVID-19, and support the CDC, NIH, and the Public Health and Social Services Emergency Fund. As a follow-up to the CPRSAA, the Coronavirus Response and Relief Supplemental Appropriations Act (CRRSAA)(3) was enacted in December of 2020, approving over $900 billion in coronavirus assistance funds, $8.75 billion of which went to the CDC, and $300 million of which was reserved specifically for "high-risk and under-served populations, including racial and ethnic minority populations and rural communities [29]." The American Rescue Plan Act (ARPA) enacted in March 2021 allocated additional funding to the CDC, and together with the CRRSAA, the CDC can use these funds to support IIS programs and operations [29]. Future policies might continue to support improvement of data systems. In the fiscal year 2021, the Centers for Disease Control and Prevention (CDC) received $16.25 billion for planning, administering, and tracking COVID-19 vaccinations, and a portion of the funds was used to support IISs. On November 30, 2021, the House of Representatives passed the Immunization Infrastructure Modernization Act of 2021 (H.R. https://doi.org/10.38105/spr.0cg04tczoe It supports early- to late-stage vaccine development with a mission to prevent HIV globally. Funding is both public and private, with NIAID as the primary funding source backing the majority of HVTN vaccine clinical trials. The HVTN has clinical trial sites in 16 countries, which has proven especially useful in numerous HIV vaccine trials including RV144 and HVTN702. The clinical trial infrastructure and network of collaborating institutions that includes biotech companies, pharmaceutical In the phase 1 trial, researchers are testing the vaccine candidate on a small cohort of healthy individuals to evaluate any safety concerns. They will determine whether vaccine recipients have developed antibodies in response to the vaccine as well as a few other immune-related markers. This will be the first mRNA vaccine against HIV to be tested in a MIT Science Policy Review | August 31, 2023 | vol. 4 | pg. 34 Saiz and Farenhem https://doi.org/10.38105/spr.0cg04tczoe Under the principles of distributive justice, equality-based sharing involves equal sharing of benefits amongst all parties, contribution-based sharing assigns a greater share of benefits to parties who contributed during the vaccine development and clinical trial process, and need-based sharing gives more benefits to those who are worse-off regardless of whether they participated in the vaccine development phase. Identifying countries and individuals in need poses a challenge in itself. A researcher from the qualitative interview study commented, “...everybody will tell you he has a need but we have to scrutinize and say this is a real need at this point...,” [31] highlighting how addressing need is not black and white. Quantitative metrics of the severity of HIV/AIDS in a given area are important in assigning need, but they may not always capture the severity of the epidemic, especially in the case of marginalized sub-populations in need such as sex workers and injection-drug users from whom survey statistics may be more difficult to obtain. For need-based sharing to proceed effectively, policymakers must carefully decide the metrics on which they base their assessment of need. A qualitative study [31] designed to elucidate themes and opinions regarding vaccine sharing in Tanzania provides valuable insight into how vaccines should best be shared when available. Tanzania has hosted numerous HIV vaccine trials to date, hence the location of the study. By conducting interviews of 37 selected participants including researchers, institutional review board members, a policymaker, HIV/AIDS advocates, and community advisory board members, the study found that HIV vaccines—when available—can be shared fairly under the principles of distributive justice. In the context of contribution-based sharing, reciprocity to participants and host countries is the idea that people, and at a higher level, the countries that participated in vaccine development or trials should reap the benefits first. Enormous financial contributions to research and development, such as those supplied by the NIH, are less feasible for poorer countries. According to contribution-based sharing principles, enrolling participants in trials, which entails risk, pain, inconvenience, and time, may be viewed as an equal contribution, and thus entitles those participants to equal benefits as participants who contributed monetarily. Financial agreements with pharmaceutical companies stating that when a vaccine becomes available, disadvantaged countries will receive appropriate access are an example of implementing contribution-based sharing. Equality-based sharing seemed the most controversial in the 2022 interview-based study [31]. https://doi.org/10.38105/spr.0cg04tczoe Article Article Nations Programme on HIV/AIDS proposed that by 2020, 90% of all people living with HIV will know their HIV status, 90% of all people diagnosed with HIV will receive ART, and 90% of people receiving ART will have viral suppression [33]. This program was started in 2014 with a goal end date of the end of 2020. The UN provided an update in September 2020, stating that "81% of people living with HIV knew their HIV status," and that 67% of those living with HIV were on ARTs [34]. Though this was not the goal that had been initially set, these statistics were seen as "remarkable gains," and progress continues to be made in many nations. In pursuit of a rapid solution, the COVID-19 pandemic familiarized people worldwide with mRNA vaccines, which have become increasingly relevant given the development of current experimental HIV mRNA vaccines. mRNA vaccines, however, may face resistance to widespread adoption because they are unfamiliar to people who might receive them and there are fewer longitudinal data supporting their long-term safety by nature of their recent development. Vaccine sharing: If and when an HIV vaccine becomes available, how it is shared will determine the extent of its efficacy. Amongst entities with competing needs and claims—for example, pharmaceutical companies who invested money and resources into the development of a vaccine, governments who used public funds to invest in research and vaccine trials, individuals who participated in vaccine trials, or individuals who may or may not be particularly at risk for HIV—the principles of “distributive justice” lay a foundation for attaining fairness in distributing vaccines [31, 32]. Under needs-based principles, countries that did not meet the 90-90-90 goal would be given priority when vaccines become available, but need-based sharing may be viewed as unfair from the perspective of countries that have supplied large amounts of funding, such as the U.S., which has invested over $100 billion in global HIV/AIDS response—the largest amount of any nation [35]. Countries that did not meet their 90-90-90 goals, such as Tanzania, Togo, South Africa, and several others are designated PEPFAR countries and are already receiving financial aid to address the AIDS crisis [36, 37]. New 95-95-95 goals have been set for 2025, laid out in a strategic vision document [38], as countries continue to make progress towards eradicating HIV/AIDS. https://doi.org/10.38105/spr.0cg04tczoe 550)(1), which would give authorization to the Health and Human Services secretary to improve IISs. H.R. 550 would have authorized a grant program for state, local, territorial, and tribal (SLTT) government agencies to improve and standardize IIS technology, but as of January 2023, it did not pass the Senate and is now dead. Holistically, the COVID-19 pandemic demonstrated the speed at which governments can supply emergency funding to propel vaccine candidates all the way through clinical trials to recipients. The swiftness at which funding, resources, and the healthcare workforce was mobilized during the COVID-19 pandemic was unprecedented and showcased what is possible, though whether this magnitude of a concerted effort will be seen again—particularly if underserved communities and those in underdeveloped nations are at risk—remains to be seen. New legislation directing resources at improving data MIT Science Policy Review | August 31, 2023 | vol. 4 | pg. 35 Saiz and Farenhem Saiz and Farenhem Saiz and Farenhem https://doi.org/10.38105/spr.0cg04tczoe According to the principles of distributive justice, the vaccine can be shared to those in need (need-based), to those who contributed to vaccine development by funding research or participating in clinical trials (contribution-based), or to all countries equally (equality-based). troubling given that 69% of new HIV cases occur in Black and Hispanic/Latino individuals [39] and that the highest incidence of HIV occurs in the South [41]. Disparities in access to and use of PrEP exist to a greater extent in low and middle-income countries, such as in sub-Saharan Africa, which is home to two-thirds of all people living with HIV globally [42]. should precede vaccine availability so that a rapid response can be executed when vaccines are ready for distribution. More interview-based studies to unpack the ethics of vaccine sharing would undoubtedly be worthwhile in helping craft policies that maximize efficacy while minimizing unfairness. Further, more studies will need to be conducted to determine the true need of different populations beyond the current metrics. The cost of the injectable PrEP drug CAB-LA [43, 44]—which more effectively prevents HIV infection than the oral form and alleviates some issues of adherence to the therapy [45]—was initially priced in the United States at $22,000 by its developer ViiV Healthcare, 60 times more expensive than generic tenofovir disoproxil-based PrEP drugs. This presents a virtually insurmountable barrier to access, especially where it is needed most, such as in sub-Saharan Africa, where an estimated 86.4% of people earn less than $6.85 per day (a widely used international poverty line) [46]. In July 2022, ViiV Healthcare signed a voluntary agreement with the Medicines Patent Pool to allow certain generic drug manufacturers to produce and supply generic versions of CAB-LA in 90 countries, which will help more at-risk individuals gain access to preventative therapy. Future policies may incorporate awareness of the prohibitive cost of therapies compared to alternatives (e.g. injectable CAB-LA compared to oral PrEP) and downstream barriers to clinical implementation such as delivery to various geographical regions, necessity of a prescription to obtain the drug, adherence to therapy, and the limited data on CAB-LA in pregnant and breastfeeding women [45]. Lessons from HIV resource disparities: While a vaccine might still be in the future, there are numerous aspects to contemplate regarding HIV treatment approaches and disparities. These aspects could serve as predictors of the inequities that will need attention when a vaccine is eventually introduced. https://doi.org/10.38105/spr.0cg04tczoe It entails that everyone should have equal access to the vaccine. All people are arguably at risk of HIV in some way or another, so under equality-based principles, everyone deserves equal access. Equal access, however, is unrealistic, given that there is an upper limit in the logistics of vaccine production, distribution, and administration. There is also the controversy of reaping benefits having made no contribution to vaccine development or trials. Some study participants expressed concern over considering contributors to vaccine discovery equal to those who made no contribution. Some entity—a government, company, donors, etc.—must bear a disproportionate cost to provide vaccines for those who claim a right to the vaccine but make no contribution. Need-based sharing may also address the issue of HIV vaccine distribution. If vaccines are first distributed to countries with the most at-risk populations and where the spread of infection is highest, the epidemic may reach a sooner end. The “90-90-90” treatment target proposed by the Joint United Ultimately, funds must flow through different entities and several branch points to produce a vaccine that can then be distributed in a number of ways (Figure 2). Policies MIT Science Policy Review | August 31, 2023 | vol. 4 | pg. 36 Saiz and Farenhem Saiz and Farenhem https://doi.org/10.38105/spr.0cg04tczoe Article Figure 2: Flow of funds from inception to vaccine distribution. Funding is derived from multiple sources and distributed to academic or government research institutions and biotechnology companies, who use the funds for research and development. Once a vaccine candidate is ready for testing, funds then flow into clinical trials, where the vaccine is tested on cohorts of patients. If successful, manufacturers then mass produce the vaccine, which must subsequently be distributed. According to the principles of distributive justice, the vaccine can be shared to those in need (need-based), to those who contributed to vaccine development by funding research or participating in clinical trials (contribution-based), or to all countries equally (equality-based). Figure 2: Flow of funds from inception to vaccine distribution. Funding is derived from multiple sources and distributed to academic or government research institutions and biotechnology companies, who use the funds for research and development. Once a vaccine candidate is ready for testing, funds then flow into clinical trials, where the vaccine is tested on cohorts of patients. If successful, manufacturers then mass produce the vaccine, which must subsequently be distributed. Saiz and Farenhem Citation Saiz, A. C., Farenhem, K. L. HIV vaccines: Lessons from basic research, clinical trials, and public policy. MIT Science Policy Review 4, 31–40 (2023). https://doi.org/10.38105/ spr.0cg04tczoe. https://doi.org/10.38105/spr.0cg04tczoe From the start of the AIDS pandemic, disparities in access to healthcare and stigmatization of at-risk groups have prevented many patients from being diagnosed and receiving appropriate care. With the advancement of HIV research in the mid-1990s and early 2000s, ARTs became the standard for care in HIV treatment. However, a 2001 national study conducted by the HIV Research Network (HIVRN) found that Black and Hispanic/Latino HIV-positive individuals were significantly less likely to receive ARTs than white HIV-positive individuals [39]. These disparities have continued to this day. A 2018 study found that 75% of prescriptions for PrEP, which drastically reduces HIV transmission rates, were written to white men, who are gay or bisexual, and predominantly to those who live in the Northeast or on the West Coast [40]. This is particularly MIT Science Policy Review | August 31, 2023 | vol. 4 | pg. 37 Saiz and Farenhem Conclusion While scientific advances are necessary for the treatment and prevention of HIV, there are many other factors necessary for successful vaccine development and distribution. During the COVID-19 pandemic, technological innovation, preexisting clinical trial infrastructure, distribution networks, data management, health care, and financial support were obligatory for a successful, rapid vaccination campaign. As evidenced by both the HIV and COVID-19 pandemics, local, state, and federal policies, private companies, philanthropists, non-governmental organizations, and community outreach efforts are a vital part of the equation to realize an HIV vaccine from development all the way to administration and follow-up. Many domestic and global efforts have been established in order to mitigate these discrepancies in care. In 2023, the NIH’s budget for scientific research around HIV and AIDS was $3.2 billion, while an even greater amount, an additional $4.5 billion, was budgeted for preventative, health, and community-related programs to increase access to HIV resources and care [47]. Disparities in HIV resources affect people already living with HIV as well. Over 10 million HIV-positive individuals around the world do not have access to ARTs [42]. However, a 2021 study in the journal AIDS estimated that for $2 billion a year, 66,308 mother-to-child transmissions, 241,811 HIV-related deaths, and 631,398 new HIV infections per year could be prevented [48]. The Joint United Nations Programme on HIV/AIDS (UNAIDS) highlighted an increasing and disturbing inequality: only 52% of HIV-positive children have access to appropriate care, and that that trend is getting worse. Another rising disparity is in gender: adolescent girls and young women (15-24 years old) are three times more likely than men and boys of the same age to acquire HIV in sub-Saharan Africa [42]. HIV continues to pose a major health risk, particularly to those most vulnerable to the virus due to demographic or geographic factors, despite the existence of some preventative and therapeutic interventions. Policies that improve healthcare, technology, logistics, education, and awareness on which successful vaccine development rely is vital for both the development and equitable distribution of a future HIV vaccine. https://doi.org/10.38105/spr.0cg04tczoe Article Article In the proposal for the FY2024 budget, the Biden administration requested $237 million—which would grow to $9.8 billion over a 10-year period—to expand access to PrEP and related health and community services “for uninsured and under-insured individuals at high risk of HIV” in the United States [47]. The proposal highlights the “fragmented patchwork” of current PrEP access tools for uninsured individuals and states that only 9% of Black people in the United States who could benefit from taking PrEP actually do. For Hispanic and Latino individuals, only 16% do [47]. Notably, this initiative was proposed for FY2023 but was not included in the final budget. resources and healthcare—two factors that may drastically impact the distribution of and access to a future HIV vaccine. Implementing programs to mitigate the current issues of HIV resource and education disparities is, therefore, critical, not only to reduce transmission and improve quality of life for those with HIV, but also to lay the foundation for an equitable vaccine rollout. Acknowledgments The authors would like to thank their editorial team, Hannah LeBlanc and Steven Cheng, for their helpful discussions, as well as the reviewers for constructive feedback. BioRender was used to create Figure 1. Many regions have seen increases in annual HIV infections during the last 10 years, including Eastern Europe and Central Asia, the Middle East and North Africa, and Latin America [40]. However, even in the regions where this was not the case, there are some alarming warning signs. In eastern and southern Africa, the number of HIV tests conducted has dropped each year since its peak in 2018, and the number of people receiving ARTs there has halved since 2017 [42]. Though there are likely many causes of these trends, it should be noted that this drop in testing and ART use was concurrent with a drop in the supply of HIV-related financial resources (both from domestic and international assistance) to low- and middle-income nations, which peaked in 2017 and began to decrease in the subsequent years [42]. Saiz and Farenhem References [1] CDC. New HIV diagnoses and people with diagnosed HIV in the us and dependent areas by area of residence. https://www. cdc.gov/hiv/basics/statistics.html. [20] Gray, G. E. et al. Vaccine efficacy of ALVAC-HIV and bivalent subtype C gp120–MF59 in adults. The New England Journal of Medicine 384, 1089–1100 (2021). https://doi.org/10. 1056/NEJMoa2031499. [2] UNAIDS. 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The Journal of Infectious Diseases 215, 1255–63 (2017). https://doi.org/10.1093/ infdis/jix099 Open Access This MIT Science Policy Review article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission These disparities and backsliding trends pose a serious risk to the suppression of HIV, and the development of a vaccine would not, on its own, solve this, particularly if the vaccine is not financially accessible. An inexpensive vaccine is critical to reaching those most at-risk for HIV and has potential to overcome some of the disparities in access to an HIV vaccine. Many of the present-day inequities in HIV prevention and treatment stem from a lack of access to educational MIT Science Policy Review | August 31, 2023 | vol. 4 | pg. 38 Saiz and Farenhem Saiz and Farenhem https://doi.org/10.38105/spr.0cg04tczoe https://doi.org/10.38105/spr.0cg04tczoe Article Article directly from the copyright holder. 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Predictive Capacity of Boar Sperm Morphometry and Morphometric Sub-Populations on Reproductive Success after Artificial Insemination
Animals
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Predictive Capacity of Boar Sperm Morphometry and Morphometric Sub-Populations on Reproductive Success after Artificial Insemination Vinicio Barquero 1 , Eduardo R. S. Roldan 2, Carles Soler 3, Jesús L. Yániz 4 , Marlen Camacho 1 and Anthony Valverde 1,* o Barquero 1 , Eduardo R. S. Roldan 2, Carles Soler 3, Jesús L. Yániz 4 , Marlen Camacho 1 th V l d 1 * Vinicio Barquero 1 , Eduardo R. S. Roldan 2, Carles Soler 3, Jesús L. Yániz 4 , Marlen Camac and Anthony Valverde 1,* 1 Animal Reproduction Laboratory, School of Agronomy, Costa Rica Institute of Technology, San Carlos Campus, Alajuela 223-21002, Costa Rica; vinicio1196@gmail.com (V.B.); mcamacho@tec.ac.cr (M.C.) 2 Department of Biodiversity and Evolutionary Biology, Museo Nacional de Ciencias Naturales (CSIC), 28006 Madrid, Spain; roldane@mncn.csic.es 2 Department of Biodiversity and Evolutionary Biology, Museo Nacional de Ciencias Naturales (CSIC), 28006 Madrid, Spain; roldane@mncn.csic.es 3 Department of Cellular Biology, Functional Biology and Physical Anthropology, University of Valencia, Campus Burjassot, C/Dr Moliner 50, 46100 Burjassot, Spain; carles.soler@uv.es 4 BIOFITER Research Group, Higher Polytechnic School of Huesca, Institute of Environmental Sciences of Aragón (IUCA), University of Zaragoza, Ctra. Cuarte s/n, 22071 Huesca, Spain; jyaniz@unizar.es * Correspondence: anvalverde@tec.ac.cr; Tel.: +506-2401-3223 3 Department of Cellular Biology, Functional Biology and Physical Anthropology, University of Valencia, Campus Burjassot, C/Dr Moliner 50, 46100 Burjassot, Spain; carles.soler@uv.es 4 BIOFITER Research Group, Higher Polytechnic School of Huesca, Institute of Environmental Sciences of Aragón (IUCA), University of Zaragoza, Ctra. Cuarte s/n, 22071 Huesca, Spain; jyaniz@unizar.es * Correspondence: anvalverde@tec.ac.cr; Tel.: +506-2401-3223 Simple Summary: The efficiency of swine production measured as litter size influences the prof- itability of the pig industry. Furthermore, sow fertility potential depends in part on the boar semen quality and reproductive efficiency. The objective of this study is to compare boar sperm head size and morphometric features of shape to evaluate their relationships with reproductive success after artificial insemination (AI). A morphometric analysis of boar ejaculate reveals morphometrically separate sub-populations. The differences between sub-populations are displayed for sperm head size. In addition, sperm clustering into sub-populations did not have a predictive capacity on lit- ter size variables. Nevertheless, the morphometric variables of the sperm may have a predictive, albeit reduced, capacity regarding litter size variables. The results of this study therefore open up possibilities for future assessments of fertility. animals animals 1. Introduction The efficiency of pig livestock production, measured as piglets born alive, total born per litter, and the number of piglets weaned by a sow per year, influence the profitability of the pig industry [1]. Sow fertility potential depends in part on the semen quality of the boar [2]. The ability to identify sub-fertile boars to improve reproductive efficiency has its basis in economic profitability [3]. The classical routine evaluation of boar fertility has traditionally been based on the assessment of semen variables, including seminal volume, sperm concentration, motility, and morphology [3]. Boars of different breeds, lines, or crossbreeds can produce ejaculates of different volumes, sperm concentrations, motilities, and kinematic patterns [4,5]. Sperm morphometry differs according to the breed of the male [6]. It has been reported that considerable variability exists in sperm morphometry among males from the same population [7–10]. Furthermore, the sperm morphometry variability of different ejaculates from the same animal has been documented [11]. Interest in the size and shape morphometry of sperm heads has led to intense research in recent years [12,13]. The morphometric variables of the size and shape of the sperm head exhibit a relevant variability among species, among males of different species [14,15], and between breeds or selected lines within a species [6,10]. Some studies have described a correlation between the fertility of males used for artificial insemination (AI) and sperm morphome- try [12], and head size has been associated with factors predisposing fertility [16]. Other authors refer to an association between the head size and shape of the spermatozoa and semen or ejaculate variables [17–19]. The total spermatozoa in the ejaculate have been associated with the morphometric variables of spermatozoa: ejaculates with a low sperm concentration have smaller, shorter, and narrower head sizes and a smaller head area than those of ejaculates with a high sperm count [8]. Current computer-assisted sperm morphometric analysis CASA-Morph systems can be used to analyze individual sperm morphometrics more accurately, and this information can be submitted to a multivariate procedure such as cluster analysis for an overview of distinct sperm patterns grouped into sub-populations (SPs) or clusters [20]. Some authors have indicated that the distri- bution of spermatozoa in each sperm sub-population can vary among males, and some of these sub-populations have been correlated with sperm quality [21].   Citation: Barquero, V.; Roldan, E.R.S.; Soler, C.; Yániz, J.L.; Camacho, M.; Valverde, A. Predictive Capacity of Boar Sperm Morphometry and Morphometric Sub-Populations on Reproductive Success after Artificial Insemination. Animals 2021, 11, 920. https://doi.org/10.3390/ani11040920 Citation: Barquero, V.; Roldan, E.R.S.; Soler, C.; Yániz, J.L.; Camacho, M.; Valverde, A. Predictive Capacity of Boar Sperm Morphometry and Morphometric Sub-Populations on Reproductive Success after Artificial Insemination. Animals 2021, 11, 920. https://doi.org/10.3390/ani11040920 Abstract: The aim of the study was to compare the morphometric features of sperm head size and shape from the Pietrain line and the Duroc × Pietrain boar crossbred terminal lines, and to evaluate their relationship with reproductive success after artificial insemination of sows produced from crossbreeding the York, Landrace and Pietrain breeds. Semen samples were collected from 11 sexually mature boars. Only ejaculates with greater than 70% motility rate and <15% of abnormal sperm were used for artificial inseminations (AI) and included in the study. Samples were analyzed using an ISAS®v1 computer-assisted sperm analysis system for eight morphometric parameters of head shape and size (CASA-Morph). Sub-populations of morphometric ejaculates were characterized using multivariate procedures, such as principal component (PC) analysis and clustering methods (k- means model). Four different ejaculate sub-populations were identified from two PCs that involved the head shape and size of the spermatozoa. The discriminant ability of the different morphometric sperm variables to predict sow litter size was analyzed using a receiver operating characteristics (ROC) curve analysis. Sperm head length, ellipticity, elongation, and regularity showed significant predictive capacity on litter size (0.59, 0.59, 0.60, and 0.56 area under curve (AUC), respectively). The morphometric sperm sub-populations were not related to sow litter size. Academic Editor: Juan Carlos Gardón Poggi Received: 7 March 2021 Accepted: 22 March 2021 Published: 24 March 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// Keywords: sperm; CASA-morph; fertility; sperm subpopulations; boar; sow; litter size creativecommons.org/licenses/by/ Animals 2021, 11, 920. https://doi.org/10.3390/ani11040920 https://www.mdpi.com/journal/animals 2 of 15 Animals 2021, 11, 920 1. Introduction Several studies have examined the association between sperm head size and fertility in pigs [6,16,22] and species such as cattle [23], buffalo [24], sheep [25], goats [26], horses [27], dogs [28]. or humans [29]. A limited number of studies have focused on the size and head shape of sperm and the characterization of sperm sub-populations on litter size in livestock species such as pigs [30,31]. Even today, the biological relevance and direct biological meaning of sperm sub-populations remain controversial. The aim of the present study was to compare the morphometric features of the head size and shape of spermatozoa from the Pietrain and Duroc × Pietrain boar crossbred terminal lines, and to evaluate their relationships with the litter size of sows produced from crossbreeding of the York, Landrace, and Pietrain breeds. 2.2. Fertility Trial A total of 816 triple-artificial inseminations, performed with homospermic ejaculates, were evaluated on 272 sows. These AIs were conducted aleatory with 40 ejaculates from eleven males. That means that each genetic line of females was randomly inseminated with each genetic line of males. Each ejaculate was used in the first 3 days post-collection, and only those used to inseminate at least three females were evaluated. The mean of sows inseminated per boar was 24.7 ± 10.1 females. These AI resulted in 3.99 ± 3.16 farrowings from each female. Parameters measured at the time of farrowing were: litter size as total piglets born (TPB) per litter, piglets born alive (PBA), piglets born dead (PBD), number of mummies (MP), and litter weight (LW; kg). 2.4. Sample Preparation for Morphometric Analysis Duplicate samples for morphometric analysis were prepared from the ejaculates of each commercial line. After being mixed, 10 µL of each sample was spread on a glass slide and subsequently air-dried. The slides were stained using the Diff-Quik® kit (Medion Diagnostics, Düdingen, Switzerland), following the instructions of the manufacturer. All the slides were identified and then analyzed in a double-blind scheme. 2. Materials and Methods 2.1. Animals The experiment was conducted at a commercial swine farm (Agropecuaria Los Sag- itarios S.A., Alajuela, Costa Rica) during 2019–2020 in the northwest of Costa Rica (Río Cuarto, 10◦20′32′′ N, 84◦12′55′′ W, Alajuela, Costa Rica, Central America) following the laws and regulations controlling experiments on live animals in Costa Rica. This study was performed following ethical principles, and with the approval of the Committee of Centro de Investigación y Desarrollo de la Agricultura Sostenible para el Trópico Húmedo at the Costa Rica Institute of Technology (CIDASTH-ITCR) according to Section 08/2020, article 1.0, DAGSC-100-2020. Eleven sexually mature and healthy boars from two com- mercial terminal male lines (ML: Duroc × Pietrain (n = 8) and Pietrain boars (n = 3)), 20.9 ± 3.0 (Pietrain) and 24.1 ± 9.8 (Duroc × Pietrain) months of age at the beginning of the experiment and of known fertility were used as semen donors in this study. For the study, the breeding boars were housed individually in well-ventilated pens with an Animals 2021, 11, 920 3 of 15 average temperature of 25.6 ± 2.94 ◦C during the time of the experiment. The females came from four crossbred genetic lines (FL: York (Y), Landrace (L) and Pietrain (P), with the crossing schemes YLP-50 ( 1 4 Y × 1 4 L × 1 2 P), YLP-75 (1/8 Y × 1/8 L × 3/4 P), YLP-87.5 (1/16 Y × 1/16 L × 7/8 P), and Y-L-50 ( 1 2 Y × 1 2 L)). All of the females were bred on the farm and they came from maternal crossing schemes. Animals were fed with a standard breeder mixture (made on the farm) containing maize, soybean meal, mineral mixture, and common salt, as ingredients to fulfill the nutrient requirements according to the Nutrient Requirements of Swine [32]. Pregnant sows were provided with 2.5 kg of concentrated feedstuff in the first 2/3 of the gestation period and 3 kg in the final third. Males consumed 2.5 kg of concentrated feedstuff per day and were provided with water ad libitum. The gilts inseminated were 7 months of age, with a minimum weight of 145 kg. 2.3. Collection and Examination of Semen Semen samples were collected in the morning, once per week, using the “gloved-hand” technique [33], and immediately placed into a water bath at 37 ◦C at the farm laboratory. In all cases, the sperm-rich fractions were collected and diluted with a commercial ex- tender (Zoosperm ND5; Import-Vet, Barcelona, Spain). Insemination doses contained 3.7 ± 1.3 × 109 spermatozoa. From each boar, 3.64 ± 0.81 ejaculates were obtained. Sam- ples from each ejaculate were evaluated for motility, and only ejaculates with at least 70% motile spermatozoa and 85% morphologically normal spermatozoa were used. The concentration was measured with Spermacue (Minitube, GmbH, Tiefenbach, Germany), following established protocols. Samples were stored at 17 ◦C and then transported to the laboratory in the same refrigerated conditions used for commercial distribution (17 ◦C). A volume of one milliliter (1 mL) of mixed sample was placed in an Eppendorf® tube (Sigma-Aldrich, St. Louis, MO, USA) and maintained at 37 ◦C for 30 min before use. 2.5. Assessment of Sperm Variables Microscope slides were analyzed for sperm head morphometry using the ISAS® v1 (Integrated Semen Analysis System, Proiser R+D, Valencia, Spain). The equipment comprised of a UB203 microscope (UOP/Proiser R+D) equipped with a bright-field 100× objective and a 3.3× photo-ocular. A digital video camera (Proiser 782 m, Proiser R+D) was mounted on the microscope to capture the images and transmit them to the computer. The array size of the video frame grabber was 746 × 578 × 8 bit, providing a resolution of the analyzed images of 0.084 µm/pixel in both axes, and 256 gray levels. The resolution of the images was 0.08 µm per pixel in both the horizontal and vertical axes. The sperm heads were captured randomly in different fields, and only those that overlapped with Animals 2021, 11, 920 4 of 15 background particles or other cells so as to interfere with the subsequent image processing were rejected. An Initial erroneous definition of the sperm head boundary was corrected by varying the analysis factor of the system. When it was not possible to obtain a correct boundary, the sperm head was deleted from the analysis. y p y For the analysis of motility, ISAS® D4C20 disposable counting chambers (Proiser R+D., Paterna Spain) were used after being pre-warmed to 37 ◦C. After homogenization of the samples, a volume of 3 µL was distributed along the counting chamber race by capillarity to fill it completely. Analyses were conducted using the CASA-Mot system of an ISAS® v1 (Proiser R+D S.L., Paterna, Spain). The frame rate used was 50 Hz, with the final resolution of the images being 746 × 578 pixels. The camera was attached to a UB203 microscope (UOP/Proiser R+D) with a 1× eyepiece and a 10× negative phase contrast objective (AN 0.25) and an integrated heated stage was maintained at a constant temperature of 37 ± 0.5 ◦C. The CASA settings used were: a particle area between 10 and 80 µm2, and connectivity of 11 µm. The percentage of total motile cells and progressive motility (%) corresponded to the spermatozoa swimming forward quickly in a straight line. The parameters defining progressive motility were straightness (STR) ≥45%, and average path velocity (VAP) ≥25 µm/s, defined as the average velocity over a smoothed cell path. p y g y p A single technician carried out the assessments of sperm morphology. 2.5. Assessment of Sperm Variables Sperm were classified as having normal or abnormal morphologic features following WHO strict criteria [34]. A total of 200 sperm were analyzed per slide, and 100 sperm from each of two different locations on the slide were assessed. If the difference between the percentage of normal sperm in the two areas was 5 percentage points or lower, the mean value was calculated. 2.7. Statistical Analysis The data obtained for the analysis of all sperm parameters were first assessed for normality and homoscedasticity using the Shapiro–Wilks and Levene tests. A normal prob- ability plot was used to assess normal distribution. Multivariate analyses were performed to identify sperm sub-populations from the set of ejaculate sperm morphometric data. All of the values for the morphometric variables were standardized to avoid any scale effect or variables with larger scales from dominating how clusters were defined. Thus, all variables were considered by the algorithm to be of equal importance. The standardization used was Z-score, and it transformed data by subtracting the mean value for each field from the values of the file, and then dividing by the standard deviation of the field, resulting in data with a mean of zero and a standard deviation of one. Differences between means were analyzed using a Bonferroni test. Results are presented as mean ± standard deviation (s.d.). Statistical significance was considered at p < 0.05. All data were analyzed using the IBM SPSS software package version 23.0 for Windows (SPSS Inc., Chicago, IL, USA). 2.6. Morphometric Analysis Images from about 200 spermatozoa from each sample were captured and analyzed to obtain eight morphometric variable values. Following the criteria of Boersma [35], the sperm heads were measured on each slide for four primary parameters of head size, length (L, µm), width (W, µm), area (A, µm2), and perimeter (P, µm); and four derived dimensionless parameters of head shape, ellipticity (L/W), rugosity (4πA/P2), elongation ((L −W)/(L + W)), and regularity (πLW/4A). Data from each individual sperm cell were saved in a database compatible with Excel® (Microsoft Corporation, Redmond, Washington, DC, USA) by the software for further analysis. 2.7.1. Multivariate Procedures A subset of the data was created using the means per ejaculate of all eight morphome- tric variables. The first process carried out was a principal component analysis (PCA) of the morphometric data to derive a small number of linear combinations that still retained as much information as possible from the original variables. The number of principal Animals 2021, 11, 920 5 of 15 components (PC) used in the next part of the analysis was determined using the Kaiser criterion, namely selecting only those with an eigenvalue (variance extracted of each PC) > 1. Furthermore, Bartlett’s sphericity test and the Kaiser–Meyer–Olkin (KMO) measure were performed [36]. As a rotation method, the varimax method with Kaiser normalization was used [37]. An analysis was conducted to classify the ejaculates into a reduced number of sub- populations (clusters), based on the scores obtained from the factor analysis. This was accomplished in two phases combining hierarchical and non-hierarchical clustering proce- dures. The process to perform a non-hierarchical analysis was conducted with a k-means model that used Euclidean distances from the quantitative variables after the standard- ization of these data, so the cluster centers were the means of the observations assigned to each cluster [38]. The multivariate k-mean cluster analysis was made to classify the ejaculates into a reduced number of sub-populations (clusters) according to their mor- phometric variables. In the final process, to determine the optimal number of clusters, the final centroids were clustered hierarchically using the Ward method [39]. Thus, the clustering procedure enabled the identification of sperm sub-populations because each cluster contributed to a final cluster formed by the ejaculate linked to the centroids. Then, ANOVA procedures were applied to evaluate statistical differences in the distributions of observations (individual spermatozoa) within the sub-populations, and then a generalized linear model (GLM) procedure was used to determine the effects of the genetic lines of the boar and sow breeds on the mean morphometric variable values defining the different sperm sub-populations (e.g., the cluster centers). 3.1. Overall Semen Variables The sperm concentration, the volume of semen, and the total spermatozoa in the ejaculate were 374.23 ± 129.24 × 106/mL, 231.98 ± 63.08 mL, and 82.04 ± 23.73 × 109, respectively. The sperm concentration (million/mL) was 378.63 ± 134.98 in the Duroc × Pietrain crossbreed and 361.00 ± 112.35 in the Pietrain. The total motility (%) of the boar samples was 77.36 ± 11.17, with an overall range of 35.05–93.69%. The progressive motility of the sperm (%) was 63.76 ± 11.96. The average total motility (%) for the Duroc × Pietrain and the Pietrain boars was 81.28 ± 7.76, and 65.61 ± 11.73, respectively. The progressive motility (%) was 67.00 ± 10.05 (Duroc × Pietrain), and 54.04 ± 12.19 (Pietrain). 2.7.2. ROC Analysis The diagnostic test with a dichotomous outcome (positive/negative fertility test results) of the different morphometric semen variables to predict litter size variables was analyzed using receiver operating characteristic (ROC) curve analysis. This diagnostic test evaluation used sensitivity and specificity as measures of test accuracy when compared with a standard status (farrowing). The sensitivity (true positive rate) and specificity (true negative rate) of each morphometric parameter varied across the different thresholds, and the sensitivity was inversely related to the specificity. The plot of sensitivity versus the 1-specifity is called the receiver operating characteristic (ROC) curve and the area under the curve (AUC). AUC varies from 0.5 (test with no discriminatory ability) to 1 (perfect discriminatory ability). A ROC was also used to calculate the elective breaking point (cut-off value) of each morphometric sperm variable. The analysis may also be used to determine the optimal cut-off value (optimal decision threshold). 3.2. Morphometric Variables ometric variables (mean ± s.d.) of boar sperm size and head shape of in different crossbred males and AI Table 1. Morphometric variables (mean ± s.d.) of boar sperm size and head shape of in different crossbred males and AI doses use in sows. Boar Sows Variable Pietrain D × P YLP-50 YLP-75 YLP-87.5 Y-L-50 Length 8.62 ± 0.60 α 8.77 ± 0.53 β 8.70 ± 0.52 a 8.78 ± 0.55 c 8.74 ± 0.53 b 8.75 ± 0.56 b Width 4.46 ± 0.23 α 4.54 ± 0.22 β 4.50 ± 0.23 a 4.53 ± 0.22 c 4.51 ± 0.23 b 4.51 ± 0.22 b Area 34.20 ± 2.47 α 35.46 ± 2.39 β 34.94 ± 2.33 a 35.38 ± 2.53 c 35.08 ± 2.46 b 35.07 ± 2.28 b Perimeter 23.84 ± 1.13 α 24.34 ± 1.06 β 24.15 ± 1.03 a 24.31 ± 1.11 c 24.23 ± 1.05 b 24.19 ± 1.06 ab Ellipticity 1.94 ± 0.17 1.94 ± 0.14 1.94 ± 0.15 1.94 ± 0.14 1.95 ± 0.15 1.94 ± 0.15 Rugosity 0.76 ± 0.04 0.75 ± 0.04 0.75 ± 0.04 0.75 ± 0.03 0.75 ± 0.04 0.75 ± 0.04 Elongation 0.32 ± 0.04 0.32 ± 0.03 0.32 ± 0.02 0.32 ± 0.03 0.32 ± 0.03 0.32 ± 0.04 Regularity 0.88 ± 0.03 0.88 ± 0.04 0.88 ± 0.03 0.88 ± 0.04 0.88 ± 0.04 0.88 ± 0.03 AI: artificial insemination, s.d.: standard deviation, length (L, µm), width (W, µm), area (A, µm2), perimeter (P, µm), ellipticity (L/W), rugosity (4πA/P2), elongation ((L −W)/(L + W)), regularity (πLW/4 A). Y: York, L: Landrace, P: Pietrain, D: Duroc. YLP-50 = ( 1 4 Y × 1 4 L × 1 2 P), YLP-75 = (1/8 Y × 1/8 L × 3/4 P), YLP-87.5 = (1/16 Y × 1/16 L × 7/8 P), Y-L-50: 1/2 Y × 1/2 L. α,β Different letters indicate differences between crossbred males. a–c Different letters indicate differences between crossbred females. p < 0.05. Table 1. Morphometric variables (mean ± s.d.) of boar sperm size and head shape of in different crossbred males and AI doses use in sows. Table 1. Morphometric variables (mean ± s.d.) of boar sperm size and head shape of in different crossbre doses use in sows. 3.2. Morphometric Variables There was an animal effect on the sperm head size variables (p < 0.05; Figure 1; supplementary Table S1). The sperm head size from the Duroc × Pietrain boars was, in fact, larger than the sperm head size of the Pietrain boars. The sperm from the Duroc × Pietrain boar ejaculates had a larger head perimeter (0.5 µm) and head area (1.26 µm2) than the sperm from the Pietrain boar ejaculates (p < 0.05). The Duroc × Pietrain ejaculates Animals 2021, 11, 920 6 of 15 contained longer (0.15 µm) and wider (0.08 µm) sperm than did the Pietrain ejaculates (p < 0.05). There were no differences in the shape of the sperm heads between the boar breeds and the AI semen doses used for females insemination (Table 1). j g p ( μ ) ( μ ) sperm from the Pietrain boar ejaculates (p < 0.05). The Duroc × Pietrain ejaculates con- tained longer (0.15 μm) and wider (0.08 μm) sperm than did the Pietrain ejaculates (p < 0.05). There were no differences in the shape of the sperm heads between the boar breeds and the AI semen doses used for females insemination (Table 1). and the AI semen doses used for females insemination (Table 1). Figure 1. Box and whisker plot of distribution of boar sperm head size morphometric variables. Each box contains the central 50% of the observations and the whisker contains the central 95%. a–f Boxes labelled with different letters indicate differences between boars. p < 0.05. Figure 1. Box and whisker plot of distribution of boar sperm head size morphometric variables. Each box contains the central 50% of the observations and the whisker contains the central 95%. a–f Boxes labelled with different letters indicate differences between boars. p < 0.05. Figure 1. Box and whisker plot of distribution of boar sperm head size morphometric variables. Each box contains the central 50% of the observations and the whisker contains the central 95%. a–f Boxes labelled with different letters indicate differences between boars. p < 0.05. Figure 1. Box and whisker plot of distribution of boar sperm head size morphometric variables. Each box contains the central 50% of the observations and the whisker contains the central 95%. a–f Boxes labelled with different letters indicate differences between boars. p < 0.05. 3.3. Fertility Traits The mean fertility rate was 69.6 ± 21.67%. There were no differences between the male lines for this variable. The Sows inseminated with the Duroc × Pietrain semen had a larger (p < 0.05) total number born per litter (10.53 ± 3.94) than those inseminated with the Pietrain semen (9.72 ± 4.24). In the Duroc × Pietrain crossbreed, the number of piglets born alive (PBA = 9.46 ± 3.70) and piglets born dead (PBD = 0.80 ± 1.11) were higher than those of the Pietrain boars (PBA = 8.89 ± 3.77, PBD = 0.65 ± 0.95; p < 0.05). Litter weight at birth and number of mummies were lower for the Pietrain boars (p < 0.05; Table 2). There was an animal effect on litter size variables and litter weight at birth (p < 0.05; Figure 2; supplementary Table S2). (mean ± s.d.) of litter size and piglet mortality of according to source of boar semen and in Table 2. Fertility variables (mean ± s.d.) of litter size and piglet mortality of according to source of boa different lines of sows. Table 2. Fertility variables (mean ± s.d.) of litter size and piglet mortality of according to source of boar semen and in different lines of sows. Boar Sows Variable Pietrain D × P YLP-50 YLP-75 YLP-87.5 Y-L-50 Total born per litter 9.72 ± 4.24 α 10.53 ± 3.94 β 10.12 ± 3.54 a 10.37 ± 4.03 b 10.01 ± 3.89 a 10.76 ± 4.55 c Piglets born alive 8.89 ± 3.77 α 9.46 ± 3.70 β 8.71 ± 3.39 a 9.26 ± 3.53 b 9.25 ± 3.64 b 9.64 ± 4.30 c Piglets born dead 0.65 ± 0.95 α 0.80 ± 1.11 β 1.13 ± 1.08 d 0.88 ± 1.33 c 0.55 ± 1.00 a 0.77 ± 0.69 b Number of mummies 0.19 ± 0.35 α 0.26 ± 0.64 β 0.28 ± 0.6 b 0.23 ± 0.56 a 0.22 ± 0.51 a 0.35 ± 0.75 c Litter weight at birth 14.23 ± 5.74 α 15.58 ± 5.59 β 13.95 ± 4.75 a 15.44 ± 5.43 c 15.16 ± 5.72 b 15.05 ± 6.27 b s.d.: standard deviation. Y: York, L: Landrace, P: Pietrain, D: Duroc. 3.3. Fertility Traits YLP-50 = ( 1 4 Y × 1 4 L × 1 2 P), YLP-75 = (1/8 Y × 1/8 L × 3/4 P), YLP-87.5 = (1/16 Y × 1/16 L × 7/8 P), Y-L-50: 1/2 Y × 1/2 L. Litter weight at birth (kg). α,β Different letters indicate differences between crossbred males. a–d Different letters indicate differences between crossbred females. p < 0.05. s.d.: standard deviation. Y: York, L: Landrace, P: Pietrain, D: Duroc. YLP-50 = ( 1 4 Y × 1 4 L × 1 2 P), YLP-75 = (1/8 Y × 1/8 L × 3/4 P), YLP-87.5 = (1/16 Y × 1/16 L × 7/8 P), Y-L-50: 1/2 Y × 1/2 L. Litter weight at birth (kg). α,β Different letters indicate differences between crossbred males. a–d Different letters indicate differences between crossbred females. p < 0.05. Fertility traits such as total born per litter (10.76 ± 4.55), piglets born alive (9.64 ± 4.30), and the number of mummies (0.35 ± 0.75; p < 0.05) were higher in the Y-L-50 crossbred females than in the other lines. The YLP-87.5 crossbred sows presented a lower (p < 0.05) number of piglets born dead (0.55 ± 1.00). The YLP-50 sows had fewer piglets born alive (p < 0.05). The litter weight at birth was higher in the YLP-75 sows (Table 2). 3.2. Morphometric Variables AI: artificial insemination, s.d.: standard deviation, length (L, µm), width (W, µm), area (A, µm2), perimeter (P, µm), ellipticity (L/W), rugosity (4πA/P2), elongation ((L −W)/(L + W)), regularity (πLW/4 A). Y: York, L: Landrace, P: Pietrain, D: Duroc. YLP-50 = ( 1 4 Y × 1 4 L × 1 2 P), YLP-75 = (1/8 Y × 1/8 L × 3/4 P), YLP-87.5 = (1/16 Y × 1/16 L × 7/8 P), Y-L-50: 1/2 Y × 1/2 L. α,β Different letters indicate differences between crossbred males. a–c Different letters indicate differences between crossbred females. p < 0.05. g y ( ), g (( ) ( )), g y ( ) , , , ( 4 1 4 L × 1 2 P), YLP-75 = (1/8 Y × 1/8 L × 3/4 P), YLP-87.5 = (1/16 Y × 1/16 L × 7/8 P), Y-L-50: 1/2 Y × 1/2 L. α,β Different letters indicate differences between crossbred males. a–c Different letters indicate differences between crossbred females. p < 0.05. 7 of 15 Animals 2021, 11, 920 The indices used to evaluate the differences in the dimension of sperm from ejaculates indicate differences between the males in head size. The percentage of variation of the morphometric traits between the ejaculates with the lowest and highest sperm head sizes were 5.02% for length, 6.17% for width, 5.31% for the area, 4.09% for perimeter, 9.76% for ellipticity, and 3.90% for rugosity. The sperm doses used on the YLP-75 sows evaluated had a larger sperm head size (p < 0.05) than the sperm doses on the YLP-50 sows (0.44 µm2 larger on average). There were no differences (p > 0.05) for the sperm head size variables between the YLP-87.5 and Y-L-50 female lines (Table 1). 3.4. Sub-Population Structure Results from the principal component analysis revealed two PC factors. PC1 was referred to as “head shape,” or long, stretched, tubiform cells, which were represented by the elongation, ellipticity, head length, and rugosity (in reverse order). The larger eigenvector corresponded to elongation (0.47). PC2 was represented by the sperm head area, perimeter, and width, and was named “head size.” It is mainly related to the head area (Eigenfactor = 0.55) (Table 3). These results indicate that the shape and head size of sperm have a relatively greater effect (86.2%) on the total variance explained, and the elongation and ellipticity showed a maximum correlation (Figure 3). 8 of 15 Animals 2021, 11, 920 A i l 2021 11 Figure 2. Box plot of distribution of litter size and other litter variables for the different boars. Each box contains the central 50% of the observations, and the whisker contains the central 95%. a–f Boxes labelled with different letters indicate differ- ences between boars. p < 0.05. Figure 2. Box plot of distribution of litter size and other litter variables for the different boars. Each box contains the central 50% of the observations, and the whisker contains the central 95%. a–f Boxes labelled with different letters indicate differences between boars. p < 0.05. Figure 2. Box plot of distribution of litter size and other litter variables for the different boars. Each box contains the central 50% of the observations, and the whisker contains the central 95%. a–f Boxes labelled with different letters indicate differ- ences between boars. p < 0.05. Figure 2. Box plot of distribution of litter size and other litter variables for the different boars. Each box contains the central 50% of the observations, and the whisker contains the central 95%. a–f Boxes labelled with different letters indicate differences between boars. p < 0.05. y p ( ), p g ( ), and the number of mummies (0.35 ± 0.75; p < 0.05) were higher in the Y-L-50 crossbred females than in the other lines. The YLP-87.5 crossbred sows presented a lower (p < 0.05) number of piglets born dead (0.55 ± 1.00). The YLP-50 sows had fewer piglets born alive (p < 0.05). The litter weight at birth was higher in the YLP-75 sows (Table 2). 3.4. Sub-Population Structure Results from the principal component analysis revealed two PC factors. and ellipticity showed a maximum correlation (Figure 3). Var Exp: variance explained in each PC. Total variance explained = 86.2%. * Expresses the more important variables in each PC. Only eigenvectors > 0.4 are presented. 3.4. Sub-Population Structure PC1 was re- ferred to as “head shape,” or long, stretched, tubiform cells, which were represented by the elongation, ellipticity, head length, and rugosity (in reverse order). The larger eigen- vector corresponded to elongation (0.47). PC2 was represented by the sperm head area, perimeter, and width, and was named “head size.” It is mainly related to the head area (Eigenfactor = 0.55) (Table 3). These results indicate that the shape and head size of sperm have a relatively greater effect (86.2%) on the total variance explained, and the elongation and ellipticity showed a maximum correlation (Figure 3). Table 3. Eigenvectors of principal components (PCs) * for the morphometric variables of boar sperm size and head shape. Variable PC1 PC2 Length 0.44 Width 0.54 Area 0.55 Perimeter 0.45 Ellipticity 0.46 Rugosity −0.46 Elongation 0.47 Regularity Var Exp 50.6 35.6 Var Exp: variance explained in each PC. Total variance explained = 86.2%. * Expresses the more important variables in each PC. Only eigenvectors > 0.4 are presented. y p p g and the number of mummies (0.35 ± 0.75; p < 0.05) were higher in the Y-L-50 crossbred females than in the other lines. The YLP-87.5 crossbred sows presented a lower (p < 0.05) number of piglets born dead (0 55 ± 1 00) The YLP 50 sows had fewer piglets born alive Table 3. Eigenvectors of principal components (PCs) * for the morphometric variables of boar sperm size and head shape. and ellipticity showed a maximum correlation (Figure 3). Var Exp: variance explained in each PC. Total variance explained = 86.2%. * Expresses the more important variables in each PC. Only eigenvectors > 0.4 are presented. 9 of 15 9 of 15 Animals 2021, 11, 920 Animals 2021, 11, x ure 3. Distribution of morphometric variables after principal component analysis. Each blue point represents an ejaculate. Fo rm sub-population were identified from forty ejaculates. Figure 3. Distribution of morphometric variables after principal component analysis. Each blue point represents an ejaculate. Four sperm sub-population were identified from forty ejaculates. ure 3. Distribution of morphometric variables after principal component analysis. Each blue point represents an ejaculate. Fo m sub-population were identified from forty ejaculates. Figure 3. Distribution of morphometric variables after principal component analysis. Each blue point represents an ejaculate. Four sperm sub-population were identified from forty ejaculates. Table 3. 3.4. Sub-Population Structure Eigenvectors of principal components (PCs) * for the morphometric variables of boar sperm size and head shape. Variable PC1 PC2 Length 0.44 Width 0.54 Area 0.55 Perimeter 0.45 Ellipticity 0.46 Rugosity -0.46 Elongation 0.47 Regularity Var Exp 50.6 35.6 Four sperm subpopulations (SPs) with different morphometric patterns were obtained from the morphometry traits. Summary data for the morphometrics and SP fertility are presented in Table 4. They can be summarized as follows: sub-population 1 (SP1) included ejaculates with lower values for ellipticity (1.83 ± 0.03) and the widest heads (4.69 ± 0.06). This population represented 17.5% of the total sperm from ejaculates. Sub-population 2 (SP2) contained the highest values for head area and perimeter, with values of 35.77 ± 1.24, and 24.48 ± 0.44, respectively. About 42.5% of the spermatozoa from ejaculates were assigned to this sub-population. Sub-population 3 (SP3) included 20.0% of the sperm from all ejaculates, and was represented by shorter length (8.49 ± 0.22), with a smaller area (34.16 ± 1.07) and head perimeter (23.72 ± 0.51) values. This population had intermediate head shape values indicated by the ellipticity, elongation, and regularity values. Sub- population 4 (SP4) contained 20.0% of the spermatozoa from the total ejaculate population, and these spermatozoa had the highest values for head length (8.88 ± 0.13), ellipticity (2.03 ± 0.02), and elongation (0.34 ± 0.01) (Table 4). Var Exp: variance explained in each P important variables in each PC. Only 3.5. Predictive Capacity of Fertility Variable SP1 SP2 SP3 SP4 Proportion of all ejaculates (%) 17.50 42.50 20.00 20.00 Length 8.57 ± 0.13 b 8.81 ± 0.18 a 8.49 ± 0.22 b 8.88 ± 0.13 a Width 4.69 ± 0.06 a 4.58 ± 0.07 b 4.43 ± 0.04 c 4.36 ± 0.07 d Area 35.63 ± 0.69 a 35.77 ± 1.24 a 34.16 ± 1.07 b 34.65 ± 0.92 b Perimeter 24.04 ± 0.27 b 24.48 ± 0.44 a 23.72 ± 0.51 b 24.20 ± 0.32 b Ellipticity 1.83 ± 0.03 c 1.93 ± 0.04 b 1.91 ± 0.05 b 2.03 ± 0.02 a Rugosity 0.78 ± 0.01 a 0.76 ± 0.01 b 0.76 ± 0.01 b 0.75 ± 0.01 c Elongation 0.29 ± 0.01 c 0.32 ± 0.01 b 0.31 ± 0.01 b 0.34 ± 0.01 a Regularity 0.88 ± 0.01 a 0.88 ± 0.01 a 0.87 ± 0.00 b 0.88 ± 0.01 a s.d.: standard deviation. Number of ejaculates = 40. Length (L, µm), width (W, µm), area (A, µm2), perimeter (P, µm), ellipticity (L/W), rugosity (4πA/P2), elongation ((L −W)/(L + W)), regularity (πLW/4A). a–d Different letters indicate differences between ejaculate sub-populations for morphometric variables. p < 0.05. Table 5. Cut-off values of morphometric sperm variables significantly related to litter size variables calculated from receiver operating characteristic (ROC) curves. Variable Cut-Off Value Sensitivity (%) Specificity (%) Area ROC p-Value Total born per litter Area 35.18 67.19 27.08 0.54 0.11 Perimeter 24.26 68.66 28.26 0.55 0.10 Piglets born alive Area 35.18 67.19 27.08 0.53 0.17 Perimeter 24.26 68.66 28.26 0.54 0.13 Ellipticity 1.94 67.55 26.45 0.54 0.13 Piglets born dead Regularity 0.88 73.02 32.19 0.56 0.04 Number of mummies Length 8.71 71.74 31.34 0.59 0.02 Ellipticity 1.92 73.08 33.62 0.59 0.02 Elongation 0.31 73.08 33.62 0.60 0.02 Length (L, µm), width (W, µm), area (A, µm2), perimeter (P, µm), ellipticity (L/W), rugosity (4πA/P2), elongation ((L −W)/(L + W)), regularity (πLW/4A). Table 4. Morphometry of sperm heads, head shape, and fertility variables (mean ± s.d.) of the four ejaculate sub-populations (SPs) defined from boar semen samples. Var Exp: variance explained in each P important variables in each PC. Only 3.5. Predictive Capacity of Fertility Variable SP1 SP2 SP3 SP4 Proportion of all ejaculates (%) 17.50 42.50 20.00 20.00 Length 8.57 ± 0.13 b 8.81 ± 0.18 a 8.49 ± 0.22 b 8.88 ± 0.13 a Width 4.69 ± 0.06 a 4.58 ± 0.07 b 4.43 ± 0.04 c 4.36 ± 0.07 d Area 35.63 ± 0.69 a 35.77 ± 1.24 a 34.16 ± 1.07 b 34.65 ± 0.92 b Perimeter 24.04 ± 0.27 b 24.48 ± 0.44 a 23.72 ± 0.51 b 24.20 ± 0.32 b Ellipticity 1.83 ± 0.03 c 1.93 ± 0.04 b 1.91 ± 0.05 b 2.03 ± 0.02 a Rugosity 0.78 ± 0.01 a 0.76 ± 0.01 b 0.76 ± 0.01 b 0.75 ± 0.01 c Elongation 0.29 ± 0.01 c 0.32 ± 0.01 b 0.31 ± 0.01 b 0.34 ± 0.01 a Regularity 0.88 ± 0.01 a 0.88 ± 0.01 a 0.87 ± 0.00 b 0.88 ± 0.01 a s.d.: standard deviation. Number of ejaculates = 40. Length (L, µm), width (W, µm), area (A, µm2), perimeter (P, µm), ellipticity (L/W), rugosity (4πA/P2), elongation ((L −W)/(L + W)), regularity (πLW/4A). a–d Different letters indicate differences between ejaculate sub-populations for morphometric variables. p < 0.05. values of morphometric sperm variables significantly related to litter size variables calculated from receiver cteristic (ROC) curves. Table 5. Cut-off values of morphometric sperm variables significantly related to litter size variables calculated from receiver operating characteristic (ROC) curves. Variable Cut-Off Value Sensitivity (%) Specificity (%) Area ROC p-Value Total born per litter Area 35.18 67.19 27.08 0.54 0.11 Perimeter 24.26 68.66 28.26 0.55 0.10 Piglets born alive Area 35.18 67.19 27.08 0.53 0.17 Perimeter 24.26 68.66 28.26 0.54 0.13 Ellipticity 1.94 67.55 26.45 0.54 0.13 Piglets born dead Regularity 0.88 73.02 32.19 0.56 0.04 Number of mummies Length 8.71 71.74 31.34 0.59 0.02 Ellipticity 1.92 73.08 33.62 0.59 0.02 Elongation 0.31 73.08 33.62 0.60 0.02 Length (L, µm), width (W, µm), area (A, µm2), perimeter (P, µm), ellipticity (L/W), rugosity (4πA/P2), elongation ((L −W)/(L + W)), regularity (πLW/4A). Var Exp: variance explained in each P important variables in each PC. Only 3.5. Predictive Capacity of Fertility Four sperm subpopulations (SPs) with different morphometric patterns were ob- tained from the morphometry traits. Summary data for the morphometrics and SP fertility are presented in Table 4. They can be summarized as follows: sub-population 1 (SP1) in- cluded ejaculates with lower values for ellipticity (1.83 ± 0.03) and the widest heads (4.69 ± 0.06). This population represented 17.5% of the total sperm from ejaculates. Sub-popu- lation 2 (SP2) contained the highest values for head area and perimeter, with values of 35.77 ± 1.24, and 24.48 ± 0.44, respectively. About 42.5% of the spermatozoa from ejaculates i d t thi b l ti S b l ti 3 (SP3) i l d d 20 0% f th The morphometric variables of the sperm with significant results in the ROC curve analysis are presented in Table 5. Sperm head length, ellipticity, elongation, and regularity showed significant, albeit limited, predictive capacity on the litter size variables (range: 0.56–0.60 AUC). Similarly, the sperm subpopulations showed limited predictive capacity on the litter size variables (data not shown). Cut-off points, with their sensitivities and specificities, are also presented in Table 5. Among the sperm shape variables, the best cut-off points to identify ejaculates with low fertility potential in relation to the number of mummies were 8.71 µm for head length, 1.92 for ellipticity, and 0.31 for elongation. Animals 2021, 11, 920 10 of 15 Table 4. Morphometry of sperm heads, head shape, and fertility variables (mean ± s.d.) of the four ejaculate sub-populations (SPs) defined from boar semen samples. 4. Discussion The head shape also can affect the hydrodynamics of the spermatozoa, as sperm with elongated heads can move faster than those with more elliptical heads [51], and elongated heads have a relationship with male fertility rates [52]. Some authors have suggested that sperm with higher ellipticity values (head length/width) presented a lower progressiveness [53]. Our results indicate that there was a greater progressiveness in the Duroc × Pietrain boars than in the Pietrain boars, but no differences were found between the ellipticity of these two male lines. In humans, it has been observed that morphologically normal spermatozoa showed a faster acrosome reaction than the tapered, large, and small-headed types of spermatozoa [54,55]. Thus, morphological structure and functionality have a close relationship [12,55]. p g y p The outcome of this work indicated that crossbreeding influenced the head size and shape of boar sperm cells. The sperm from the Pietrain-line ejaculates had shorter and narrower heads, with a smaller head area and perimeter than sperm from the Duroc × Pietrain line. There are genetic factors to modeling shape and sperm head size [6,7,41]. The present study shows differences between the genetic lines of males for sperm head size. In rams, the distribution of sperm sub-populations had been associated with intra- and inter- male differences [56]. While determining that morphometric differences occur between two male lines is biologically notable, it can be difficult to clearly connect these morphometric measurements to the boar semen used in assessments of sow fertility when artificially inseminated. In our study, this was most evident when the head shape morphometry was reviewed. Studies in humans demonstrated that the size and normal shape of sperm heads affect functions, such as the acrosome reaction [57] and the binding zone to the zona pellucida of the oocyte [58], which could affect the potential fertility of the male [59]. p y p y Sperm head size has also been related to fertility. Accordingly, variability in sperm head size has been correlated with variation in the chromatin structure of the cell nu- cleus [23]. Other authors have suggested that minor variations in the shape of sperm heads can be associated with changes in the chromatin structure in the spermatozoa nucleus, which can result in reduced fertility [60]. 4. Discussion Fertility is a complex trait in which a wide range of sperm characteristics may be involved [40]. These fertility parameters can be expressed by several variables with dis- tributions that can be continuous—where any value in the range of the distribution is possible (farrowing rate), or discrete—where only specific values can be returned (piglets born alive). The results of the present study indicate that the Pietrain x Duroc crossbreed had larger sperm heads than the Pietrain boars. Similar results have been described in the larger spermatozoa head length, area, and perimeter of crossbred boars, as compared with purebred boars [41]. The morphometric variables related to the head size of the sperm were also significantly different among the individual Duroc × Pietrain or Pietrain boars, and between the Duroc × Pietrain and Pietrain boars. In stallions, differences in sperm head size within breeds [42,43] and between stallions [44] have been reported. Moreover, our results also showed that the sperm head size increased as the sperm concentration of the ejaculate increased, in agreement with previous studies in pigs [45]. In other species, such as dogs, it has been found that sperm concentration can influence the head size dimensions [46]. The head size and shape of the sperm may affect their motility [47] and Animals 2021, 11, 920 11 of 15 11 of 15 fertilization capacity [45]. Overall, the percentage of motile sperm presented by the Pietrain males was lower than that of the Duroc × Pietrain boars. Sperm length has been positively correlated with motility [48], and an inverse rela- tionship has been described between sperm length and the effective time of maintenance of sperm cell motility, and thus its capacity for oocyte fertilization [49]. In pigs, lower- fertility boars also showed more elongated sperm heads [22]. Our study reports that boars with a larger litter size had significantly less elongated spermatozoa. Nevertheless, the mortality of piglets was greater in these males. This observation is in agreement with a previous study of Pietrain boars, in which the sperm heads of high-fertility boars were less elongated and smaller than those of lower fertility boars [22]. Although the sperm head morphometric parameters explained a variation in the litter size of 7.7%, these results are partially explained by factors affecting the female [50]. 4. Discussion When the multivariate analysis was applied to the ejaculates analyzed for fertility variables and the spermatozoa size and head shape, discrete sub-populations (cluster centers) were generated based on the set number of two standardized principal components. In the analysis of sub-populations of sperm head dimensions, significant differences in the values for length, width, area, and perimeter were found between the sub-populations of Duroc × Pietrain and Pietrain boar sperm heads. The differences in the cluster populations were analyzed across all boar semen morphometric variables for size, head shape, and semen doses used in AI. Several studies have described the presence of different sub-populations within an ejaculate [11,19,61–67]. These sub-populations may be affected by external factors associated with semen, such as extender type or species [68,69]. Even the sperm sub-population distribution can vary depending on the statistical multivariate procedure used [70]. In boars, different morpho- metric sperm sub-populations were found and were categorized according to head size as large, small elongated, and small round, and these variables could have a functional involvement [31]. Past studies have associated sperm head morphometry and fertility variables in boars [22], male goats [26,71], stallions [27], rams [52], and rabbits [72], in which Animals 2021, 11, 920 12 of 15 12 of 15 subsequent fertility was reduced with the lowest head size variables. When comparing male lines, we found that that boars with lower sperm head sizes generally had higher litter sizes. These findings were verified when we analyzed the sperm sub-populations in their ejaculate. The sperm sub-populations SP2 and SP3 presented more minor variations amongst each other than the sub-populations SP1 and SP4 did. Thus, there was less unifor- mity regarding the sperm head size variables between SP1–SP4, which suggests the idea that possibly the sperm competition between these sub-populations supports low levels of sperm competition, which could result in poor semen quality, as has been described in several studies on rodents [73,74]. 5. Conclusions We have shown that morphometric analysis of boar ejaculates reveals morphometri- cally separate populations. Differences between sub-population sperm head sizes were displayed. Sperm morphometric variables may have a predictive capacity on the litter size variables. Clustering the sperm into sub-populations did not have a predictive capacity on the litter size variables. Supplementary Materials: The following are available online at https://www.mdpi.com/2076-261 5/11/4/920/s1, Table S1: morphometric variables (mean ± SEM) of the sperm size and sperm head shape of individual boars. Table S2: fertility variables (mean ± SEM) of litter size and mortality of piglets born after artificial insemination with the semen of different boars. Author Contributions: Conceptualization, A.V., E.R.S.R., J.L.Y. and C.S.; methodology, V.B., A.V.; software, V.B.; validation, V.B.; formal analysis, A.V., M.C.; investigation, V.B., A.V.; resources, A.V.; data curation, A.V., M.C.; writing—original draft preparation, A.V., V.B.; writing—review and editing, V.B., E.R.S.R., J.L.Y., A.V.; visualization, E.R.S.R., C.S., J.L.Y., A.V.; supervision, A.V.; project administration, A.V.; funding acquisition, A.V. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Fundación para el Fomento y Promoción de la Investi- gación y Transferencia de Tecnología Agropecuaria de Costa Rica (FITTACORI) and the Costa Rica Institute of Technology (Vice-Chancellor’s office of Research and Extension; VIE (Vicerrectoría de investigación y Extensión); Project 5402-2151-1015). The funders had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript. Institutional Review Board Statement: The study was conducted according to the approval of the ethical principles of the Committee of Centro de Investigación y Desarrollo de la Agricultura Sostenible para el Trópico Húmedo at the Costa Rica Institute of Technology (CIDASTH-ITCR) according to Section 08/2020, article 1.0, DAGSC-100-2020. Data Availability Statement: The data presented in this study are available within the article and/or its Supplementary Materials. Acknowledgments: The authors thank the Costa Rica Institute of Technology (ITCR) and the Fun- dación para el Fomento y Promoción de la Investigación y Transferencia de Tecnología Agropecuaria de Costa Rica (FITTACORI) for financing this study. The authors are grateful to the staff of the Agropecuaria Los Sagitarios S.A. farm for supplying the boar ejaculates. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 1. Gadea, J. Sperm factors related to in vitro and in vivo porcine fertility. Theriogenology 2005, 63, 431–444. [CrossRef] [PubMed] 2. Tsakmakidis, I.A.; Lymberopoulos, A.G.; Khalifa, T.A.A. Relationship between sperm quality traits and field-fertility of porcine semen. J. Vet. Sci. 2010, 11, 151–154. [CrossRef] [PubMed] 3. Rodriguez, A.L.; van Soom, A.; Arsenakis, I.; Maes, D. Boar management and semen handling factors affect the quality of boar extended semen. Porc. Health Manag. 2017, 3, 1–12. 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Reclaiming the systems approach to paediatric safety
Archives of disease in childhood
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Authors 1. C Ronny Cheung (corresponding author) • Evelina London Children’s Hospital, Guy’s & St Thomas’ NHS Foundation Trust, Westminster Bridge Rd, London, SE1 7EH, UK 2. Damian Roland • SAPPHIRE Group, Health Sciences, Centre for Medicine, University of Leicester, Leicester, UK • Paediatric Emergency Medicine Leicester Academic (PEMLA) Group, Children’s Emergency Department, Leicester Hospitals, Leicester, UK 3. Peter Lachman • International Society for Quality in Healthcare, Dublin, Republic of Ireland 1. C Ronny Cheung (corresponding author) 1. C Ronny Cheung (corresponding author) • Evelina London Children’s Hospital, Guy’s & St Thomas’ NHS Foundation Trust, Westminster Bridge Rd, London, SE1 7EH, UK 2. Damian Roland • SAPPHIRE Group, Health Sciences, Centre for Medicine, University of Leicester, Leicester, UK • Paediatric Emergency Medicine Leicester Academic (PEMLA) Group, Children’s Emergency Department, Leicester Hospitals, Leicester, UK 3. Peter Lachman • International Society for Quality in Healthcare, Dublin, Republic of Ireland y g ( ) • Evelina London Children’s Hospital, Guy’s & St Thomas’ NHS Foundation Trust, Westminster Bridge Rd, London, SE1 7EH, UK 2. Damian Roland • Evelina London Children’s Hospital, Guy’s & St Thomas’ NHS Foundation Trust, Westminster Bridge Rd, London, SE1 7EH, UK • SAPPHIRE Group, Health Sciences, Centre for Medicine, University of Leicester, Leicester, UK • Paediatric Emergency Medicine Leicester Academic (PEMLA) Group, Children’s Emergency Department, Leicester Hospitals, Leicester, UK 3. Peter Lachman • International Society for Quality in Healthcare, Dublin, Republic of Ireland Reclaiming the systems approach to paediatric safety Reclaiming the systems approach to paediatric safety Abstract: Over the past 20 years there has been a rapid growth in the understanding of the complexities of patient safety. The science of patient safety has learnt from other complex industries, and has started to view safety science as a system problem, rather than relating to individual errors in single processes or by individual people. However, with the development of sophisticated safety tools, emphasis has been placed on individual responsibility for interventions, aimed at reducing defects in a presumed linear or step-wise process rather than within the broader context of the system in which these processes occur. This paper will outline a broader view that focuses paediatric safety as a system issue, rather than simply the sum of its constituent tools. Introduction Prior to the emergence of the patient safety movement as a distinct science, it was assumed that the safety of patients was an outcome of good professional acumen, and that if healthcare providers could individually perform well then their patients would remain safe at all times. It is now 20 years since the publication of To Err is Human,1 the first major review of healthcare safety in the USA. In the United Kingdom, the publication Organisation with a Memory2 in 2000 supported the view that patient safety required a wider system approach. Both documents reframed safety and error in healthcare as an It is now 20 years since the publication of To Err is Human,1 the first major review of healthcare safety in the USA. In the United Kingdom, the publication Organisation with a Memory2 in 2000 supported the view that patient safety required a wider system approach. Both documents reframed safety and error in healthcare as an organisational or system issue rather than one of individual error, whether of omission or of commission. Over the past 20 years, there has been major progress in the understanding of patient safety and the complexity of the systems involved in providing healthcare. In a recent review of the state of patient safety in 2018, Bates and Singh conclude that “Highly effective interventions have since been developed and adopted for hospital-acquired infections and medication safety, although the impact of these interventions varies because of their inconsistent implementation and practice.”3 organisational or system issue rather than one of individual error, whether of omission or of commission. Abstract: Over the past 20 years, there has been major progress in the understanding of patient safety and the complexity of the systems involved in providing healthcare. In a recent review of the state of patient safety in 2018, Bates and Singh conclude that “Highly effective interventions have since been developed and adopted for hospital-acquired infections and medication safety, although the impact of these interventions varies because of their inconsistent implementation and practice.”3 Within paediatrics, the National Patient Safety Agency made the first attempt in the United Kingdom to detail the extent of healthcare-derived harm among children4. The problems identified remain a challenge – namely communication, deterioration, Within paediatrics, the National Patient Safety Agency made the first attempt in the United Kingdom to detail the extent of healthcare-derived harm among children4. The problems identified remain a challenge – namely communication, deterioration, delayed or missed diagnosis, infections and medication harm. This is despite well- tested theories and interventions being available for many of these. In this paper, we explore the theories of patient safety and provide principles to tackle the challenge ahead. The evolution of patient safety theories The original approach to patient safety was essentially limited to risk management and review of adverse events. This included the introduction of root cause analysis and failure mode effects analysis, which aimed to understand the causation of harm. Measures of harm such as the Paediatric Global Trigger Tool were developed,5 which provided greater insight into paediatric patient safety and allowed the development of interventions to address single patient issues such as prescribing, pressure ulcers and infection control. Interventions were effective but often slow and reactive, and learning often delayed. The next leap in understanding came from human factors and ergonomics, which originated in engineering and aviation. Human factors acolytes consider safety as part of a complex system of interactions. While acknowledging the differences between the aircraft cockpits and the clinical environment, these theories postulate that improving safety requires a focus on the interactions between humans, and between humans and their working environment. Interventions such as Team STEPS6 or Systems Engineering Initiative for Patient Safety (SEIPS) model focused on the team interaction and culture, and the interplay of the environment, tools and technology and the people involved (both patients and providers) as a means to achieve safe outcomes.7 Building on these theories, the concept of reliability in healthcare was introduced from the study of “High Reliability Organisations (HRO)” in diverse industries such as nuclear power, the military and air traffic control.8,9 Although these organisations are general highly complex, with many interdependencies and working to tight time pressures, safety remains core to their business.10 Rather than reacting to events, they generate new safety solutions proactively. They incorporate human factors and ergonomics to design their processes and systems, to remain error free. Healthcare’s adoption of high reliability principles - that is, aiming for minimal defects or scope for error through development of standardised tools, processes and interventions to prevent predictable adverse events – led to the introduction of care bundles and standardisation of care pathways, which have achieved considerable success, for example in the elimination of central line associated bloodstream infections.11,12 nuclear power, the military and air traffic control.8,9 Although these organisations are general highly complex, with many interdependencies and working to tight time pressures, safety remains core to their business.10 Rather than reacting to events, they generate new safety solutions proactively. The evolution of patient safety theories They incorporate human factors and ergonomics to design their processes and systems to remain error free nuclear power, the military and air traffic control.8,9 Although these organisations are general highly complex, with many interdependencies and working to tight time pressures, safety remains core to their business.10 Rather than reacting to events, Healthcare’s adoption of high reliability principles - that is, aiming for minimal defects or scope for error through development of standardised tools, processes and interventions to prevent predictable adverse events – led to the introduction of care bundles and standardisation of care pathways, which have achieved considerable success, for example in the elimination of central line associated bloodstream infections.11,12 Initially, the development of discrete, proven safety interventions brought the promise of reliable improvement in safety that could be replicated everywhere, irrespective of setting, provided they were implemented consistently and predictably. But over time, it became clear that this was overly simplistic, and there has since been a move from focusing on individual responsibility, and towards an understanding of safety in the context of the complexity inherent in healthcare. A case in point is the introduction of interventions in adult intensive care across the UK to decrease central line infections, replicating the work of Pronovost in Michigan.10 The programme failed because the concept of context and local environment had not been adequately addressed. It was assumed that the simple roll-out of an intervention proven to work in one setting would achieve the same outcome in another. Dixon-Woods and others have highlighted that implementation of practice occurs through many routes, and simple translation of one intervention to a different clinical environment is unlikely to have identical effects.13 Amalberti and Vincent go further still in outlining the limitations of this linear, “process-defect” approach in healthcare. They argued that healthcare is composed of many interconnected processes of varying complexity, and therefore the context in which clinical care is delivered should be the primary factor in determining the approach required.14 For instance, many highly predictable clinical care processes (e.g. blood transfusions or radiotherapy) should have the goal of being a highly reliable services with clear operating systems, while others such as routine surgery should aim for reliability with some scope for adaptation to changing circumstances. Still others, such as emergency medicine, may need to be even more adaptable, even as they continue to embrace the underlying principles of reliability theory. The evolution of patient safety theories There is now an understanding that the achievement of safety requires a broader lens which encompasses both the clinical process and safety culture and environmental context.15,16 John Launer reflected on the pragmatic application of complexity theory thus: “…in a world where prediction can never be certain, are there nevertheless some general rules that can reduce uncertainty, so that our actions stand a better chance of achieving their intended results?”17 Box 1 illustrates some considerations to challenge the simplistic, linear approach to healthcare safety. Box 1: Dealing with a complex system (from Launer 2018)17 • Resist the temptation to focus on an isolated problem. Instead, look for interconnections within the system. • Look for patterns in the behaviour of a system, not just at events. • Be careful when attributing cause and effect. It’s rarely that simple. Box 1: Dealing with a complex system (from Launer 2018)17 Box 1: Dealing with a complex system (from Launer 2018)17 • Resist the temptation to focus on an isolated problem. Instead, look for interconnections within the system. • Look for patterns in the behaviour of a system, not just at events. • Be careful when attributing cause and effect. It’s rarely that simple. • Keep in mind the system is dynamic and it doesn’t necessarily respond to intended change as predicted Where we are now Although there is a requirement for individual accountability, there is a recognition now that a safe service must go beyond a linear, mechanistic approach and embrace the broader system. This starts with the clinical team as a “clinical micro system” with its own culture and set of processes.18 .Systems theory is a scientifically rigorous approach which incorporates all other theories such as proactive design for safety, using human factors and ergonomics approaches and reliability methodologies, in order to optimise outcomes.7,19 Despite this, there remains an understandable desire to secure evidence to support individual safety interventions that can be easily implemented. We illustrate this potential pitfall, and the importance of a systems approach, with two examples. Understanding systems: the example of Paediatric Early Warning Systems (PEWS) Understanding systems: the example of Paediatric Early Warning Systems (PEWS) The focus on PEWS - structured tools which aggregate an individual patient’s risk of requiring urgent intervention to prevent morbidity or mortality (based on physiological observations such as heart rate, respiratory rate and blood pressure) - is based on evidence that patient lives can be saved by recognising (and reversing) early deterioration in hospital.20 As with many interventions in healthcare, there is often significant delay between the root causes of harm being identified (e.g. delayed recognition of deterioration in hospital) and adequate interventions to address them being implemented.21 One reason for this is the tendency to view solutions as individual interventions and failing to understand that identifying deterioration is a complex and multifactorial exercise.22 It is obviously tempting to implement a focused, defined, and instantly auditable intervention, rather than engaging with the complex, cultural factors within a healthcare system. But this approach ignores the critical factors that determine the performance of PEWS: communication, cultural hierarchy and organisational factors.23 Evaluation of a specific score without consideration of these system factors is therefore at best limited, and at worst, misleading. The differing approaches to the design of PEWS also illustrates the requirement to apply design principles and ergonomics in safety science. There is evidence that score-based PEWS tools (where cumulative scores assigned to vital signs are used to identify thresholds for escalation of care) are subject to significantly greater errors in completion and interpretation than “Track and Trigger” tools (where breaching thresholds for any individual vital signs leads to escalation, obviating the need for adding together numerical scores).24,25,26 This crucial interface between the tool and the humans who interact with it, particularly in highly stressful and busy “live” clinical environments, is too often ignored in studies which simply evaluate the tools from an isolated statistical perspective, based on reviewing clinical notes retrospectively.27 This was emphasised in the EPOCH study, the largest prospective trial of an early warning system in children, in which the “BedsidePEWS” scoring system failed to demonstrate improvement in mortality in live use, despite being previously validated using retrospective clinical notes data.28 Increasingly there is a recognition that research into the efficacy of PEWS requires evaluation of the context within which any single score is used (including the way in which the tools is designed to allow for ergonomic and human factors), rather than simply the score itself. There is also a more fundamental point to consider. Understanding systems: the example of Paediatric Early Warning Systems (PEWS) PEWS appear to have inherent face validity, have been utilised for over 20 years and have spread rapidly.29 Despite this, it is not clear what direct role the scores themselves have had in this, given that inpatient mortality is decreasing across all healthcare systems in any case.30,31 The difficulty for an organisation is that, having introduced PEWS, it is tempting to believe that they have found a solution to the underlying problem. This may fuel the continued roll-out of PEWS with minimal sense-checking, not only around the underlying causative factors, but also the underlying tenets of any adaptive change process needed to implement improvement.32,33 Box 2 – Factors to consider before introducing a paediatric early warning score (from Roland, 2012)33 1. What is the patient group the EWS will be used on? 2. What outcome are you looking to alter? 3. What type of EWS would you like to introduce? 4. Is there a current system you could employ? 5. How will you engage and be responsive to the concerns of the stakeholders? 6. How will you monitor its effect? Box 2 – Factors to consider before introducing a paediatric early warning score (from Roland, 2012)33 1. What is the patient group the EWS will be used on? 2. What outcome are you looking to alter? 3. What type of EWS would you like to introduce? 4. Is there a current system you could employ? 5. How will you engage and be responsive to the concerns of the stakeholders? 6. How will you monitor its effect? Box 2 – Factors to consider before introducing a paediatric early warning score (from Roland, 2012)33 1. What is the patient group the EWS will be used on? 2. What outcome are you looking to alter? 3. What type of EWS would you like to introduce? 4. Is there a current system you could employ? 5. How will you engage and be responsive to the concerns of the stakeholders? 6. How will you monitor its effect? The importance of safety culture: Safety Huddles As more technical interventions for patient safety were developed, the quest for transferability means the experience of PEWS has been mirrored elsewhere. As noted previously, a key component of any success is the understanding of the context and the culture of the organisation, clinical team and the individual. Safety huddles amply demonstrate this principle.34,35 This safety tool exploits the concept of situational awareness, which acts on many levels and applies not only to the actions of individual staff with patients, but to the co-ordination of multiple hospitals by a senior management team. Safety huddles bring together multiple staff, of different specialty and grade, to assess risk and formulate plans for a given clinical area. Huddles have been demonstrated to have an impact on the outcomes of children but, like PEWS, risk being seen as on off-the-shelf solution that can be delivered in any setting.36 The evidence from the Situation Awareness for Everyone (SAFE) national programme of huddle implementation in paediatric departments in the UK is clear: while a huddle may allow information to be exchanged in flattened hierarchical fashion, this will only be effective if the organisation genuinely espouses the underlying principles.37 It would be perfectly possible, for instance, to undertake huddles which were no different to the more traditional “command-and-control” model, with one individual dictating the flow of conversation. The effectiveness of huddles, and that of other safety initiatives (such as focused handover tools e.g. SBAR), is entirely dependent on the personnel involved understanding the underlying principle and rationale for use, rather than simply enforced on a reluctant workforce in the form of yet another change endeavour. Safety tools in context Network learning has demonstrated that organisations can improve their safety culture by working together and by learning from each other at scale.38 The key insight is that it is not only the technical tools that are important, but rather the beliefs and attitudes of the clinical teams. Indeed, even the most perfect technical safety tool will fail if poorly applied, in an unreceptive environment. Safety science should keep focused on examining the factors that contribute to a high performing system in the round (complex and “dirty” though that may be) rather than concentrating too narrowly on individual tools, which might seem easier to evaluate but with much less meaningful results if done in isolation. We would rightly be accused of poor medicine if we initiated antihypertensive therapies (such as diet, exercise or medication) for our patients without exploring the patient’s circumstances as a whole - their comorbidities, their family support, their social and educational background. Nor, as clinicians and scientists, are we naïve enough to believe that we can extrapolate drug trial outcomes to clinical outcomes without viewing them through the lens of individual patient characteristics. In clinical practice, the treatment of tuberculosis with multiple antibiotic therapy, while proven to be biologically efficacious in clinical trials, did not always work as intended, with negative consequences for individual patients and more drastic ones for the wider population through the development of drug resistance. Of course, there could be no therapy at all without effective drugs. But human and behavioural factors (such as family support, social stigma, or the limits of human memory and routines) were critical to the success of the treatment regime, leading to the introduction of directly observed therapy which was the key to unlocking the theoretical benefit of these treatments.39 Conclusion Conclusion Evaluating PEWS, huddles, electronic prescribing or other tools in isolation (or worse still, simply by running historical databases to test statistical significance of individual tools in vitro) rather than as part of a greater system manned by human beings, subject to cultural and behavioural influences, risks falling into the same trap as those initial pioneers in tuberculosis. Just as we have all moved from an organ- or treatment- specific model of patient care towards a holistic, multidisciplinary model for treating our patients, so must we move back towards understanding safety as a complex interconnected whole, rooted in the culture and environment in which the tools act. 1 Corrigan JM, Kohn LT, Donaldson MS editors. To err is human: building a safer health system. Washington (DC), USA: National Academies Press;1999. 1 Corrigan JM, Kohn LT, Donaldson MS editors. To err is human: building a safer health system. Washington (DC), USA: National Academies Press;1999. 2 Donaldson L. Organisation with a memory. Department of Health, England. 2000. 2 Donaldson L. Organisation with a memory. Department of Health, England. 2000. https://webarchive.nationalarchives.gov.uk/20130105144251/http://www.dh.gov.uk/pr Safety tools in context Future evaluations of safety interventions must take into account wider human and system factors which inevitably affect their performance in real life. Paediatricians as clinicians must also take a lead in improving the safety of the care they deliver on a systems basis. This means measuring harm and adverse events in real time, studying processes in their clinical team or microsystem using a human factors approach and actively fostering a culture of safety. Much progress has been achieved over the past twenty years. Embracing the understanding of systems rather than a linear model of safety and improvement, allied with the potential of health informatics and technology40 will be critical if we are to move paediatric safety to the next level. 2 Donaldson L. Organisation with a memory. Department of Health, England. 2000. https://webarchive.nationalarchives.gov.uk/20130105144251/http://www.dh.gov.uk/pr od_consum_dh/groups/dh_digitalassets/@dh/@en/documents/digitalasset/dh_4065 086.pdf Accessed January 3 2019. od_consum_dh/groups/dh_digitalassets/@dh/@en/documents/digitalasset/dh_4065 086.pdf Accessed January 3 2019. 3 Bates D, Singh H. Two Decades Since To Err Is Human: An Assessment of Progress And Emerging Priorities in Patient Safety. Health Affairs, 2018: 37(11):1736–1743 4 National Patient Safety Agency. Review of Patient Safety for Children and young people. London, UK. 2009. Crown Stationery.. 4 National Patient Safety Agency. Review of Patient Safety for Children and young people. London, UK. 2009. Crown Stationery.. 5 Chapman SM, Fitzsimons J, Davey N, et al. Prevalence and severity of patient harm in a sample of UK-hospitalised children detected by the Paediatric Trigger Tool BMJ Open 2014;4:e0050 66. doi: 10.1136/bmjopen-2014-005066 6 Team Steps. Agency for Healthcare Research and Quality https://www.ahrq.gov/teamstepps/index.html . Accessed 3 January 2019. 7 Carayon P, Schoofs Hundt A, Karsh BT et al. Work system design for patient safety: the SEIPS model. Qual Saf Health Care 2006;15:i50–i58. doi:10.1136/qshc.2005.015842 safety: the SEIPS model. Qual Saf Health Care 2006;15:i50–i58. 8 Weick KE, Sutcliffe KM. Managing the unexpected. 2007. Jossey-Bass, Hoboken, NJ, USA. 8 Weick KE, Sutcliffe KM. Managing the unexpected. 2007. Jossey-Bass, Hoboken, NJ, USA. NJ, USA. 9 Chassin MR, Loeb JM. High-reliability health care: getting there from here. Millbank Quarterly. 2013 Sep 91(3): 459-490 10 Hudson P. Applying the lessons of high risk industries to health care. Qual Saf Health Care 2003;12(Suppl 1):i7–i1 11 Pronovost P, Needham D, Berenholtz S, et al. An intervention to decrease catheter-related bloodstream infections in the ICU. New England Journal of Medicine. 2006; 355(26):2725-2732 12 Lachman P, Yuen, S. Using care bundles to prevent infection in neonatal and paediatric ICUs Current Opinion in Infectious Diseases. 2009;22(3):224–228 9 Chassin MR, Loeb JM. High-reliability health care: getting there from here. Millbank Quarterly. 2013 Sep 91(3): 459-490 9 Chassin MR, Loeb JM. High-reliability health care: getting there from here. Millbank Quarterly. 2013 Sep 91(3): 459-490 10 Hudson P. Applying the lessons of high risk industries to health care. Qual Saf Health Care 2003;12(Suppl 1):i7–i1 11 Pronovost P, Needham D, Berenholtz S, et al. An intervention to decrease catheter-related bloodstream infections in the ICU. New England Journal of Medicine. 2006; 355(26):2725-2732 12 Lachman P, Yuen, S. Using care bundles to prevent infection in neonatal and paediatric ICUs Current Opinion in Infectious Diseases. 2009;22(3):224–228 11 Pronovost P, Needham D, Berenholtz S, et al. An intervention to decrease catheter-related bloodstream infections in the ICU. New England Journal of Medicine. 2006; 355(26):2725-2732 12 Lachman P, Yuen, S. od_consum_dh/groups/dh_digitalassets/@dh/@en/documents/digitalasset/dh_4065 086.pdf Accessed January 3 2019. Using care bundles to prevent infection in neonatal and paediatric ICUs Current Opinion in Infectious Diseases. 2009;22(3):224–228 12 Lachman P, Yuen, S. Using care bundles to prevent infection in neonatal and paediatric ICUs Current Opinion in Infectious Diseases. 2009;22(3):224–228 13 Dixon-Woods M, Leslie M, Tarrant C, Bion J. Explaining Matching Michigan: an ethnographic study of a patient safety program. Implementation Science 2013;8:70. doi:10.1186/1748-5908-8-70. 13 Dixon-Woods M, Leslie M, Tarrant C, Bion J. Explaining Matching Michigan: an ethnographic study of a patient safety program. Implementation Science 2013;8:70. doi:10.1186/1748-5908-8-70. 14 Vincent C Amalberti R. Safer Healthcare - Strategies for the Real World 2016. Springer. 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Effect of a Pediatric Early Warning System on All-Cause Mortality in Hospitalized Pediatric Patients: The EPOCH Randomized Clinical Trial. JAMA. 31 Chapman SM, Wray J, Oulton K, et al. ’Death is not the answer’: the challenge of measuring the impact of early warning systems. Arch Dis Child Published Online First: 14 September 2018. doi:10.1136/archdischild-2018-315392) od_consum_dh/groups/dh_digitalassets/@dh/@en/documents/digitalasset/dh_4065 086.pdf Accessed January 3 2019. 2018;319(10):1002–1012. Warning System on All-Cause Mortality in Hospitalized Pediatric Patients: The EPOCH Randomized Clinical Trial. JAMA. 2018;319(10):1002–1012. Warning System on All-Cause Mortality in Hospitalized Pediatric Patients: The EPOCH Randomized Clinical Trial. JAMA. 2018;319(10):1002–1012. 29 Roland D, Oliver A, Edwards E, et al . Use of paediatric early warning systems in Great Britain: has there been a change of practice in the last 7 years? Arch Dis Child 2014;99:26–9. 30 Joffe AR, Anton NR, Burkholder SC. Reduction in hospital mortality over time in a hospital without a pediatric medical emergency team: limitations of before-and-after study designs. Arch Pediatr Adolesc Med 2011;165:419–23. 31 Chapman SM, Wray J, Oulton K, et al. ’Death is not the answer’: the challenge of measuring the impact of early warning systems. Arch Dis Child Published Online First: 14 September 2018. doi:10.1136/archdischild-2018-315392) 32 http://www.ihi.org/resources/Pages/HowtoImprove/default.aspx (Last accessed 1 October 2018) 40Classen D, Li M, Miller S, Ladner D. An Electronic Health Record–Based Real- Time Analytics Program for Patient Safety Surveillance And Improvement. Health Affairs. 2018; 37(11):1805–1812 32 http://www.ihi.org/resources/Pages/HowtoImprove/default.aspx (Last accessed 1 October 2018) 33 Roland D. 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Int J Qual Health Care, 2018; 30(1): 44-49 https://doi.org/10.1093/intqhc/mzx162 38 Health Foundation. Effective Clinical Networks. 2014. https://www.health.org.uk/sites/health/files/EffectiveNetworksForImprovement.pdf (Last accessed 1 October 2018) 39 Weis SE, Slocum PC, Blais FX et al. The effect of directly observed therapy on the rates of drug resistance and relapse in tuberculosis, N Engl J Med , 1994; 330: 1179-84. 38 Health Foundation. Effective Clinical Networks. 2014. https://www.health.org.uk/sites/health/files/EffectiveNetworksForImprovement.pdf (Last accessed 1 October 2018) 39 W i SE Sl PC Bl i FX t l Th ff t f di tl b d th 38 Health Foundation. Effective Clinical Networks. 2014. https://www.health.org.uk/sites/health/files/EffectiveNetworksForImprovement.pdf (Last accessed 1 October 2018) 39 Weis SE, Slocum PC, Blais FX et al. The effect of directly observed therapy on the rates of drug resistance and relapse in tuberculosis, N Engl J Med , 1994; 330: 1179-84. 39 Weis SE, Slocum PC, Blais FX et al. The effect of directly observed therapy on the rates of drug resistance and relapse in tuberculosis, N Engl J Med , 1994; 330: 1179-84. 40Classen D, Li M, Miller S, Ladner D. An Electronic Health Record–Based Real- Time Analytics Program for Patient Safety Surveillance And Improvement. Health Affairs. 2018; 37(11):1805–1812 40Classen D, Li M, Miller S, Ladner D. An Electronic Health Record–Based Real- Time Analytics Program for Patient Safety Surveillance And Improvement. Health Affairs. 2018; 37(11):1805–1812
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Improving Quality of Service Provisioning of Optimised Cuckoo Search Ad Hoc on-demand Distance Vector Routing Scheme for Cognitive Radio Ad Hoc Networks.
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Improving Quality of Service Provisioning of Optimised Cuckoo Search Ad Hoc on-demand Distance Vector Routing Scheme for Cognitive Radio Ad Hoc Networks. Improving Quality of Service Provisioning of Optimised Cuckoo Search Ad Hoc on-demand Distance Vector Routing Scheme for Cognitive Radio Ad Hoc Networks. Ramahlapane Lerato Moila  (  moilaramahlapane@gmail.com ) University of Limpopo - Tur§oop Campus: University of Limpopo https://orcid.org/0000-0002-8012- 9909 Mthulisi velempini  University of Limpopo, Tur§oop campus Research Keywords: Cognitive radio network, Cuckoo search algorithm, Network simulator, Quality of service Posted Date: October 29th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-952534/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Ramahlapane Lerato Moila  (  moilaramahlapane@gmail.com ) Mthulisi velempini University of Limpopo, Tur§oop campus IMPROVING QUALITY OF SERVICE PROVISIONING OF OPTIMISED CUCKOO SEARCH AD HOC ON-DEMAND DISTANCE VECTOR ROUTING SCHEME FOR COGNITIVE RADIO AD HOC NETWORKS. Ramahlapane Lerato Moila (corresponding author); Mthulisi Velempini moilaramahlapane@gmail.com; mthulisi.velempini@ul.ac.za Department of computer science, University of Limpopo, South Africa, Polokwane, 0727. Ramahlapane Lerato Moila (corresponding author); Mthulisi Velempini moilaramahlapane@gmail.com; mthulisi.velempini@ul.ac.za Department of computer science, University of Limpopo, South Africa, Polokwane, 0727. Research Research Keywords: Cognitive radio network, Cuckoo search algorithm, Network simulator, Quality of service Posted Date: October 29th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-952534/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Abstract Spectrum mobility, cloud computing and the Internet of Things (IoTs) create large data sets, while the demand for more spectrum is increasing. Unfortunately, the spectrum is a scarce resource which is being underutilized by licensed users. The cognitive radio network, also known as intelligent radio, is a network that can adjust to environment changes and, detect available channels. It has emerged as a promising solution for the underutilization of the licensed spectrum and overcrowded free spectrum. Furthermore, given spectrum mobility, frequent link breakages impact negatively on the delivery of packets and the performance of the network. Hence there is need to address the routing problem. We therefore investigated which control methods can be utilized to improve the QoS provisioning in CRAHNs to minimize the signal overhead and to increase the achievable throughput. The study integrated the QoS requirements with optimized cuckoo search (OCS) algorithm to enhance the ad hoc on-demand distance vector (AODV) algorithm to establish a scheme we refer to as OCS-AODV. NS 2 simulation were run on Linux operating system. The comparative results show that the proposed scheme performed well in terms of end-to-end delay and throughput. However, the scheme does not backup alternative paths which can be used in the event of link breakages. The route discovery has to be re-initiated again. Though the route discovery process is faster because of the capability of the CS technique, it still degrades the performance of the scheme. words: Cognitive radio network, Cuckoo search algorithm, Network simulato Keywords: Cognitive radio network, Cuckoo search algorithm, Network simulator, Quality of service. 1. Introduction Quality of Service (QoS) aware technologies that seek to guarantee the provisioning of the requirements of high-priority applications and traffic under the constraints of limited network capacity [1]. The goal of QoS provisioning is to achieve a more deterministic network behavior, so that data can be delivered efficiently while network resources are utilized efficiently. The fundamental of cognitive radio ad hoc network (CRAHN) is to deliver the QoS requirements while the unlicensed users access network channels opportunistically when the spectrum is idle [2]. CRAHN has to ensure that interference with the licensed user’s activities is avoided. Therefore, the need to meet QoS requirements of unlicensed users is a challenge [3]. The spectrum mobility and the Internet of Things (IoT) have generated large volumes of data, which require the more bandwidth and spectrum for data transmission. Unfortunately, the spectrum is a scarce resource which is underutilized by licensed users while unlicensed spectrum is overcrowded. Cognitive networks are spectrum-agile devices that changes their configurations based on the spectral environment [4]. Cognitive network has emerged as a promising solution to the underutilization and overcrowding of the spectrum by allowing unlicensed users to utilize these free channels opportunistically when they are idle. This technique is expected to reduce contention on the channels and minimize the interference and improve the average channel efficiency [5]. There are various accessible spectrum ranges, such as global system for mobile (GSM), Television, wireless local area network (WLAN), military, and long-term evolution (LTE) as depicted in figure 1 that cause spectral inefficiency. Figure 1 In ad hoc networks, each node is able to perform routing functions but due to the unique characteristics of a dynamic environment and frequent changes in the topology. The network is characterized by limited and varying bandwidth and power limitations of mobile devices and the lack of centralized infrastructure, it is a challenge to meet QoS requirements [6]. Furthermore, routing problem requires further consideration given the fact that current routing protocols designed for traditional networks are not suitable for CRAHNs because of node and spectrum mobility [7]. The study is motivated by the potential of cognitive radios, which are designed to address the spectrum scarcity. The study investigates the efficient routing strategies that can be applied to CRAHNs to improve the QOS and the flexibility of the routing protocols while ensuring that they remain reliable and stable. The study proposes an optimized cuckoo search ad hoc on-demand distance vector (OCS-AODV) scheme which is a meta-heuristic optimization algorithm designed to solve the routing problem and improve QoS [8]. Cuckoos are parasite birds that ordinarily uses other birds’ nests to lay their eggs. They replace the host eggs with their own in an endeavor to ensure that their eggs are not detected. The algorithm was originally created in 2009 by Deb and Yang and the cuckoo search algorithm is an NP- hard algorithm which was designed to solve complicated and hard problems efficiently. 2. Literature review In [9] the authors proposed a discrete variants of the cuckoo search optimization (DCSO) scheme, namely the levy and the random walk approach to achieve improvement on route discovery in ad hoc networks. The proposed scheme was compared to a genetic algorithm (GA). The simulation results show that the proposed scheme achieved a better performance under various network scenarios. However, the proposed algorithm performs poorly for real time applications. It incurs a lot of delay experienced during random movement made by each node and there seems to be more challenges experience during node movement [10]. The study may be optimized for complex cases where the nodes are moving randomly in an area with obstacles and QoS sensitive users. In [2], the authors proposed a routing protocol called TIHOO for VANETs. The routing scheme intelligently utilizes fuzzy and cuckoo methods. The scheme is effective in large search space because it selects stable and optimal paths among the available paths by calculating the fitness function based on criteria such as path lifetime, path stability and the availability of the adequate buffer space [11]. The simulation results show that the proposed scheme performs better than ANT and AODV in terms of throughput and packet delivery ratio. However, when the mobile nodes increase, the source node generates more packets causing a delay and overwhelms the receiving node. The scheme is not designed for QoS and routing overhead [12]. In [13], the authors proposed a hybrid computational intelligent scheme known as TCSA, which is the extension of the CSA. It is designed to solve the multi-constrained QoS routing problem experienced by ad hoc networks. The results proved that the proposed scheme TCSA outperformed the PSO and CSA schemes in terms of packet delivery ratio (PDR), end-to-end delay, path success ratio. The proposed scheme is effective and efficient. Furthermore, the scheme can be enhanced to consider the network robustness and real time applications as they are crucial when it comes to time deadline and constraints. QoS routing problem experienced by ad hoc networks. The results proved that the proposed scheme TCSA outperformed the PSO and CSA schemes in terms of packet delivery ratio (PDR), end-to-end delay, path success ratio. The proposed scheme is effective and efficient. Furthermore, the scheme can be enhanced to consider the network robustness and real time applications as they are crucial when it comes to time deadline and constraints. 2. Literature review In [14], the authors proposed an improved AODV routing scheme to solve the end-to- end delay and improve achievable throughput. The simulation results show that the proposed scheme was able to reduce routing overhead through the selection of the best route to the destination. However, the scheme increases packet drop rate. In trying to solve the high packet drop rate the authors in [15] proposed a virtual ad-hoc routing designed to minimizes energy consumption in discovering the alternative paths. The simulation results show that the scheme seems to have managed to reduce energy consumption at the cost of increased routing overhead associated with the discovery alternative routes. The scheme is therefore not effective and hasn’t effectively addressed the routing problem. A routing scheme was proposed in [16] which is cluster-based multipath routing which was designed to reduce the link failures caused by spectrum and node mobility, cloud computing and the Internet of Things (IoT). The experiments results show that the scheme was able to solve the link failure problem however, poor selection of gateway nodes is a challenge. Hence there is still a need to design an algorithm optimized for the discovery of optimum route [17]. Improving the routing problem would mean increased PDR, fewer packet losses, less delay, and increased achievable throughput. 3. Results We evaluate the proposed scheme in terms of the end-to-end delay. The comparative results are there presented analyzed in this Section. The proposed scheme incurred the least delay and guaranteed the delivery of most packets within deadline constraints. The other schemes incurred delay relating to the retransmission of lost packets which was not the case with the OCS-AODV. The retransmission protocols in the other schemes, increased the amount of delay. In figure 2, we observed that the existing schemes incur longer delay caused by the network congestions as a result of packet losses. The delay is dependent on the network’s state; when the network performance degrades, the delay problem worsens. As a result, packets are buffered for longer periods until they are timed out. Dropped packets will require to be retransmitted. The proposed scheme avoids retransmissions and reduces delay in the network by ensuring that quality routes are selected. The CS-DSDV protocol experiences the worst delay when the number of nodes increases. The performance of CS-DSDV performance is affected by the delay in route repair. Secondly, when a link is broken, the route cannot be repaired locally. Thus, the route discovery and repair incur a lot of delay. Frequent route updates incur a lot of overhead. Figure 2 In figure 3, we observed that the proposed scheme had a lower delay, few packets dropped and higher PDR. These three factors helped the OCS-AODV scheme improve its performance as it was able to achieve higher throughput than ACO-AODV and CS-DSDV. The throughput of CS-DSDV was affected by delays and periodic route updates. The scheme experiences a lower throughput due to network bottlenecks and high overhead cost. The ACO-AODV suffers from lengthy search and very slow convergence in route discovery process. The study observed that the proposed scheme OCS-AODV is optimized for QoS constraints; it ensures that a path chosen is energy efficient, stable and has sufficient network resources required by an application. While the DSDV depletes the battery power due to periodic updates of routing tables. Figure 3 Figure 4 presents the delay results, and the results show that when the number of nodes increases, the delay also increases. Applying the optimised CS algorithm to the AODV had a significant impact on the selection of the best route. We observed that the OCS-AODV algorithm incurred less delay due to the selection of reliable paths with sufficient network resources. The longer delay in CS-DSDV is due to frequent updates of its routing tables, which consumes a lot of battery power and bandwidth even when the network is idle. Topological changes cause the CS-DSDV to take long to converge and degrades in high dynamic network environment. The OCS-AODV has a fast convergence rate and selects optimal paths faster. Furthermore, the configuration of the fitness function of the AODV protocol, considering parameters such as available buffer, helps in identifying candidate paths for de-selection in endeavour to avoid congested paths. Figure 4 Figure 5 presents the throughput results of a network scenario with high mobility. The proposed scheme OCS-AODV outperformed the CS-DSDV and ACO-AODV protocols. CS-DSDV is a proactive protocol which takes long time to select routs and which also suffers from slow to convergence. As a result, the protocol achieves low throughput. Periodic route updates also degrade throughput. The OCS-AODV converges faster which improves its performance. The study also observed that all the routing protocols are degraded by congestion. However, the OCS-AODV still outperforms the other protocols in such congested and highly mobile network scenarios. 5. Conclusion CRAHNs is a network is a promising technology which addresses the spectrum scarcity problem. The IoT smart devices have increased the need for spectrums which affects the QoS provisioning. This study focused on the factors which affects the QoS routing and proposed an OCS-AODV scheme which utilizes an effective search technique and provides quality solutions. The scheme selects paths with sufficient battery life to prolong the network lifetime during data transmissions. Based on the experimental results of this study, we conclude that the OCS-AODV is a QoS aware routing protocol which performs well in CRAHNs. 4. DISCUSSIONS The study observed that the DSDV is not suitable for ad hoc networks and highly mobile network scenarios due to a need to freshen routes on regular basis. Based on the results, the study observed that the proposed OCS-AODV scheme in dynamic environment obtained less delay even when the number of nodes increased. CS- DSDV incurs longer delays as it attempts to discover alternative routes after the existing routes become outdated and when link failures are experienced. The OCS-AODV utilizes the CS scheme search space, which is very fast and efficient in establishing paths. It also avoids disruptions of services as nodes with sufficient power are considered. As a result, shorter delays and increased achievable throughput are incurred. The proposed also losses less packets in the event of link failures. Less delay is incurred as a result while achievable throughput increases. In a high mobile environment, the scheme is effective due to the number of backup available paths. When a link breaks the backup path is immediately utilized. 6.1 Quality of service challenges In a conventional system, QoS requirements are met using either overprovisioning or the network traffic engineering techniques. However, the fist technique is not suitable for ad hoc networks due to limited resources and the second one is sub optimum for the ad hoc environment [18]. For example, the buffering approach cause the network to drop and loose packets when packets are generated at higher rate than they are consumed resulting in the producer and consumer challenge [19]. Redesigning and optimizing the traffic engineering technique for ad hoc networks is a challenge. Most applications require QoS support to meet guarantee reliability required to solve the unbalanced traffic in order to address the mobility routing challenge characterized by node and spectrum mobility [20]. Due to the unique nature of ad hoc networks, is susceptible to high incidents of node failures, high energy consumption, node mobility and frequent partitioning of the network. When a node initiates a route, it may not secure sufficient resources to guarantee QoS requirements. Meeting QoS requirements in routing involves the selection of paths with sufficient network resources [21]. However, there are challenges in providing the QoS in ad hoc networks, such as hidden terminal problem, lack of coordination, insecure medium, limited resources, frequent network topologically changes, error prone radio channel and imprecise state information [22]. Figure 6 Figure 6 is a basic model that depict the effects of increasing number of nodes on the provisioning of QoS requirements [23]. As more users join the network, the network resources are depleted. The objective of QoS aware routing protocols is to improve the provisioning of QoS requirements of different class of applications. Figure 6 is a basic model that depict the effects of increasing number of nodes on the provisioning of QoS requirements [23]. As more users join the network, the network resources are depleted. The objective of QoS aware routing protocols is to improve the provisioning of QoS requirements of different class of applications. 6.3 Adaptation of cuckoo search algorithm for cognitive radio ad hoc network. Cuckoo Search study which was adapted for CRAHNs designed to improve the routing issue through the provisioning of QoS requirements. A fixed number of local area networks (LANs) referred to as host nests with existing solutions (mobile nodes) is assumed. A new solution node (cuckoo egg) exists within each LAN and acts as a gateway node which links a LAN to other LANs. A LAN with a gateway node is regarded the best fit as the gateway is regarded to be providing a better network lifetime. The gateway is used for external transmission and for meeting QoS requirements. If the cuckoo egg is detected by the host bird, it is rejected. This means that the nest in this case the LAN does not have a solution or gateway and it cannot be used for the next generations. This means that in our context, a given LAN cannot communicate with other LANs. We therefore assume that there always exists a solution in every LAN. 6.2 Cuckoo search algorithm overview Cuckoo search algorithm is inspired by natural selection and was first developed by Yang and Deb in 2009. The cuckoo birds lay eggs in the host bird’s nest and leave them to be incubated by the host bird. The cuckoo eggs can be blue, earthly coloured, green or grey or with various spots and patterns. When the cuckoo lays its egg in another host birds’ nest, it replaces the host bird’ eggs with its own to ensure that its eggs are not detected. Therefore, the Cuckoo eggs should be of the same colour and texture like the host eggs for them to be undetected. 6.4 Proposed methodology We propose a routing scheme called an optimized cuckoo search ad hoc on-demand distance vector (OCS-AODV). The routing scheme combines the AODV and OCS attributes to improve the efficiency of the routing protocol. The proposed scheme was evaluated according to route stability of each link, the throughput and the end-to-end delay. To reduce the routing overheads as the network traffic increase, this study establishes multiple paths and calculate their fitness function values to ensure that a selected path meets the QoS requirements. The scheme reduces the routing overhead, increases the achievable throughput and reduces the end-to-end delay to guarantee data transmission while it addresses the routing problem. The OCS-AODV routing scheme is based on a wide area network (WAN) whereby each node within the LAN stores neighbourhood data. The communication between two nodes in different LANs takes place through gateway nodes – the Cuckoo eggs. 6.5 Implementation steps  Initialization step – there exist a network with mobile nodes, and a new gateway node. Considering N to be the number of nodes in CRAHNs, with number of nodes (n), step size α, discovering probability (Pα) and the maximum number of iterations to be an end state.  Initialization step – there exist a network with mobile nodes, and a new gateway node. Considering N to be the number of nodes in CRAHNs, with number of nodes (n), step size α, discovering probability (Pα) and the maximum number of iterations to be an end state.  Generating gateway nodes using levy flight – to generate gateway node using levy flight distribution.  Generating gateway nodes using levy flight – to generate gateway node using levy flight distribution.  Generating gateway nodes using levy flight – to generate gateway node using levy flight distribution.  Population of nodes – mobile nodes are generated randomly. Figure 7 denotes the LANs within the WAN and each LAN has one gateway node.  Population of nodes – mobile nodes are generated randomly. Figure 7 denotes the LANs within the WAN and each LAN has one gateway node.  Population of nodes – mobile nodes are generated randomly. Figure 7 denotes the LANs within the WAN and each LAN has one gateway node. igure 7 Figure 7 Figure 7  Best and worse network – a queue is formed by orchestrating the best and worse nodes using the levy flight values, where the best network is passed on to the next generation – considered for routing purposes in our case.  Best and worse network – a queue is formed by orchestrating the best and worse nodes using the levy flight values, where the best network is passed on to the next generation – considered for routing purposes in our case.  Significance of Gateways in the network – gateway nodes facilitate communication to take place between two nodes belonging to different LANs. They form a network backbone in a WAN while networking LANs to ensure connectivity within the WAN. Cognitive radio devices must have three qualities to achieve optimal spectrum utilization such as sensing spectrum, meeting the QoS requirements and the utilization of the capabilities of cognitive radios. The primary goal of QoS is to manage packet loss and reduce latency and jitter within a network. QoS policies ensure QoS of different classes of data are met. 7. Proposed cuckoo search algorithm One of the latest nature-inspired search algorithms, which was proposed by Deb and Yang in 2009 [26], known as cuckoo search (CS). To apply the CS as an optimization tool, there are three principles that needs to be considered such as:  Cuckoo bird lays its egg in some random chosen nest at any given time.  A nest containing the cuckoo egg would be viewed as the best nest and would be considered for the next generations.  Fixed number of nests would be accessible, Pa shows the probability of finding the cuckoo egg such that Pα ∈[0,1]. The lower the number the lesser the chance of finding the cuckoo egg. Assumptions Assumptions Assumptions  We assume that the cuckoo egg within the host bird’s nest cannot be detected.  Each nest must have a cuckoo egg. 7.1 Significance of cuckoo search in CRAHNs. We have studied how the cuckoo bird multiplies its species and this study applies the cuckoo strategy to improve the routing problem. There is a fixed number of local area networks forming a WLAN, each LAN has a gateway node, which facilitates communication between two mobile nodes. The study investigated different QoS routing metrics, CRAHN protocols, their shortcomings and quality before proposing a routing scheme that integrates AODV and OCS to improve the routing issue. To reduce the frequently occurring routing overheads as the network traffic increases, the study establishes multiple paths and calculate their fitness function values to ensure that a chosen path meets the QoS requirements. The proposed scheme, the OCS-AODV is a WLAN scheme consisting of several LANs, whereby each node within the LAN is responsible for constructing data about it current state. The step size is as follows: The step size is as follows: The step size is as follows: (1) 𝛼 = 𝛼max – (N_iter/N_itertotal) x (𝛼max - 𝛼min) 𝛼 = 𝛼max – (N_iter/N_itertotal) x (𝛼max - 𝛼min) 𝛼 = 𝛼max – (N_iter/N_itertotal) x (𝛼max - 𝛼min) Where 𝛼max and 𝛼min represent the maximum and minimum of the step size because the global best solution cannot be found unless there is many iterations which results in consuming a lot of energy associated with searching. When the value of 𝛼 is high, according to the step length causes the generated solution to be further from the current local best solution and it cannot guarantee convergence to the optimal solution. Hence, a proper step size is important to find global best solution. The N_iter and N_itertotal represents the current number of iterations, and total number of iterations. The probability that the cuckoo egg can be detected is Pa, which is associated with the fitness of the solution, declaring a nest having higher fitness value closer to current global optimum has a higher probability to be selected as the population of the next generation. New cuckoo eggs can be generated using the levy flight distribution as follows: Xi (t+1) = Xit + α ⊕ Lévy (λ), (2 Xi (t+1) = Xit + α ⊕ Lévy (λ), (2) Xi (t+1) = Xit + α ⊕ Lévy (λ), (2) Where 𝛼 > 0, is the development measure identified with the sizes of the problem of interests. The levy flight gives an arbitrarily walk while the irregular advance length is drawn from a duty distributed which is denoted by Lévy ̴ u = t−λ where λ ∈ (0, 3]. (3) Lévy ̴ u = t−λ where λ ∈ (0, 3]. Lévy ̴ u = t−λ where λ ∈ (0, 3]. (3) (3) Which consist of both the infinite mean and variance. However, the next location is based on the current location. SN = ∑ + 𝑁−1 𝑖=1 XN = SN-1 + XN, (5) 7.2 Random walk Random walk is a process that consists of taking a series of consecutive random steps. Mathematically, can be denoted as, say SN denotes the sum of each consecutive random step say Xi, then the SN can form a random walk represented as follows: SN = ∑ 𝑋𝑖 𝑁 𝑖=1 = X1 + ... + XN (4) SN = ∑ 𝑋𝑖 𝑁 𝑖=1 = X1 + ... + XN (4) Where the Xi is an arbitrary step that is drawn from irregular dispersion and the relationship could be written as a recursive formula denoted by: Where the Xi is an arbitrary step that is drawn from irregular dispersion and the relationship could be written as a recursive formula denoted by: (5) SN = ∑ + 𝑁−1 𝑖=1 XN = SN-1 + XN, where the next step depends on the current state SN-1 and the transition of Xn is based on the existing state to reach the targeted state. Hence the step size and the Wt is a variable step. Utilising the Mantegna’s algorithm to ascertain the progression length is indicated by the: s= u/ |v|1/b (6) s= u/ |v|1/b (6) where the β is a boundary between the range [1,2] interval. The incentive for β is given to be 1.5; where the estimations of u and v are drawn from the normal distribution denoted as: where the β is a boundary between the range [1,2] interval. The incentive for β is given to be 1.5; where the estimations of u and v are drawn from the normal distribution denoted as: u ~ N (0, σ2u), v ~ N (0, σ2v), (7 (7) where N denotes the normal distribution; σu and σv can be calculated using the following equation: σu = { 𝛤(1+𝛽)sin (𝜋𝛽/2) 𝛤[1+𝛽 2 ] 𝛽2 exp(𝛽−1)/2} 1/β, σv = 1 (8) (8) where the Γ denotes the gamma function. The discovery of the new route is performed using the matrix P, and its probability is computed as follows: where the Γ denotes the gamma function. The discovery of the new route is performed using the matrix P, and its probability is computed as follows: Pij = {1, 𝑖𝑓 𝑟𝑎𝑛𝑑 (0,1) < 𝑃𝑎 0, 𝑜𝑡ℎ𝑒𝑟𝑤𝑖𝑠𝑒, (9) Pij = {1, 𝑖𝑓 𝑟𝑎𝑛𝑑 (0,1) < 𝑃𝑎 0, 𝑜𝑡ℎ𝑒𝑟𝑤𝑖𝑠𝑒, (9) Where Pij is the probability of finding node i and j within the matrix P. 7.2 Random walk The P𝛼 value is contrasted within (0,1) to determine whether the local random walk is considered or not and the rand is a random number in [0,1] after the discovery of probabilities, new nodes are generated using equation (10). new_nodet = nodest + S * P (10) new_nodet = nodest + S * P (10) I dditi S d t th l l t i t i d d i new_nodet = nodest + S * P ( In addition, S denotes the local step size matrix produced using: new_nodet = nodest + S * P (10) In addition, S denotes the local step size matrix produced using: In addition, S denotes the local step size matrix produced using: (11) S = rand () * (nodes (randperm1 (n, :) – nodes (randperm2 (n), : ) (11) Where the rand ( ) value is the random number generator with the interval 0 and 1, randomperm1 and randperm2. 7.3 Performance metrics QoS varies from one application to another for example, multimedia depends on metrics such as delay, throughput, packet loss rate, scalability and overhead communication. The cuckoo search optimisation is concerned with certain reproduction behaviour because it pertains improved methods as opposed to meta- heuristics algorithms. QoS varies from one application to another for example, multimedia depends on metrics such as delay, throughput, packet loss rate, scalability and overhead communication. The cuckoo search optimisation is concerned with certain reproduction behaviour because it pertains improved methods as opposed to meta- heuristics algorithms. The objective function of the QoS routing is formulated as follows: The objective function of the QoS routing is formulated as follows: Minimise C (H(x, S)) = Cc + δ1Cb + δ2Cd + δ3Cdj + δ4Cpl. (12) Minimise C (H(x, S)) = Cc + δ1Cb + δ2Cd + δ3Cdj + δ4Cpl. ( (12) There are only two significance reasons for combining the cost optimisation and the multi constrained routing: There are only two significance reasons for combining the cost optimisation and the multi constrained routing: I. QoS routing issues ordinarily are figured as far as various rules and the multicast structure should recognise a reasonable way from source to destination. II. The most important objective is to minimise the network resource utilisation Summing the above two points, it can be denoted by the following formula: C (H(x, y)) = ∑ 𝐶 e∈H(x,S) (x) + ∑ 𝐶 𝑛∈𝐻(𝑥,𝑆) (y) (13 (13) The study considers two QoS routing metrics which are throughput and end-to-end delay as shown is equation 14 and 15 respectively. Bandwidth (R(x, y)) = min (bw(x)) , x∈R (x,y) (14) (14) And the end-to-end delay which is the amount of allowed limit delay that each branch of network can incur such as: Delay (R(x, y)) = ∑ 𝑑𝑙 e∈R(x,y) (x) + ∑ 𝑑𝑙 𝑛∈𝑅(𝑥,𝑦) (y) (15) Delay (R(x, y)) = ∑ 𝑑𝑙 e∈R(x,y) (x) + ∑ 𝑑𝑙 𝑛∈𝑅(𝑥,𝑦) (y) (15) 7.4 Simulation parameters Delay (R(x, y)) = ∑ 𝑑𝑙 e∈R(x,y) (x) + ∑ 𝑑𝑙 𝑛∈𝑅(𝑥,𝑦) (y) (15) (15) List of abbreviations AODV: ad hoc on demand distance vector; CRAHN: cognitive radio ad hoc network; CS-DSDV: cuckoo search-destination sequence distance vector; CRCN: cognitive radio cognitive network; DCSO: discrete cuckoo search optimization; GSM: global system for mobile; GA: genetic algorithm; IoTs: internet of things; LTE: long-term evolution; LANs: local area networks; OCS: optimized cuckoo search; PSO: Particle swarm optimization; PDR: packet delivery ratio; QoS: quality of service; TCSA: Tuned cuckoo search algorithm; WLAN: wireless local area network. Declarations We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We attest that this paper has not been published in whole elsewhere and is prepared following the instructions to authors. Availability of data and materials The datasets used or analyzed during the current study are available from the corresponding author on reasonable request. 7.4 Simulation parameters The simulation experiments were conducted using the network simulator version 2.31. It was patched with cognitive radio cognitive network (CRCN). Using the CRCN patch is able to address the radio spectrum scarcity, avoids intentional radio jamming scenarios, switching the power saving protocol, improve the satellite communications and improves quality of service. The duration of the simulation was set to 100 simulation seconds, the mobile nodes ranges from 10 to 100, the mobility of each mobile node was set to 30 km/h. The network resources are only assigned to nodes that meet the QoS requirements, this strategy ensures that resources are not assigned to nodes that compromises the availability of resources. Table 1 shows the parameters and their values which were utilized for the experiment. Table 1: Simulation parameters Authors contributions RL carried out the whole study and M as an advisor and supervisor. Funding This work is based on the research supported in part by the National Research Foundation of South Africa (Grant Number: 1141155) and Council of Scientific and Industrial Research. The authors declare that they have no competing interests. The authors declare that they have no competing interests. 8. References 1. T. Kurokawa & N. Hayashibara. “Performance Evaluation of Data Replication Protocol Based on Cuckoo Search in Mobile Ad Hoc Networks” ScienceDirect, Vol. 11, September 2020. 2. D. Mahato, J. Sandhu & G. Dutta. “Distributed Routing for Underwater Wireless Sensor Networks Using Cuckoo Search-Ant Colony Optimization” International conference on distributed computing and networking, Vol.39, Pp. 1-5, January 2020. 2. D. Mahato, J. Sandhu & G. Dutta. “Distributed Routing for Underwater Wireless Sensor Networks Using Cuckoo Search-Ant Colony Optimization” International conference on distributed computing and networking, Vol.39, Pp. 1-5, January 2020. 3. J. Chaochuan, Y. Ting, W. Chuanjiang, F. Binghui & H. Fugui. “An Improved Adaptive Cuckoo Search Algorithm Based on the Population Feature and Iteration Information” International Journal of Communication Networks and Distributed Systems, Vol. 24, February 2020. 4. M. Usman, M. Jan, X. He & P. Nanda, “QASEC: A Secured Data Communication Scheme for Mobile Ad-Hoc Networks”, ScienceDirect, Vol. 109, pp. 604-610, August 2020. 5. A. Malar, M. Koslgan, N. Krishnamoorthy, S. Prabhu & K. Venkatachalam, “Multi Constraints Applied Energy Efficient Routing Technique Based on Ant Colony Optimization Used for Disaster Resilient Location Detection in Mobile Ad-Hoc Network” Journal of Ambient Intelligence and Humanised Computing, February 2020. 6. H. Riasudheen, K. Selvamani, S. Mukherjee & I. Divyasree, “An Efficient Energy-Aware Routing Scheme for Cloud-Assisted MANETs in 5G.” Ad Hoc Networks, Vol. 97, 2020. 7. R. Thiagarajan, M. Babu, & M. Moorthi, “Quality of Service Based Ad Hoc On- demand Multipath Distance Vector Routing Protocol in Mobile Ad Hoc Network,” Journal of Ambient Intelligence and Humanized Computing, Vol.13, Pp. 1-9, 2020 8. D. Lakew, U. Sa’ad, N. Dao, W. Na, & S. Cho, “Routing in Flying Ad Hoc Networks: A Comprehensive Survey,” IEEE Communications Surveys & Tutorials. Vol. 2, 2020. 9. K. Muralidhar, & K. Madhavi, “Approaches to Address the Operational Limitations of MANETs through Ad Hoc Mobile Cloud Computing Paradigm,” International Journal of Interactive Mobile Technologies, Vol. 14, 2020. 10. K. Yitayih, & M. Libsie “Towards Developing Enhanced Cluster-Based QoS- Aware Routing in MANET” Journal of Computer Networks and Communications, 2020. 11. S. Yahiabadi, B. Barekatain & K. Raahemifar. “TIHOO: An Enhanced Hybrid Routing Protocol in Vehicular Ad-hoc Networks” EURASIP Journal on Wireless Communications and Networking, 2019. 12. A. Rama, R. Satyananda & V. Valli., “Multipath Selection Based on Fractional Cuckoo Search Algorithm for QoS Aware Routing in MANET.” Sensor Review, 2018. 13. M. Mareli & B. Twala. 8. References “An adaptive Cuckoo Search Algorithm for Optimisation” applied computing and informatics, Vol. 14 issues 2 July 2018. 14. P. Aimtongkham, T. Nguyen & ChakchaiSo-In, “Congestion Control and Prediction Schemes Using Fuzzy Logic System with Adaptive Membership Function in Wireless Sensor Networks Wireless,” Communications and Mobile Computing, Vol.20, 2018. 15. A. Rama, R. Satyanand, V. Valli, "Multi-Path Selection Based on Fractional Cuckoo Search Algorithm for QoS Aware Routing in MANET," Sensor Review, 2018. 16. D. Saravanan & T. Sangeetha.,” Enhancing Network Performance Using Genetic Algorithm in FACE Routing Protocol”, International Journal of Innovative Research in Science, Engineering and Technology 2017. 17. A. Kout, S. Labed, S. Chikhi & E. Bourennane., “AODVCS, A New Bio-Inspired Routing Protocol Based on Cuckoo Search Algorithm for Mobile Ad Hoc Networks” wireless network, Mar 2017. 18. S. Thakur & B. Batra. “Improvement in AODV for Congestion Avoidance Using Bio-inspired Techniques,” International Journal of Emerging Trends of Technology in Computer Science, Vol. 6, issue 5, 2017. 19. P. Damaraju & S. Jena. “Dynamic Quality of service stability-based multicast routing for Manet” Scientific journal of Impact factor, Vol. 4, issue 7, July 2017. 20. N. Kaur & A. Verma. “Efficient path establishment algorithm in Manets using bio-inspired techniques” international journals of advanced research in computer science and software engineering, 2017. 21. S. Rajalakshmi & R. Maguteeswaran, “Quality of Service Routing in Manet Using a Hybrid Intelligent Algorithm Inspired by Cuckoo Search” the scientific world journal, Vol. 2015. 22. A. Rama & R. Satyananda, “Multi-Path Selection Based on Fractional Cuckoo Search Algorithm for QoS Aware Routing in MANET”, Computer Communications (Networks), Vol. 39, 2018. 23. D. Saravanan & T. Sangeetha.,” Enhancing network performance using genetic algorithm in FACE routing protocol”, International Journal of Innovative Research in Science, Engineering, and technology, 2017. 24. K. RahimiZadeh & P. Kabiri, “Trust-based routing method using a mobility- based clustering approach in mobile ad hoc networks” security and communication networks, Vol. 7, issue 11, 2013. 25. R. Moila & M. Velempini, “Optimisation of Cuckoo Search Algorithm to improve Quality of Service Routing in Cognitive Radio Ad Hoc Networks” IEEE EnergyConference, 2020. 26. X. Yang & S. Deb, “Cuckoo search via L´evy flights”, in: Proc. Of World Congress on Nature & Biologically Inspired Computing (NaBIC 2009), December 2009, India. IEEE Publications, USA, pp. 210-214 (2009). 8. References Figures Figures Figures Figure 1 is the illustration of the spectrum channels, showing that the GSM, LTE and WLAN are overcrowded whereas the military and television channels are underutilised. Figure 1 Figure 1 is the illustration of the spectrum channels, showing that the GSM, LTE and WLAN are overcrowded whereas the military and television channels are underutilised. is the illustration of the spectrum channels, showing that the GSM, LTE and WLAN are overcrowded whereas the military and television channels are underutilised. Figure 2 Figure 2 depicts the incorporation of the quality of service support to users. Figure 3 Shows a network demonstration of existing nodes and cuckoo nodes illustration of the LANs in WLAN. depicts the incorporation of the quality of service support to users. Figure 3 Figure 3 Shows a network demonstration of existing nodes and cuckoo nodes illustration of the LANs in WLAN. Shows a network demonstration of existing nodes and cuckoo nodes illustration of the LANs in WLAN. Figure 4 is the illustration of the end-to-end delay results, where it shows the proposed scheme OCS-AODV outperforming the CS-DSDV and ACO-AODV routing protocols. Figure 4 Figure 4 is the illustration of the end-to-end delay results, where it shows the proposed scheme OCS-AODV outperforming the CS-DSDV and ACO-AODV routing protocols. Figure 5 is the illustration of the throughput results, where it shows the proposed scheme OCS-AODV generating a higher throughput than the CS-DSDV and ACO-AODV routing protocols. Figure 5 is the illustration of the throughput results, where it shows the proposed scheme OCS-AODV generating a higher throughput than the CS-DSDV and ACO-AODV routing protocols. Figure 6 is the illustration of the end-to-end delay results in a higher environment, where it shows the proposed scheme OCS-AODV generating a higher throughput than the CS-DSDV and ACO-AODV routing protocols. Figure 6 is the illustration of the end-to-end delay results in a higher environment, where it shows the proposed scheme OCS-AODV generating a higher throughput than the CS-DSDV and ACO-AODV routing protocols. is the illustration of the end-to-end delay results in a higher environment, where it shows the proposed scheme OCS-AODV generating a higher throughput than the CS-DSDV and ACO-AODV routing protocols. Figure 7 is the illustration of the throughput results in a higher environment, where it shows the proposed scheme OCS-AODV generating a higher throughput than the CS-DSDV and ACO-AODV routing protocols. Figure 7 is the illustration of the throughput results in a higher environment, where it shows the proposed scheme OCS-AODV generating a higher throughput than the CS-DSDV and ACO-AODV routing protocols. is the illustration of the throughput results in a higher environment, where it shows the proposed scheme OCS-AODV generating a higher throughput than the CS-DSDV and ACO-AODV routing protocols.
https://openalex.org/W2909408517
https://europepmc.org/articles/pmc6351941?pdf=render
English
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Adherence to Treatment in Stroke Patients
International journal of environmental research and public health/International journal of environmental research and public health
2,019
cc-by
6,907
Received: 30 November 2018; Accepted: 10 January 2019; Published: 11 January 2019 Abstract: Background: Compliance with medication in patients who have suffered stroke is usually not-optimal. This study aims to measure the level of compliance with the treatment and to identify socio-demographic, clinical, and subjective factors related to the long-term compliance of stroke patients with their treatment. Methods: 140 patients (66.4% males) suffered an ischemic stroke at least six months old, participated in the survey. Compliance was measured using the Medication Adherence Report Scale and the quality of life by the Stroke Specific Quality of Life questionnaire. Furthermore, the Beliefs about Medicines Questionnaire and the Brief Illness Perception Questionnaire on perceptions about the disease were assessed. The doctor–patient relationship was assessed by the Common-Sense Model of Self-Regulation questionnaire and the family support was assessed by the FSS scale. Univariate and multivariate analysis was employed to identify the significant factors affecting compliance in these stroke patients. Results: In 68.6% of patients the compliance was classified as optimal, in 25.7% as partial and as poor in 5.7%; the last two categories were treated as sub-optimal compliance in multivariate analysis. The high compliance was related to patient’s mental state (OR:3.94 95% CI: 1.84–4.46), the perception medication necessity (OR:1.26 95% CI: 1.01–1.56), and the doctor–patient communication (OR:1.76 95% CI: 1.15–2.70). Men showed a lower compliance than women, as well as increased concerns about taking medication (OR: 0.83, 95% CI: 0.69–0.99). Paradoxically, the work /productivity related quality of life was inversely associated with compliance (OR (95% CI): 0.44 (0.23 to 0.82)). Conclusions: The perception of medication necessity and the doctor–patient communication are manageable factors associated with compliance in treating patients who have suffered stroke. In addition, rehabilitation and return to work programs should consider these factors when providing support to those persons. Keywords: stroke; recovery; return to work; rehabilitation; compliance; beliefs; Greece International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health Int. J. Environ. Res. Public Health 2019, 16, 196; doi:10.3390/ijerph16020196 Emmanouela Cheiloudaki 1 and Evangelos C. Alexopoulos 1,2,* 1 School of Social Sciences, Hellenic Open University, 26335 Patra, Greece; litsachi@hotmail.com 2 Occupational Health Department, Metropolitan General Hospital, 15562 Athens, Greece * Correspondence: ecalexop@med.uoa.gr 1 School of Social Sciences, Hellenic Open University, 26335 Patra, Greece; litsachi@hotmail.com 2 Occupational Health Department, Metropolitan General Hospital, 15562 Athens, Greece * Correspondence: ecalexop@med.uoa.gr Keywords: stroke; recovery; return to work; rehabilitation; compliance; beliefs; Greece 1. Introduction Stroke often leads to death or permanent disability, causing functional or neurological deficits and affects the quality of life of both patients and their families [1,2]. Reduced cognitive function in stroke patients has been associated with reduced ability in daily life. Returning to work and sustaining employment emerge as key goals of rehabilitation and recovery by working-age stroke survivors. Successful return to work after stroke is a major factor in the achievement of high subjective well-being and life satisfaction [3,4]. Stroke related costs are enormous for patients, their families and the society and its rising frequency constitutes a major challenge for health policymakers [5]. The control of the reversible risk factors like diabetes, hyperlipidemia, atrial fibrillation, smoking, and hypertension reduce morbidity and mortality and improve quality of life. Effective tertiary prevention is based on the strict application of the guidelines given by the doctor to the patient which are mainly related to changes in lifestyle and specific medication [6]. However, studies have shown Int. J. Environ. Res. Public Health 2019, 16, 196; doi:10.3390/ijerph16020196 www.mdpi.com/journal/ijerph 2 of 11 Int. J. Environ. Res. Public Health 2019, 16, 196 that secondary management of risk factors is not optimal [7]. Compliance with medication constitutes a primary factor of treatment success since suboptimal compliance is a risk factor for secondary stroke or even death [8]. that secondary management of risk factors is not optimal [7]. Compliance with medication constitutes a primary factor of treatment success since suboptimal compliance is a risk factor for secondary stroke or even death [8]. The purpose of the study was to investigate the factors affecting the compliance to treatment of patients who have suffered ischemic stroke. Several factors related to the patient, to his/her beliefs about the disease and its treatment and his/her relationship with the doctor, were investigated. 2.2.1. MARS-5 Questionnaire (Medical Adherence Report Scale) The MARS-5 questionnaire on the adherence to medication, is widely used in stroke patients [9] asks respondents to rate the frequency of performing each of five behavioral aspects of non-compliance, on a five-point scale with the options: always, often, sometimes, rarely, never. Each answer is assigned the values 1 to 5 with: Always = 1, often = 2, sometimes = 3, seldom = 4, and never = 5. Scores are summed up to a scale ranging from 5 to 25, with higher scores indicating higher levels of patient compliance to treatment. In this research compliance under 23 were considered as sub-optimal. However, we have used in a limited extent a trichotomized compliance variable by dividing suboptimal compliance category in two levels: partial compliance (i.e., MARS-5 score 20–23) and poor or no compliance (i.e., MARS-5 score below 23). The Cronbach alpha coefficient was 0.650. 2.1. Study Design The survey was conducted in the neurology-neurosurgery outpatient clinic of the general hospital of Chania, Crete, during a four-month period (November 2015–February 2016). All patients visited the clinic during this period and fulfilling the eligibility criteria were asked to participate in the study, by giving their informed consent. Eligibility criteria included: (1) Patients who have suffered a first—non-hemorrhagic—stroke at least six months before the interview; (2) patients able to communicate in the Greek language; and (3) patients mentally capable of giving consent and able to comprehend and answer the questionnaire (e.g., patients with dementia and psychiatric disorders were excluded). The research protocol was submitted and approved by both the Hospital Ethics Committee and the Clinic director (Registration number: 16488/8-12-2015), under the conditions of patients’ anonymity and confidentiality and the exclusive use of the study results for research purposes. Participation was voluntary, the stress imposed on patients was limited to a minimum level, while not disrupting the operation of the clinic. Following a brief interview to ensure eligibility and provision of adequate information about the study, the questionnaires were completed by personal interview with patients. From 208 eligible patients 140 agreed to participate (response rate 67.3%) and gave their informed consent. The main reasons for refusals were reservations or fear and previous negative experience on research participation. 2.2. Research Tools 2.2.5. Brief Illness Perception Questionnaire (BIPQ) The BIPQ questionnaire is related to the perceptions of patients about ill-health and is the short version of the Revised Illness Perceptions Questionnaire [16,17]. It is designed to test cognitive and emotional ideas about the disease, describing the process by which individuals respond to a perceived threat to health. 5 questions test the effects of cognitive dimensions of disease (IPQ1- Consequences, IPQ2-Timeline of Disease, IPQ3-Personal Check, IPQ4-Therapeutic Monitoring, IPQ5-Identity) and the remaining three the emotional ones (IPQ6-Concern, IPQ7-Consistency, IPQ8-Emotional Effect). Each question rated from 0 to 10. The total score ranges from 8–80 while questions 3, 4, and 7 are calculated by a reverse scale [16]. An open-ended ninth question asks the respondent to indicate the three most important factors he/she believes has caused his/her illness. This questionnaire has been validated in Greek language [18]. 2.2.2. Stroke Specific Quality of Life Questionnaire (SS-QOL) The questionnaire has been extensively used to assess the quality of life in various sub-groups of patients who have suffered stroke and is validated in Greek language [10,11]. The scale consists of 49 questions assess the quality of life of patients with stroke during the last week, including physical, emotional, and social aspects of life. The questions (q) are grouped into 12 sectors and concerns: personal care (SC, 5q), vision (V, 3q), speech (L, 5q), mobility (M, 6q), work/productivity (W, 3q), upper limb function (UE, 5q), mental status (T, 3q), personality (P, 3q), mood (MD, 5q), family participation (FR, 3q), social participation (SR, 5q), and energy (E, 3q). The options in each of the 49 questions depends on the problem level and the rating scale ranges from 1.0 (worst quality of life) to 5.0 (optimum quality of life). The total score is calculated as the average score of the individual 3 of 11 Int. J. Environ. Res. Public Health 2019, 16, 196 sectors and values less than 4.2 are associated with a significant reduction in quality of life after stroke. The Cronbach coefficients were quite high for all subscales and total SSQOL: SSQOL-SC: 0.927, SSQOL-V: 0.851, SSQOL-L: 0.974, SSQOL-SSQOL-M: 0.908, SSQOL-W; 0.935, SSQOL-UE: 0.955, SSQOL-T: 0.827, SSQOL-P: 0.835, SSQOL-MD: 0.774, SSQOL-FR: 0.843, SSQOL-SR: 0.909, SSQOL-E: 0.938, SSQOL-TOTAL: 0.970. 2.2.3. Julkunen Family Support Scale (FSS) The scale of family support is intended to record the perceived support a person receives from his family and has been validated and used widely in Greece [12,13]. It consists of 13 items (seven are scored inversely) in a Likert scale ranging from 1 (strongly disagree) to 5 (strongly agree). A higher score corresponds to an increased perceived family support. The Cronbach’s alpha was 0.772. Because the scale is intended for people living with or in very close proximity with their family, people who live alone handled as missing values in our analysis. 2.2.4. Beliefs about Medicines Questionnaire (BMQ) The questionnaire assesses cognitive representations of patients about their medication [14] and has been validated in Greek language version [15]. It consists of two parts: (a) a specific part which assesses the beliefs of patients about the medicines prescribed for their personal use, and (b) a general part which assesses beliefs about medicines in general. In this study only, the specific part was used. The specific part consists of two subscales; five questions assess patients’ beliefs on the necessity of prescribed medication and six questions assess their concerns about possible negative effects. Respondents indicate the degree of agreement with each statement in a five-point Likert scale where 1 = strongly disagree and 5 = strongly agree and the rating sum ranges from 5 to 25 for the first subscale and from 6 to 30 for the second one. The higher scores indicating stronger beliefs on the concepts (necessity and concerns) represented by each subscale. The Cronbach’s alpha values were low for the dimension of necessity (0.560) and high for the dimension of concerns (0.779); for the whole specific part was 0.695. 2.3. Statistical Methods The mean and standard deviation were used to describe the continuous variables while the frequency and the % frequency were used for discrete variables. Pearson chi square test was used to test the association between discrete variables and independent samples t-test was used for continuous variables comparison between two groups or the one-way ANOVA for more than two groups comparison. Univariate logistic regression was used to assess crude’s Odds ratios and multiple logistic regression was applied to estimate the weighted OR ratios (adjusted OR). OR is related to per point increase of the independent variable scale. In this research, we have used compliance mainly as dichotomized variable but in a limited extent a trichotomized variable was entered a univariate analysis to explore if the trends are constant as a kind of sensitivity analysis. So, optimal compliance is the same both in dichotomized and trichotomized variable (i.e., MARS-5 scores 14 or 25). In dichotomized variable sub-optimal compliance include all scores below 24. In trichotomized variable scores 20–23 were considered as partial compliance while scores below 20 as poor or no compliance. The statistical analysis was done using IBM SPSS Statistics for Windows, Version 21.0 (IBM Corp, Armonk, NY, USA). 2.2.6. CS-SRM Questionnaire The patient-doctor relationship and communication were tested with the CS-SRM questionnaire which assess whether doctors provide patients with the appropriate information to fully understand their illness and treatment [19]. The questionnaire consists of seven questions refer to specific domain of representations of the disease: causes, identification, timing, control and consequences. The scores are 1 for yes/agree, 0 for no/disagree, and a non-rating option of “do not know” or “not applicable”. The total score ranging from 0–7, with higher scores indicating good communication between doctor and patient [20]. The Cronbach alpha value was 0.561. Int. J. Environ. Res. Public Health 2019, 16, 196 4 of 11 3. Results The higher mean values were reported for vision related subscale (4.7), self-care (4.7), the upper limb function (4.5) and speech (4.5). The lower values recorded in the social participation (3.9), energy (3.9), and personality (3.4) SS-QOL subscales. The family support total score (FSS scale) ranged from 27–64 with a mean of 59.6 and a median of 61.0. Concerning respondents’ beliefs on the three most important factors causing their illness anxiety, unhappiness and hypertension were reported followed by diabetes, thrombophilia, and lifestyle. The mean score of perceived necessity of medication was 18.8 (range 5–25) and of the perceived concerns 11.1 (range 6–30). In perceptions of respondents about their illness (IPQ) the largest average value was monitored in the therapeutic control scale (8.2 ± 1.7) following by personal control (7.5 ± 1.6) and consistency (7.0 ± 1.6) scales. The perception of the doctor patient relationship as perceived by the patient CS-SMR questionnaire was high (mean 5.9 and a median of 6.0) (Table 2). Table 2. Study variables and its relationship with compliance. 3. Results The study sample consisted of 140 patients who have suffered a first ischemic stroke. 93 (66.4%), were male, 16 (11.4%) were living alone and 52 (37.1%) were smokers. The mean age of patients was 64.2 (± 9.0) years old with an age range of 35–79 years (Table 1). Out of the 16 people who living alone, four had no children, 2 had children in other city and 10 in the same city. 52 (37.1%) patients, were current smokers, more among males compared to females (p = 0.055). Age and education did not differ significantly between men and women but more women (23.4%) were living alone compared with men (5.4%, p = 0.002). Table 1. Patients characteristics. Study Variable Males (n = 93) Females (n = 47) All (n = 140) Mean SD Mean SD Mean SD p Age (years) 65.1 8.5 62.3 9.7 64.2 9.0 0.088 Number of children 2.3 1.9 2.1 1.0 2.3 1.1 0.222 n % n % n % Living status Living alone 5 5.4 11 23.4 16 11.4 0.002 Living with others 88 94.6 36 76.6 124 88.6 Children residence Same house 15 17.4 4 9.3 19 14.7 0.436 Same city 64 74.4 36 83.7 100 77.5 Another city 7 8.1 3 7.0 10 7.8 Education Primary 37 39.8 23 48.9 60 42.9 0.388 Secondary 36 38.7 18 38.3 54 38.6 Higher 20 21 6 12.8 26 18.6 Smoker Current 38 40 14 29.8 52 37.1 0.055 Never 30 32 25 53.2 55 39.3 Ex- 25 26 8 17.0 33 23.6 Support in medication Never 67 72 36 76.6 103 73.6 0.564 Always/sometimes 26 28 11 23.4 37 26.4 Table 1. Patients characteristics. The median number of daily medicines was 4, ranging from 1–12 drugs. In the present study, 73.6% of patients reported no support in taking their medication while 37 patients were supported; mainly by their spouse (70.3%). Females hold mainly the role of the caregiver (88.5%) compared to men (27.3%, p < 0.001) (Table 1). The mean compliance score (MARS-5 scale) was 23.7 (± 2.1), the minimum score was 10 and 96 patients (68.6%) were considered as optimally compliant. 5 of 11 Int. J. Environ. Res. Public Health 2019, 16, 196 The quality of life total score (SSQOL scale) was on average 4.3 (± 0.7) with a range from 2 to 4.9. 3. Results Study Variable All Patients Compliance Suboptimal Optimal n % n % p Gender (m/f) 93/47 29/15 31.2/31.9 64/32 68.8/68.1 NS Living with others/alone 124/16 35/9 28.2/56.3 89/7 71.8/43.8 0.023 Educational level (higher/lower) 26/114 5/39 19.2/34.2 21/75 80.8/65.8 NS * Current smoker (no/yes) 88/52 27/17 30.7/32.7 61/35 69.3/67.3 NS Mean SD Mean SD Mean SD Age (y) 64.2 9.0 63.8 10.1 64.3 8.5 NS Disease duration (y) 4.8 5.2 3.3 3.2 0.048 SSQOL Self-care (SC) 4.7 0.7 4.8 0.5 4.6 0.7 NS Vision (V) 4.7 0.6 4.5 0.8 4.8 0.4 0.012 Language (L) 4.5 0.9 4.5 0.8 4.5 0.9 NS Mobility (M) 4.2 0.9 4.4 0.6 4.1 1.0 0.125 Work/Productivity (W) 4.1 1.0 4.3 0.7 4.0 1.1 0.111 Upper Extremity function (UE) 4.5 0.9 4.7 0.6 4.5 0.9 NS Thinking (T) 4.2 1.0 3.6 1.1 4.4 0.8 <0.001 Personality (P) 3.4 1.1 3.2 1.2 3.5 1.1 0.169 Mood (MD) 4.3 0.7 4.2 0.9 4.4 0.7 0.164 Family roles (FR) 4.4 0.9 4.3 1.1 4.5 0.9 NS Social roles (SR) 3.9 1.0 3.8 1.0 4.0 1.0 NS Energy (E) 3.9 1.2 3.6 1.4 4.0 1.1 0.119 All subscales 4.3 0.7 4.2 0.7 4.3 0.7 NS FSS 59.6 5.1 58.2 8.0 60.2 3.2 0.080 BMQ Necessity 18.8 2.8 18.0 3.7 19.1 2.3 0.038 BMQ Concern 11.1 4.3 12.6 4.6 10.4 3.9 0.004 IPQ 39.0 11.7 39.8 10.7 38.6 12.2 NS CS-CRM 5.9 1.3 5.07 1.48 6.29 1.08 <0.001 In bold: p < 0.05; NS: p > 0.20; * educational level had three subcategories. Table 2. Study variables and its relationship with compliance. In the univariate analysis of the relation of socio-demographic and disease duration on compliance, living status was of importance (OR: 3.27, 95% CI: 1.13 to 9.46). 56.3% of patients living alone did not comply optimally compared to others (28.2%). This is anticipated to partially capture the relation with the marital status. Compliance was also related to disease duration. Compliant patients had significantly less mean illness duration of 3.3 years compared with the non-compliant ones (4.8 years). Gender, age, the number of children, educational level, smoking status, support in taking medication, did not found to be significantly related with compliance (Table 2). Compliance was related to sight subscale of quality of life related (OR (95% CI): 2.30 (1.20 to 4.43)) and the mental state subscale OoL (OR (95% CI): 2.49 (1.64 to 3.79)) (Table 2). 3. Results Compliance had a borderline significant with the family support scale (OR (95% CI): 1.07 (0.99–1.16)) and the perceived necessity and concerns scales of medication (BMQ) with ORs (95% CI) of 1.14 (1.01 to 1.30) and 0.89 (0.82 to 0.97), respectively (Table 2). The physician–patient communication (CS-SRM scale) showed also significant relation to compliance (OR (95% CI): 2.11 (1.50–2.96)) (Table 2). Compliance was related to sight subscale of quality of life related (OR (95% CI): 2.30 (1.20 to 4.43)) and the mental state subscale OoL (OR (95% CI): 2.49 (1.64 to 3.79)) (Table 2). Compliance had a borderline significant with the family support scale (OR (95% CI): 1.07 (0.99–1.16)) and the perceived necessity and concerns scales of medication (BMQ) with ORs (95% CI) of 1.14 (1.01 to 1.30) and 0.89 (0.82 to 0.97), respectively (Table 2). The physician–patient communication (CS-SRM scale) showed also significant relation to compliance (OR (95% CI): 2.11 (1.50–2.96)) (Table 2). 6 of 11 Int. J. Environ. Res. Public Health 2019, 16, 196 As previously mentioned, we have used compliance as trichotomized variable in a univariate analysis to explore if the trends are constant as a kind of sensitivity analysis (Table 3). In the trichotomized variable sub-optimal compliance was further divided in two groups: the partial compliant group (scores 20–23) and the poor or no compliant group (scores below 20). Partial compliance was reported by 36 people and, poor by 8 individuals. Table 3 shows the comparison of optimal and partial compliance with the poor. Compared with poor compliance partial compliance was related to gender; more male patients reported poor compliance (OR: 0.11, 95% CI: 0.02 to 0.65). Increased concerns related to lower compliance (OR: 0.83, 95% CI: 0.69–0.99) as current smoking also did (OR: 0.15, 95% CI: 0.03–0.84). Living with others were less common among those with poor compliance compared to those with partial and optimal compliance (Table 3). Concerning the quality of life subscales, the single most important was that associated with vision problems with an OR 7.72 for optimal and 5.33 for partial compliance. Other important relations were found between mental status (SSQOL-T OR: 3.31), energy (SSQOL-E OR: 1.90) and mood (SSQOL-MD OR: 2.17) (Table 3). Family and social participation SSQOL subscales exhibited borderline significance while were interrelated with family support (Table 3). All variables exhibited a p-value <0.200 were included to multivariate modelling (data not shown in Table). 3. Results Environ. Res. Public Health 2019, 16, 196 In the final multivariate modeling the dichotomized outcome variable (i.e., MARS-5 scores above 23 as optimal and all the others as sub-optimal compliance) was used. All variables under study on treatment compliance exhibited a p-value <0.200 were included to univariate and multivariate modelling (Table 4). The most important relations with compliance were found for mental state (SSQOL-T score) with weighted OR 5.14 (95% CI: 2.64 to 10); good doctor–patient relationship (CS-SRM scale) with weighted OR 1.96 (95% CI: 1.35 to 2.85) and the perceived need for medication with weighted OR 1.29 (95% CI: 1.06 to 1.56) (Table 4). The work /productivity quality of life was inversely associated with compliance (OR (95% CI): 0.44 (0.23 to 0.82)) (Table 4). Table 4. Multivariate modelling of study variables on treatment compliance (optimal vs. all other) of stroke patients. Table 4. Multivariate modelling of study variables on treatment compliance (optimal vs. all other) of stroke patients. Study Variable Full Model * Final Model * OR 95% LL 95% UL p OR 95% LL 95% UL p Living with others 2.11 0.24 18.42 NS Disease duration (y) 0.88 0.76 1.03 0.115 SSQOL Vision (V) 2.30 0.66 8.04 0.191 Mobility (M) 0.46 0.11 1.98 NS Work/Productivity (W) 0.45 0.15 1.33 0.150 0.44 0.23 0.82 0.010 Thinking (T) 3.94 1.84 8.46 <0.001 5.14 2.64 10.00 <0.001 Personality (P) 0.89 0.54 1.46 NS Mood (MD) 0.75 0.22 2.54 NS Energy (E) 1.35 0.60 3.02 NS BMQ Necessity 1.26 1.01 1.56 0.038 1.29 1.06 1.56 0.010 BMQ Concern 0.90 0.78 1.04 0.156 CS-CRM 1.76 1.15 2.70 0.009 1.96 1.35 2.85 <0.001 * Full model: all variables with p < 0.2 in univariate analysis; Final model: variables retained in the final model by forward selection; in bold: p < 0.05; NS: p > 0.20. ivariate modelling of study variables on treatment compliance (optimal vs. all other) of 3. Results Compared to those with poor compliance, three variable reached statistically significant level for optimal compliance, in multivariate analysis: the medication necessity (OR: 2.18, 95% CI: 1.04 to 4.57); the medication concerns (OR: 0.51, 95% CI: 0.27 to 0.99) and in a borderline level the doctor–patient communication (OR: 3.41, 95% CI: 0.80 to 14.50). As expected, due to the low sample sizes between the poor/no and partial compliance categories, the comparison did not reach statistically significant level in any of the study variables. However, medication necessity, medication concerns and doctor–patient communication showed similar trends (OR: 1.93, 95% CI: 0.93 to 4.02; OR: 0.54, 95% CI: 0.28 to 1.05; and 1.96, 95% CI: 0.45 to 8.45, respectively). Table 3. Univariate analysis of factors affecting the level of compliance (optimal or partial vs. poor/no) of stroke patients. Table 3. Univariate analysis of factors affecting the level of compliance (optimal or partial vs. poor/no) of stroke patients. Study Variable Adherence Optimal Partial OR 95%LL 95%UL OR 95%LL 95%UL Age (years) 1.01 0.97 1.05 1.00 0.92 1.08 Gender (RC = females) 1.00 0.92 1.09 0.11 0.02 0.65 Living with others 12.71 2.61 62.05 6.20 1.16 33.17 Number of children 0.80 0.47 1.78 1.41 0.63 3.14 Education (RC = Higher) Primary-secondary 1.54 0.34 6.90 1.56 0.32 7.70 Current Smoker 0.19 0.04 1.00 0.15 0.03 0.84 Support in medication 0.33 0.08 1.44 0.33 0.07 1.62 SSQOL Self-care (SC) 1.21 0.51 2.91 2.40 0.72 7.99 Vision (V) 7.72 2.82 21.16 5.33 1.86 15.3 Language (L) 1.24 0.62 2.48 1.38 0.64 2.97 Mobility (M) 0.97 0.43 2.15 1.56 0.60 3.86 Work/Productivity (W) 1.08 0.56 2.11 1.69 0.78 3.67 Upper Extremity function (UE) 1.07 0.50 2.32 1.55 0.64 3.77 Thinking (T) 3.31 1.63 6.74 1.41 0.71 2.81 Personality (P) 1.89 0.93 3.85 1.64 0.77 3.45 Mood (MD) 2.17 1.02 4.64 1.78 0.79 4.00 Family roles (FR) 1.78 0.97 3.28 1.72 0.88 3.37 Social roles (SR) 1.80 0.98 3.32 1.76 0.91 3.41 Energy (E) 1.90 1.06 3.39 1.65 0.90 3.05 All subscales 2.18 0.92 5.16 2.31 0.89 6.05 FSS 1.13 1.01 1.26 1.06 0.96 1.18 BMQ Necessity 1.32 1.05 1.68 1.20 0.95 1.53 BMQ Concern 0.76 0.64 0.90 0.83 0.69 0.99 IPQ Total 0.98 0.92 1.04 0.98 0.92 1.05 CS-SRM 2.57 1.56 4.32 1.27 0.82 1.97 RC = Reference Category; in bold: p < 0.05; in shading: variables with p < 0.05 in multivariate multinomial analysis. 7 of 11 Int. J. 4. Discussion In this research, we have studied the compliance to treatment of 140 patients who have suffered ischemic stroke and its relation to various variables. The compliance was measured by the Medication Adherence Report Scale and was treated as dichotomous (optimal or suboptimal) or trichotomous (optimal, partial, and poor) variable. Most patients (68.6%) were categorized as optimally compliant while the 25.7% as partially compliant and the 5.7% as of poor compliance. The patients’ quality of life score (as measured by the SS-QOL questionnaire) was quite high. These high rates are not uncommon in patients with ischemic stroke and has been associated with high compliance rates, as in our study [21]. Compliance was related to less visual and mental problems which have been found to affect compliance [22]. Cognitive disorders have been shown to adversely affect treatment compliance [9,23] as well as emotional disorders such as anger, fatigue and stress, confirming the results of this research. In our study, the age of the patients was not related with compliance, although in another study, younger stroke patients reported that they often forgot some of their doses [9]. Other studies also support higher compliance rates in elderly patients [24–26]. We found that male patients showed a lower compliance than women, who hold significantly more often the caring role compared to men. This finding is consistent with previous research in which most non-compliant patients were men and often were depended in their wife’s help or relatives’ support [27]. Living alone was related to lower compliance in our study, as other studies have also shown [23,27]. Lack of family support and the feeling of conflict within the family have also found to decrease compliance in patient treatment [2,28]. 8 of 11 Int. J. Environ. Res. Public Health 2019, 16, 196 The inverse relation of compliance with time (disease duration) found in our study has been confirmed in several studies which consist a severe medical care problem in chronic disease management [27,29–32]. The inverse relation of compliance with time (disease duration) found in our study has been confirmed in several studies which consist a severe medical care problem in chronic disease management [27,29–32]. The compliant patients reported more positive beliefs about medicines, deemed them as necessary ("necessity") and less negative beliefs questioning the need and the benefits or worrying about the damage they might cause ("concerns"), as measured by the BMQ questionnaire. 4. Discussion Similar results were observed in a study from the United Kingdom, in which statistically significant correlation found between anxiety and reduced compliance [9]. In other studies, non-compliant patients reported lower scores on the need and benefits and higher ones on concerns [27,33]. We did not find a significant relation between the perception of the disease (as measured by IPQ questionnaire) and compliance. Several studies have also found stronger correlations between medication beliefs than disease perceptions [34,35]. This is also consistent with the extended model of Leventhal (2011), according to which perceptions of the disease is directly related to compliance but the beliefs about medication are often stronger [36]. The relation of compliance with the doctor–patient communication is well established. Numerous studies have emphasized that the effective communication between patient and doctor as well as the reduction of any maladaptive belief that patients have about health or illness is of large importance in patient compliance [18,37–41]. Moreover, the need to optimize care under the increasing pressures of ageing, multi-morbidity, disease chronicity is constantly increasing. Within a primary healthcare setting the implementation of chronic care model interventions improved outcomes and patient compliance with treatment. However, their implementation is restricted by various factors. Considerable resources are needed to support implementation and sustainability and human factors played essential roles since both healthcare providers should be prepared for the implementation, and patients should be supported to receive care changes [42]. In this context and under the initiative of people-centered and integrated care, the role of care manager (i.e., specially trained nurses) seem effective to empower patients to make lifestyle changes and to achieve better compliance to treatment and care recommendations [43]. A noteworthy finding in our study was that those patients reported better quality of life in the domain of work and productivity and in a lesser extent also to mobility were far less compliant. This underlies the need to focus on these patients when returning to work and to monitor them during the whole recovery process. So, in addition to the doctor–patient relationship the occupational health services, where available, should continuously assesses the progress and medication adherence of these vulnerable productive people. The specific health care system and study setting may influence generalizability of our findings. 4. Discussion The use of self-report measures for all variables under study is a limitation of our research design; however, is very common in this type of research in which is difficult to utilize additional resources in an outpatient setting. The main limitation is the low sample size given the high number of variables collected. We have treated this weakness by applying a p-value limit from univariate logistic analysis (p < 0.200) in a backward logistic regression modelling instead of a complete logistic regression model. Further research should consider these limitations in order to increase the utility of findings and application to practice. management [27,29–32]. References 1. Strong, K.; Mathers, C.; Bonita, R. Preventing stroke: Saving lives around the world. Lancet Neurol. 2007, 6, 182–187. [CrossRef] 2. DiMatteo, M.R. Variations in patient’s adherence to medical recommendations: A quantitative review of 50 years of research. Med. Care 2004, 42, 200–209. [CrossRef] [PubMed] 3. Wang, Y.; Kapellusch, J.; Gard, A. Important factors influencing the return to work after stroke. Work 2014, 47, 553–559. 4. Wolfenden, B.; Grace, M. Returning to work after stroke: A review. Int. J. Rehabil. Res. 2009, 32, 93–97. [CrossRef] [PubMed] 5. Mukherjee, D.; Patil, C.G. Epidemiology and the global burden of stroke. World Neurosurg. 2011, 76, 85–90. [CrossRef] [PubMed] 5. Mukherjee, D.; Patil, C.G. Epidemiology and the global burden of stroke. World Neurosurg. 2011, 76, 85–90. [CrossRef] [PubMed] 6. Slark, J. Adherence to secondary prevention. Strategies after stroke: A review of the literature. Br. J. Neurosci. Nurs. 2010, 6, 282–286. [CrossRef] 6. Slark, J. Adherence to secondary prevention. Strategies after stroke: A review of the literature. Br. J. Neurosci. Nurs. 2010, 6, 282–286. [CrossRef] 7. Chaudhry, H.J.; McDermott, B. Recognizing and improving patient non-adherence to statin therapy. Curr. Atheroscler. Rep. 2008, 10, 19–24. [CrossRef] 8. Wang, Y.; Wu, D.; Wang, Y.; Ma, R.; Wang, C.; Zhao, W. A survey of adherence to secondary ischemic stroke prevention. Neurol. Res. 2006, 28, 16–20. [CrossRef] 9. O’Carroll, R.; Whittaker, J.; Hamilton, B.; Johnston, M.; Sudlow, C.; Dennis, M. Predictors of adherence to secondary preventive medication in stroke patients. Ann. Behav. Med. 2011, 41, 383–390. [CrossRef] 9. O’Carroll, R.; Whittaker, J.; Hamilton, B.; Johnston, M.; Sudlow, C.; Dennis, M. Predictors of adherence to secondary preventive medication in stroke patients. Ann. Behav. Med. 2011, 41, 383–390. [CrossRef] 10. Williams, L.S.; Weinberger, M.; Clark, D.O.; Harris, L.E.; Biller, J. Development of a stroke-specific of life 10. Williams, L.S.; Weinberger, M.; Clark, D.O.; Harris, L.E.; Biller, J. Development of a stroke-specific of life scale. Stroke 1999, 30, 1362–1369. [CrossRef] 11. Kapreli, E.; Paplamata, Z.; Damplia, Z.; Strimpakos, N.; Paras, G. Reliability and responsiveness of the Greek version of the stroke-specific quality of life Questionnaire (SSQOL-GR): A Pilot Study. In Proceedings of the 3rd International Congress on Neurology and Neuroepidemiology, Abu Dhabi, UAE, 21–23 November 2013; Volume 41, pp. 223–316. 12. Tselebis, A.; Anagnostopoulou, T.; Bratis, D.; Moulou, A.; Maria ASikaras, C.; Ilias, I. 5. Conclusions According to our results adherence to treatment in patients with stroke associated with a variety of social, medical, and personal factors. The disease perceived severity, the doctor–patient relationship and the patient’s perception of medication need were all related to compliance. Interventions aimed at compliance are complex and include self-monitoring schemes, counseling and supportive care. In a patient-centered and integrated care, personal beliefs of patients should be carefully considered, and healthcare providers need to recognize the concerns patients have for medicines, stressing the need of 9 of 11 Int. J. Environ. Res. Public Health 2019, 16, 196 these benefits, making the shape simple and adjusted to the lifestyle of the patient, with a view to the reduction of a secondary stroke episode. these benefits, making the shape simple and adjusted to the lifestyle of the patient, with a view to the reduction of a secondary stroke episode. Author Contributions: Conceptualization, E.C. and E.C.A.; Data curation, E.C.; Formal analysis, E.C. and E.C.A.; Investigation, E.C.; Methodology, E.C.A.; Project administration, E.C. and E.C.A.; Supervision, E.C.A.; Visualization, E.C.A.; Writing—original draft, E.C.; Writing—review & editing, E.C.A. Author Contributions: Conceptualization, E.C. and E.C.A.; Data curation, E.C.; Formal analysis, E.C. and E.C.A.; Investigation, E.C.; Methodology, E.C.A.; Project administration, E.C. and E.C.A.; Supervision, E.C.A.; Visualization, E.C.A.; Writing—original draft, E.C.; Writing—review & editing, E.C.A. Funding: This research received no external funding. Funding: This research received no external funding. Acknowledgments: We thanks all the participants and Georgakakis George for his support. Acknowledgments: We thanks all the participants and Georgakakis George for his support. Conflicts of Interest: The authors declare no conflict of interest. References Patient characteristics associated with medication 24. Sappok, T.; Faulstich, A.; Stuckert, E.; Kruck, H.; Marx, P.; Koennecke, H.C. Compliance with secondary prevention of ischemic stroke: A prospective evaluation. Stroke 2001, 32, 1884–1889. [CrossRef] [PubMed] prevention of ischemic stroke: A prospective evaluation. 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The beliefs about medicines Questionnaire: The development and Evaluation of a New Method for Assessing the cognitive Representation of Medication. Psychol. Health 1999, 14, 1–24. [CrossRef] 15. Komninos, I.; Micheli, K.; Roumeliotaki, T.; Horne, R. Adaptation and validation of the beliefs about medicines questionnaire (BMQ) in primary care patients in Greece. Eur. J. Pers. Cent. Health Care 2013, 1, 224–231. [CrossRef] 16. Broadbent, E.; Petrie, K.; Main, J.; Weinman, J. The brief Illness Perception Questionnaire. J. Psychosom. Res. 2006, 60, 631–633. [CrossRef] 17. Moss-Morris, R.; Weinman, J.; Petrie, K.J.; Horne, R.; Cameron, L.D.; Buick, D. The revised Illness Perception Questionnaire (IPQ-R). Psychol. Health 2002, 17, 1–16. [CrossRef] 10 of 11 Int. J. Environ. Res. Public Health 2019, 16, 196 18. Karademas, E.C.; Bakouli, A.; Bastounis, A.; Kallergi, F.; Tamtami, P.; Theofilou, M. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 42. Davy, C.; Bleasel, J.; Liu, H.; Tchan, M.; Ponniah, S.; Brown, A. Factors influencing the implementation of chronic care models: A systematicliterature review. BMC Fam. Pract. 2015, 16, 102. [CrossRef] [PubMed] 42. Davy, C.; Bleasel, J.; Liu, H.; Tchan, M.; Ponniah, S.; Brown, A. Factors influencing the implementation of chronic care models: A systematicliterature review. BMC Fam. Pract. 2015, 16, 102. [CrossRef] [PubMed] 43 Ciccone M M ; Aquilino A ; Cortese F ; Scicchitano P; Sassara M ; Mola E ; Rollo R ; Caldarola P; References Congitive science speaks to the “common-sense” of chronic illness management. Ann. Behav. Med. 2011, 41, 152–163. [CrossRef] [PubMed] 37. Elliott, R.A.; Ross-Degnan, D.; Adams, A.S.; Safran, D.G.; Soumerai, S.B. 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Factors influencing the implementation of chronic care models: A systematicliterature review. BMC Fam. Pract. 2015, 16, 102. [CrossRef] [PubMed] 43. Ciccone, M.M.; Aquilino, A.; Cortese, F.; Scicchitano, P.; Sassara, M.; Mola, E.; Rollo, R.; Caldarola, P.; Giorgino, F.; Pomo, V.; et al. Feasibility and effectiveness of a disease and care management model in the primary health care system for patients with heart failure and diabetes (Project Leonardo). Vasc. Health Risk Manag. 2010, 6, 297–305. [CrossRef] [PubMed] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Dušan Zorica Mathematical Institute of the Serbian Academy of Sciences and Arts: Matematicki institut Srpske akademije nauka i umetnosti Research Article Keywords: dissipative and generative electric elements, memory type fractional constitutive models, fractional RC and RL circuits, transient and steady state regimes, frequency and asymptotic analysis Keywords: dissipative and generative electric elements, memory type fractional constitutive models, fractional RC and RL circuits, transient and steady state regimes, frequency and asymptotic analysis Posted Date: March 26th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-332858/v1 ❆❜str❛❝t ●❡♥❡r❛❧✐③❡❞❝❛♣❛❝✐t♦r ✭✐♥❞✉❝t♦r✮✐s ❝♦♥st✐t✉t✐✈❡❧②♠♦❞❡❧❡❞❜②❡①♣r❡ss✐♥❣❝❤❛r❣❡✭♠❛❣♥❡t✐❝✢✉①✮✐♥ t❡r♠s ♦❢✈♦❧t❛❣❡✭❝✉rr❡♥t✮♠❡♠♦r②❛s ❛s✉♠♦❢✐♥st❛♥t❛♥❡♦✉s ❛♥❞♣♦✇❡r t②♣❡❤❡r❡❞✐t❛r②❝♦♥tr✐❜✉t✐♦♥s ❛♥❞✐t ✐s ♣r♦✈❡❞t♦❜❡❛❞✐ss✐♣❛t✐✈❡❡❧❡❝tr✐❝❡❧❡♠❡♥t ❜②t❤❡r♠♦❞②♥❛♠✐❝❛♥❛❧②s✐s✳❖♥t❤❡❝♦♥tr❛r②✱ ❣❡♥❡r❛❧✐③❡❞❝❛♣❛❝✐t♦r ✭✐♥❞✉❝t♦r✮❛s ❛❣❡♥❡r❛t✐✈❡❡❧❡❝tr✐❝❡❧❡♠❡♥t ✐s ♠♦❞❡❧❡❞✉s✐♥❣t❤❡s❛♠❡❢♦r♠♦❢t❤❡ ❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥✱❜✉t ❜②❡①♣r❡ss✐♥❣✈♦❧t❛❣❡✭❝✉rr❡♥t✮✐♥t❡r♠s ♦❢❝❤❛r❣❡✭♠❛❣♥❡t✐❝✢✉①✮♠❡♠♦r②✳ ❚❤❡s❡❝♦♥st✐t✉t✐✈❡♠♦❞❡❧s ❛r❡✉s❡❞✐♥tr❛♥s✐❡♥t ❛♥❞st❡❛❞②st❛t❡r❡❣✐♠❡❛♥❛❧②s✐s ♦❢t❤❡s❡r✐❡s RC ❛♥❞ RL ❝✐r❝✉✐ts s✉❜❥❡❝t t♦❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡✱❛s ✇❡❧❧❛s ✐♥t❤❡st✉❞②♦❢❝✐r❝✉✐ts✬❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ✐♥❝❧✉❞✐♥❣❛s②♠♣t♦t✐❝❜❡❤❛✈✐♦r✳ ❑❡②✇♦r❞s✿❞✐ss✐♣❛t✐✈❡❛♥❞❣❡♥❡r❛t✐✈❡❡❧❡❝tr✐❝❡❧❡♠❡♥ts❀♠❡♠♦r②t②♣❡❢r❛❝t✐♦♥❛❧❝♦♥st✐t✉t✐✈❡♠♦❞❡❧s❀ ❢r❛❝t✐♦♥❛❧RC ❛♥❞RL ❝✐r❝✉✐ts❀tr❛♥s✐❡♥t ❛♥❞st❡❛❞②st❛t❡r❡❣✐♠❡s❀❢r❡q✉❡♥❝②❛♥❞❛s②♠♣t♦t✐❝❛♥❛❧②s✐s DOI: https://doi.org/10.21203/rs.3.rs-332858/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Nonlinear Dynamics on August 20th, 2021. See the published version at https://doi.org/10.1007/s11071-021-06809-1. ✯❉❡♣❛rt♠❡♥t ♦❢P♦✇❡r✱❊❧❡❝tr♦♥✐❝❛♥❞❚❡❧❡❝♦♠♠✉♥✐❝❛t✐♦♥❊♥❣✐♥❡❡r✐♥❣✱❋❛❝✉❧t②♦❢❚❡❝❤♥✐❝❛❧❙❝✐❡♥❝❡s✱❯♥✐✈❡rs✐t②♦❢◆♦✈✐ ❙❛❞✱❚r❣❉✳❖❜r❛❞♦✈✐➣❛✻✱✷✶✵✵✵◆♦✈✐❙❛❞✱❙❡r❜✐❛✱❦r✐st✐❛♥✳❤❛s❦❛❅✉♥s✳❛❝✳rs ❸ ❹❉❡♣❛rt♠❡♥t ♦❢P❤②s✐❝s✱❋❛❝✉❧t②♦❢❙❝✐❡♥❝❡s✱❯♥✐✈❡rs✐t②♦❢◆♦✈✐❙❛❞✱❚r❣❉✳❖❜r❛❞♦✈✐➣❛✹✱✷✶✵✵✵◆♦✈✐❙❛❞✱❙❡r❜✐❛✱ ❞✉s❛♥✳③♦r✐❝❛❅❞❢✳✉♥s✳❛❝✳rs ❛♥❞▼❛t❤❡♠❛t✐❝❛❧■♥st✐t✉t❡✱❙❡r❜✐❛♥❆❝❛❞❡♠②♦❢❆rts ❛♥❞❙❝✐❡♥❝❡s✱❑♥❡③❛▼✐❤❛✐❧❛✸✻✱✶✶✵✵✵ ❇❡♦❣r❛❞✱❙❡r❜✐❛✱❞✉s❛♥❴③♦r✐❝❛❅♠✐✳s❛♥✉✳❛❝✳rs ✯❉❡♣❛rt♠❡♥t ♦❢P♦✇❡r✱❊❧❡❝tr♦♥✐❝❛♥❞❚❡❧❡❝♦♠♠✉♥✐❝❛t✐♦♥❊♥❣✐♥❡❡r✐♥❣✱❋❛❝✉❧t②♦❢❚❡❝❤♥✐❝❛❧❙❝✐❡♥❝❡s✱❯♥✐✈❡rs✐t②♦❢◆♦✈✐ ❙❛❞✱❚r❣❉✳❖❜r❛❞♦✈✐➣❛✻✱✷✶✵✵✵◆♦✈✐❙❛❞✱❙❡r❜✐❛✱❦r✐st✐❛♥✳❤❛s❦❛❅✉♥s✳❛❝✳rs ❸❉❡♣❛rt♠❡♥t ♦❢P♦✇❡r✱❊❧❡❝tr♦♥✐❝❛♥❞❚❡❧❡❝♦♠♠✉♥✐❝❛t✐♦♥❊♥❣✐♥❡❡r✐♥❣✱❋❛❝✉❧t②♦❢❚❡❝❤♥✐❝❛❧❙❝✐❡♥❝❡s✱❯♥✐✈❡rs✐t②♦❢◆♦✈✐ ❙❛❞✱❚r❣❉✳❖❜r❛❞♦✈✐➣❛✻✱✷✶✵✵✵◆♦✈✐❙❛❞✱❙❡r❜✐❛✱st❡✈❛♥✳❝✈❡t✐❝❛♥✐♥❅✉♥s✳❛❝✳rs ❹❉❡♣❛rt♠❡♥t ♦❢P❤②s✐❝s✱❋❛❝✉❧t②♦❢❙❝✐❡♥❝❡s✱❯♥✐✈❡rs✐t②♦❢◆♦✈✐❙❛❞✱❚r❣❉✳❖❜r❛❞♦✈✐➣❛✹✱✷✶✵✵✵◆♦✈✐❙❛❞✱❙❡r❜✐❛✱ ❞✉s❛♥✳③♦r✐❝❛❅❞❢✳✉♥s✳❛❝✳rs ❛♥❞▼❛t❤❡♠❛t✐❝❛❧■♥st✐t✉t❡✱❙❡r❜✐❛♥❆❝❛❞❡♠②♦❢❆rts ❛♥❞❙❝✐❡♥❝❡s✱❑♥❡③❛▼✐❤❛✐❧❛✸✻✱✶✶✵✵✵ ✱ ❣ ✱ ✱ ✱ ❸❉❡♣❛rt♠❡♥t ♦❢P♦✇❡r✱❊❧❡❝tr♦♥✐❝❛♥❞❚❡❧❡❝♦♠♠✉♥✐❝❛t✐♦♥❊♥❣✐♥❡❡r✐♥❣✱❋❛❝✉❧t②♦❢❚❡❝❤♥✐❝❛❧❙❝✐❡♥❝❡s✱❯♥✐✈❡rs✐t②♦ ❞✱❚r❣❉✳❖❜r❛❞♦✈✐➣❛✻✱✷✶✵✵✵◆♦✈✐❙❛❞✱❙❡r❜✐❛✱st❡✈❛♥✳❝✈❡t✐❝❛♥✐♥❅✉♥s✳❛❝✳rs ❹ ❉✐ss✐♣❛t✐✈❡❛♥❞❣❡♥❡r❛t✐✈❡❢r❛❝t✐♦♥❛❧❡❧❡❝tr✐❝❡❧❡♠❡♥ts ✐♥ ♠♦❞❡❧✐♥❣RC ❛♥❞RL ❝✐r❝✉✐ts ♠♦❞❡❧✐♥❣RC ❛♥❞RL ❝✐r❝✉✐ts ❑r✐st✐❛♥❍❛➨❦❛✯ ❙t❡✈❛♥▼✳❈✈❡t✐➣❛♥✐♥❸ ❉✉➨❛♥❩♦r✐❝❛❹ ▼❛r❝❤✶✼✱✷✵✷✶ ✶ ■♥tr♦❞✉❝t✐♦♥ ❈♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s ❝♦rr❡s♣♦♥❞✐♥❣t♦❝❛♣❛❝✐t♦r ❛♥❞✐♥❞✉❝t♦r✱❛s ❜❛s✐❝❡❧❡♠❡♥ts ♦❢❡❧❡❝tr✐❝❝✐r❝✉✐ts✱ ❛r❡❣❡♥❡r❛❧✐③❡❞✐♥♦r❞❡r t♦✐♥❝❧✉❞❡♠❡♠♦r②❡✛❡❝ts✱s♦t❤❛t t❤❡♣❤②s✐❝❛❧q✉❛♥t✐t✐❡s ❝❤❛r❛❝t❡r✐st✐❝❢♦r t❤❡ ❡❧❡♠❡♥t ❛r❡❝♦♥♥❡❝t❡❞t❤r♦✉❣❤t❤❡❝♦♠❜✐♥❛t✐♦♥♦❢✐♥st❛♥t❛♥❡♦✉s ❛♥❞❤❡r❡❞✐t❛r②t❡r♠s✱❡✐t❤❡r ❛s q(t) = C uC(t) + Cα 0I1−α t uC(t), ✭✶✮ φ(t) = L iL(t) + Lβ 0I1−β t iL(t), ✭✷✮ ✭✶✮ φ(t) = L iL(t) + Lβ 0I1−β t iL(t), ✭✷✮ ✯❉❡♣❛rt♠❡♥t ♦❢P♦✇❡r✱❊❧❡❝tr♦♥✐❝❛♥❞❚❡❧❡❝♦♠♠✉♥✐❝❛t✐♦♥❊♥❣✐♥❡❡r✐♥❣✱❋❛❝✉❧t②♦❢❚❡❝❤♥✐❝❛❧❙❝✐❡♥❝❡s✱❯♥✐✈❡rs✐t②♦❢◆♦✈✐ ❙❛❞✱❚r❣❉✳❖❜r❛❞♦✈✐➣❛✻✱✷✶✵✵✵◆♦✈✐❙❛❞✱❙❡r❜✐❛✱❦r✐st✐❛♥✳❤❛s❦❛❅✉♥s✳❛❝✳rs ❸ ❹❉❡♣❛rt♠❡♥t ♦❢P❤②s✐❝s✱❋❛❝✉❧t②♦❢❙❝✐❡♥❝❡s✱❯♥✐✈❡rs✐t②♦❢◆♦✈✐❙❛❞✱❚r❣❉✳❖❜r❛❞♦✈✐➣❛✹✱✷✶✵✵✵◆♦✈✐❙❛❞✱❙❡r❜✐❛✱ ❞✉s❛♥✳③♦r✐❝❛❅❞❢✳✉♥s✳❛❝✳rs ❛♥❞▼❛t❤❡♠❛t✐❝❛❧■♥st✐t✉t❡✱❙❡r❜✐❛♥❆❝❛❞❡♠②♦❢❆rts ❛♥❞❙❝✐❡♥❝❡s✱❑♥❡③❛▼✐❤❛✐❧❛✸✻✱✶✶✵✵✵ ❇❡♦❣r❛❞✱❙❡r❜✐❛✱❞✉s❛♥❴③♦r✐❝❛❅♠✐✳s❛♥✉✳❛❝✳rs ✶ ✶ ❡①♣r❡ss✐♥❣t❤❡t♦t❛❧❝❤❛r❣❡q ✭t♦t❛❧♠❛❣♥❡t✐❝✢✉①φ✮✐♥t❡r♠s ♦❢❤✐st♦r②♦❢❣❡♥❡r❛❧✐③❡❞❝❛♣❛❝✐t♦r✬s ✈♦❧t❛❣❡ uC ✭✐♥❞✉❝t♦r✬s ❝✉rr❡♥t iL✮✱♦r ❛s uC(t) = 1 C q(t) + 1 Cµ 0Iµ t q(t), ✭✸✮ iL(t) = 1 L φ(t) + 1 Lν 0Iν t φ(t), ✭✹✮ ✭✸✮ ✭✹✮ ✭✹✮ ❡①♣r❡ss✐♥❣t❤❡✈♦❧t❛❣❡✭❝✉rr❡♥t✮♦♥❣❡♥❡r❛❧✐③❡❞❝❛♣❛❝✐t♦r ✭✐♥❞✉❝t♦r✮✐♥t❡r♠s ♦❢❝❤❛r❣❡✭✢✉①✮❤✐st♦r②✱ ✇❤❡r❡t❤❡❤❡r❡❞✐t❛r✐♥❡ss ✐s ❛ss✉♠❡❞❛s t❤❡♣♦✇❡r ❢✉♥❝t✐♦♥❞❡❝r❡❛s✐♥❣✐♥t✐♠❡✱t > 0✱❛♥❞t❤✉s ♠♦❞❡❧❡❞ ❜②t❤❡❘✐❡♠❛♥♥✲▲✐♦✉✈✐❧❧❡❢r❛❝t✐♦♥❛❧✐♥t❡❣r❛❧♦❢♦r❞❡r 1 −α, 1 −β, µ, ν ∈[0, 1]✱❞❡✜♥❡❞❜② 0Iξ tf (t) = tξ−1 Γ (ξ) ∗f (t) = 1 Γ(ξ) Z t 0 f(t′) (t −t′)1−ξ dt′, 0Iξ tf (t) = tξ−1 Γ (ξ) ∗f (t) = 1 Γ(ξ) Z t 0 f(t′) (t −t′)1−ξ dt′, ✇✐t❤∗❞❡♥♦t✐♥❣t❤❡❝♦♥✈♦❧✉t✐♦♥✱✇❤❡r❡C[F]✱Cα[ F s1−α ]✱❛♥❞Cµ[F sµ] ❛r❡❝❧❛ss✐❝❛❧❛♥❞❢r❛❝t✐♦♥❛❧❝❛♣❛❝✲ ✐t❛♥❝❡s✱✇❤✐❧❡L[H]✱Lβ[ H s1−β ]✱❛♥❞Lν[H sν] ❛r❡❝❧❛ss✐❝❛❧❛♥❞❢r❛❝t✐♦♥❛❧✐♥❞✉❝t❛♥❝❡s✳❚❤❡r❡❢♦r❡✱❛s t❤❡ ♣❤②s✐❝❛❧♣❤❡♥♦♠❡♥♦♥✐s ❞✉❡t♦t❤❡❞✐✛❡r❡♥t ❡✛❡❝ts ♣r❡s❡♥t ✐♥t❤❡♠❛t❡r✐❛❧✱❛s ❝✉st♦♠❛r②✱t❤❡❝♦♥st✐t✉t✐✈❡ ♠♦❞❡❧s ❛r❡❛ss✉♠❡❞❛s ❛s✉♣❡r♣♦s✐t✐♦♥♦❢t❡r♠s ❛❝❝♦✉♥t✐♥❣❢♦r t❤❡s❡❡✛❡❝ts✳■❢t❤❡❣❡♥❡r❛❧✐③❡❞❡❧❡❝tr✐❝ ❡❧❡♠❡♥t ❞✐s♣❧❛②s ✐♥st❛♥t❛♥❡♦✉s ❛♥❞♠❡♠♦r②❡✛❡❝ts✱t❤❡②♠❛②❜❡❝♦♥st✐t✉t✐✈❡❧②♠♦❞❡❧❡❞❜②t❤❡s✉♠♦❢ ❝❧❛ss✐❝❛❧❝♦♥st✐t✉t✐✈❡♠♦❞❡❧✱❛❝❝♦✉♥t✐♥❣❢♦r t❤❡✐♥st❛♥t❛♥❡♦✉s ❡✛❡❝ts✱❛♥❞❛t❡r♠❝♦♥t❛✐♥✐♥❣t❤❡✐♥t❡❣r❛❧ ♦❢♣❤②s✐❝❛❧q✉❛♥t✐t②✱❛❝❝♦✉♥t✐♥❣❢♦r t❤❡♠❡♠♦r②❡✛❡❝ts✳❚❤❡❝❧❛ss✐❝❛❧❛♣♣r♦❛❝❤✐♥♠♦❞❡❧✐♥❣❤❡r❡❞✐t❛r② ♣❤❡♥♦♠❡♥❛✇♦✉❧❞✐♥❝❧✉❞❡s❤♦rt✲t❛✐❧♠❡♠♦r②t❤r♦✉❣❤t❤❡❡①♣♦♥❡♥t✐❛❧❢✉♥❝t✐♦♥❛s t❤❡♠❡♠♦r②❦❡r♥❡❧✱ ✇❤✐❧❡✐♥t❤❡❛♣♣r♦❛❝❤✉s❡❞✐♥♣♦st✉❧❛t✐♥❣❝♦♥st✐t✉t✐✈❡♠♦❞❡❧s ✭✶✮✲✭✹✮✱t❤❡❧♦♥❣✲t❛✐❧♠❡♠♦r②✐s ♠♦❞❡❧❡❞ t❤r♦✉❣❤t❤❡♣♦✇❡r t②♣❡❤❡r❡❞✐t❛r②❦❡r♥❡❧✳ ❆❧t❤♦✉❣❤❝❤❛r❣❡✲✈♦❧t❛❣❡❛♥❞✈♦❧t❛❣❡✲❝❤❛r❣❡❝♦♥st✐t✉t✐✈❡r❡❧❛t✐♦♥s ✭✶✮❛♥❞✭✸✮✱s♦❛s ✢✉①✲❝✉rr❡♥t ❛♥❞ ❝✉rr❡♥t✲✢✉①❝♦♥st✐t✉t✐✈❡r❡❧❛t✐♦♥s ✭✷✮❛♥❞✭✹✮✱❤❛✈❡❡①❛❝t❧②t❤❡s❛♠❡♠❛t❤❡♠❛t✐❝❛❧❢♦r♠✱t❤❡②❞❡s❝r✐❜❡ ❞✐✛❡r❡♥t t②♣❡♦❢❡❧❡❝tr✐❝❡❧❡♠❡♥ts✿t❤❡❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s ✭✶✮❛♥❞✭✷✮❞❡s❝r✐❜❡t❤❡♣❛ss✐✈❡❡❧❡♠❡♥t✱ ✐✳❡✳✱t❤❡❡❧❡♠❡♥t t❤❛t ❞✐ss✐♣❛t❡s ❡❧❡❝tr✐❝❡♥❡r❣②✱✇❤✐❧❡t❤❡❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s ✭✸✮❛♥❞✭✹✮❞❡s❝r✐❜❡t❤❡ ❛❝t✐✈❡❡❧❡♠❡♥t✱✐✳❡✳✱t❤❡❡❧❡♠❡♥t t❤❛t ❣❡♥❡r❛t❡s ❡❧❡❝tr✐❝❡♥❡r❣②✱❛s ♣r♦✈❡❞✐♥❙❡❝t✐♦♥✷✳ ❚❤❡❤✐st♦r②❞❡♣❡♥❞❡♥❝❡❜❡t✇❡❡♥t❤❡♣❤②s✐❝❛❧q✉❛♥t✐t✐❡s ❞❡s❝r✐❜❡❞❜②t❤❡❝❤❛r❣❡✲✈♦❧t❛❣❡❛♥❞✢✉①✲ ❝✉rr❡♥t ❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s ✭✶✮❛♥❞✭✷✮✐s ❡♠♣❤❛s✐③❡❞❜②s♦❧✈✐♥❣t❤❡♠✇✐t❤r❡s♣❡❝t t♦t❤❡✈♦❧t❛❣❡❛♥❞ ❝✉rr❡♥t r❡s♣❡❝t✐✈❡❧②❛s uC(t) = 1 C q(t) + 1 C ˙e1−α, Cα C (t) ∗q(t), ✭✺✮ iL(t) = 1 L φ(t) + 1 L ˙e1−β, Lβ L (t) ∗φ(t), ✭✻✮ ✭✺✮ ✭✻✮ ✷ ✇✐t❤˙eξ,λ ❜❡✐♥❣t❤❡t✐♠❡❞❡r✐✈❛t✐✈❡♦❢♦♥❡✲♣❛r❛♠❡t❡r ▼✐tt❛❣✲▲❡✤❡r ❢✉♥❝t✐♦♥✱❞❡✜♥❡❞❛s eξ,λ (t) = Eξ −λtξ , ✇❤❡r❡Eξ (z) = ∞ X k=0 zk Γ (ξk + 1), t❤✉s ❡①♣r❡ss✐♥❣t❤❡♠✐♥t❤❡❢♦r♠❛♥❛❧♦❣♦✉s t♦t❤❡❝♦♥st✐t✉t✐✈❡r❡❧❛t✐♦♥s ✭✸✮❛♥❞✭✹✮✱❤❛✈✐♥❣t❤❡♠❡♠♦r② ❦❡r♥❡❧❝❤❛♥❣❡❞❢r♦♠t❤❡♣♦✇❡r t♦t❤❡▼✐tt❛❣✲▲❡✤❡r t②♣❡✳❙✐♠✐❧❛r❧②✱❜②s♦❧✈✐♥❣t❤❡✈♦❧t❛❣❡✲❝❤❛r❣❡❛♥❞ ❝✉rr❡♥t✲✢✉①❝♦♥st✐t✉t✐✈❡r❡❧❛t✐♦♥s ✭✸✮❛♥❞✭✹✮✇✐t❤r❡s♣❡❝t t♦❝❤❛r❣❡❛♥❞✢✉①r❡s♣❡❝t✐✈❡❧②❛s q(t) = C uC(t) + C ˙eµ, C Cµ (t) ∗uC(t), ✭✼✮ φ(t) = L iL(t) + L ˙eν, L Lν (t) ∗iL(t), ✭✽✮ ✭✼✮ ✭✽✮ ♦♥❡❡①♣r❡ss❡s t❤❡❝❤❛r❣❡✭✢✉①✮✐♥t❡r♠s ♦❢t❤❡✈♦❧t❛❣❡✭❝✉rr❡♥t✮❤✐st♦r②✱❛s ✐♥❝♦♥st✐t✉t✐✈❡r❡❧❛t✐♦♥s ✭✶✮ ❛♥❞✭✷✮❤❛✈✐♥❣❛❣❛✐♥t❤❡♠❡♠♦r②❦❡r♥❡❧❝❤❛♥❣❡❞✳ ▼♦r❡♦✈❡r✱t❤❡❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s ✭✶✮❛♥❞✭✼✮✱❛s t❤❡❡q✉✐✈❛❧❡♥t ❢♦r♠♦❢✭✸✮✱❝❛♥❜❡t♦♣♦❧♦❣✐❝❛❧❧② ✈✐❡✇❡❞❛s t❤❡♣❛r❛❧❧❡❧❝♦♥♥❡❝t✐♦♥s ♦❢t❤❡❝❧❛ss✐❝❛❧❛♥❞❣❡♥❡r❛❧✐③❡❞❝❛♣❛❝✐t♦r✱✇❤✐❧❡t❤❡❝♦♥st✐t✉t✐✈❡❡q✉❛✲ t✐♦♥s ✭✸✮❛♥❞✭✺✮✱❛s t❤❡❡q✉✐✈❛❧❡♥t ❢♦r♠♦❢✭✶✮✱❝❛♥❜❡t♦♣♦❧♦❣✐❝❛❧❧②✈✐❡✇❡❞❛s t❤❡s❡r✐❡s ❝♦♥♥❡❝t✐♦♥s ♦❢t❤❡❝❧❛ss✐❝❛❧❛♥❞❣❡♥❡r❛❧✐③❡❞❝❛♣❛❝✐t♦r ❛♥❞s✐♠✐❧❛r❧②t❤❡s❡r✐❡s ❝♦♥♥❡❝t✐♦♥s ♦❢t❤❡❝❧❛ss✐❝❛❧❛♥❞❣❡♥✲ ❡r❛❧✐③❡❞✐♥❞✉❝t♦r ❛r❡❞❡s❝r✐❜❡❞❜②t❤❡r❡❧❛t✐♦♥s ✭✷✮❛♥❞✭✽✮✱❛s t❤❡❡q✉✐✈❛❧❡♥t ❢♦r♠♦❢✭✹✮✱✇❤✐❧❡t❤❡ ♣❛r❛❧❧❡❧❝♦♥♥❡❝t✐♦♥s ♦❢t❤❡❝❧❛ss✐❝❛❧❛♥❞❣❡♥❡r❛❧✐③❡❞✐♥❞✉❝t♦r ❛r❡❞❡s❝r✐❜❡❞❜②t❤❡r❡❧❛t✐♦♥s ✭✹✮❛♥❞✭✻✮✱ ❛s t❤❡❡q✉✐✈❛❧❡♥t ❢♦r♠♦❢✭✷✮✳❚❤❡r❡❢♦r❡✱❞✐✛❡r❡♥t t♦♣♦❧♦❣✐❝❛❧❣❡♥❡r❛❧✐③❛t✐♦♥s ♦❢❡❧❡❝tr✐❝❡❧❡♠❡♥ts ❝❛♥st✐❧❧ ❞❡s❝r✐❜❡t❤❡s❛♠❡♣❤❡♥♦♠❡♥♦❧♦❣②♦❢♣❤②s✐❝❛❧♣r♦❝❡ss❡s ❜②❝❤♦♦s✐♥❣❛♣♣r♦♣r✐❛t❡♠❡♠♦r②❦❡r♥❡❧s✳ ❋✉rt❤❡r✱❝❧❛ss✐❝❛❧RC ❛♥❞RL ❝✐r❝✉✐ts s✉❜❥❡❝t t♦t❤❡❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡❛r❡❣❡♥❡r❛❧✐③❡❞❜②❝♦♥s✐❞❡r✐♥❣ t❤❡❝❛♣❛❝✐t♦r ❛♥❞✐♥❞✉❝t♦r ❞✐s♣❧❛②✐♥❣❤❡r❡❞✐t❛r②❡✛❡❝ts ♠♦❞❡❧❡❞❜②t❤❡♣r❡✈✐♦✉s❧②♠❡♥t✐♦♥❡❞♠❡♠♦r② t②♣❡❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s ♦❢❢r❛❝t✐♦♥❛❧♦r❞❡r r❛t❤❡r t❤❛♥❜②t❤❡❝❧❛ss✐❝❛❧♦♥❡s t❤❛t ❛r❡❧♦❝❛❧✐♥t✐♠❡✳ ❙✉❝❤❣❡♥❡r❛❧✐③❡❞❝✐r❝✉✐ts ❛r❡❛♥❛❧②③❡❞❜②t❤❡♠❡❛♥s ♦❢▲❛♣❧❛❝❡tr❛♥s❢♦r♠♠❡t❤♦❞✐♥t❤❡tr❛♥s✐❡♥t r❡❣✐♠❡ ❢♦r ❛❣✐✈❡♥❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡❛♥❞❡s♣❡❝✐❛❧❧②❢♦r t❤❡❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡❛ss✉♠❡❞❛s t❤❡❍❡❛✈✐s✐❞❡st❡♣ ❢✉♥❝t✐♦♥❛♥❞❛s ❛❤❛r♠♦♥✐❝❢✉♥❝t✐♦♥✳❚❤❡❛ss✉♠♣t✐♦♥♦❢❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡✐♥t❤❡❢♦r♠♦❢t❤❡❤❛r♠♦♥✐❝ ❢✉♥❝t✐♦♥❡♥❛❜❧❡❞t❤❡❝♦♠♣❛r✐s♦♥♦❢t❤❡tr❛♥s✐❡♥t ❛♥❞st❡❛❞②st❛t❡r❡❣✐♠❡s ❛s ✇❡❧❧❛s t❤❡❛♥❛❧②s✐s ♦❢ ❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s✳❚❤❡❝❤❛r❣❡✲✈♦❧t❛❣❡❛♥❞✢✉①✲❝✉rr❡♥t ❝♦♥st✐t✉t✐✈❡♠♦❞❡❧s ✭✶✮❛♥❞✭✷✮✱❞❡s❝r✐❜✐♥❣ t❤❡♣❛ss✐✈❡❝❛♣❛❝✐t♦r ❛♥❞✐♥❞✉❝t♦r✱❛r❡❛❧s♦✉s❡❞✐♥t❤❡tr❛♥s✐❡♥t ❛♥❞st❡❛❞②st❛t❡r❡❣✐♠❡❛♥❛❧②s✐s ♦❢t❤❡ ❢♦r❝❡❞s❡r✐❡s ❢r❛❝t✐♦♥❛❧RLC ❝✐r❝✉✐t ✐♥❬✶✹❪✳ ❈♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s ❞❡s❝r✐❜✐♥❣❜❡❤❛✈✐♦r ♦❢❡❧❡❝tr✐❝❡❧❡♠❡♥ts ✉s✐♥❣❢r❛❝t✐♦♥❛❧❝❛❧❝✉❧✉s ❢♦✉♥❞t❤❡✐r ❛♣♣❧✐❝❛t✐♦♥✐♥♠♦❞❡❧✐♥❣s✉♣❡r❝❛♣❛❝✐t♦rs✱✉❧tr❛❝❛♣❛❝✐t♦rs✱❛♥❞❡❧❡❝tr♦❝❤❡♠✐❝❛❧❞♦✉❜❧❡✲❧❛②❡r ❝❛♣❛❝✐t♦rs ✭❊❉▲❈✮✱t❤❛t ❛r❡✉s❡❞❛s ❡♥❡r❣②st♦r✐♥❣❡❧❡♠❡♥ts ✐♥✈❛r✐♦✉s ❝♦♥str✉❝t✐♦♥❞❡✈✐❝❡s✳▼♦❞❡❧s ♦❢s✉♣❡r❝❛✲ ♣❛❝✐t♦r ❛♥❞✉❧tr❛❝❛♣❛❝✐t♦r r❛♥❣❡❢r♦♠t❤❡❧✐♥❡❛r ❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s ♦❜t❛✐♥❡❞❜②❝♦♠❜✐♥✐♥❣r❡s✐st♦rs ✸ ❛♥❞❢r❛❝t✐♦♥❛❧❝❛♣❛❝✐t♦rs ❛s ✐♥❬✼✱✷✻✱✷✼❪t♦♥♦♥✲❧✐♥❡❛r ♠♦❞❡❧s ❧✐❦❡t❤❡♦♥❡♣r♦♣♦s❡❞✐♥❬✾❪✳▼♦r❡♦✈❡r✱ ❢r❛❝t✐♦♥❛❧♠♦❞❡❧s ♦❢❝❛♣❛❝✐t♦r ❤❛✈✐♥❣t❤❡❞✐✛❡r❡♥t✐❛t✐♦♥♦r❞❡rs ❡①❝❡❡❞✐♥❣t❤❡✜rst ♦r❞❡r ❛r❡❝♦♥s✐❞❡r❡❞✐♥ ❬✶✾❪✱❛❧♦♥❣✇✐t❤t❤❡✐r ❜❡❤❛✈✐♦r ❛s ❝✐r❝✉✐t ❡❧❡♠❡♥ts✳❋r❛❝t✐♦♥❛❧♦r❞❡r ❡❧❡♠❡♥ts ✜♥❞t❤❡✐r ❛♣♣❧✐❝❛t✐♦♥✐♥t❤❡ st✉❞②♦❢❝♦♠♣❧❡①❡❧❡❝tr✐❝♥❡t✇♦r❦s ❛s ✇❡❧❧✱s❡❡❬✸✹✱✹✸❪✳❚❤❡r❡✈✐❡✇♦❢s✉♣❡r❝❛♣❛❝✐t♦r✬s ❢r❛❝t✐♦♥❛❧♦r❞❡r ♠♦❞❡❧s ✐♥✈♦❧✈✐♥❣t❤❡✐r ❛♣♣❧✐❝❛t✐♦♥s ❝❛♥❜❡❢♦✉♥❞✐♥❬✸❪✳❙✉♣❡r❝❛♣❛❝✐t♦rs ❛r❡❛❧s♦✐♥✈❡st✐❣❛t❡❞❛♥❛❧②t✐❝❛❧❧② ❛♥❞❡①♣❡r✐♠❡♥t❛❧❧②❛t ❤✐❣❤❢r❡q✉❡♥❝✐❡s ✐♥❬✷❪✱✇❤✐❧❡✐♥❬✷✵❪❞✐✛❡r❡♥t ♠♦❞❡❧s ♦❢❢r❛❝t✐♦♥❛❧❝❛♣❛❝✐t♦rs ❛r❡ ♣r❡s❡♥t❡❞❛♥❞t❡st❡❞❡①♣❡r✐♠❡♥t❛❧❧②✳❊❧❡❝tr✐❝❝✐r❝✉✐ts ❝♦♥t❛✐♥✐♥❣❢r❛❝t✐♦♥❛❧♦r❞❡r ❡❧❡♠❡♥ts ❛r❡✉s❡❞t♦ ♠♦❞❡❧t❤❡❡❧❡❝tr♦❧②t❡♣r♦❝❡ss❡s ✐♥❡❧❡❝tr♦❝❤❡♠✐❝❛❧❞♦✉❜❧❡✲❧❛②❡r ❝❛♣❛❝✐t♦rs✱❛s ❞❡♠♦♥str❛t❡❞t❤r♦✉❣❤t❤❡ ❢r❡q✉❡♥❝②❛♥❛❧②s✐s ✐♥❬✶✽❪✱❛s ✇❡❧❧❛s ❜②t❤❡❛♥❛❧②s✐s ✐♥t❤❡t✐♠❡❞♦♠❛✐♥✐♥❬✷✸✱✷✽❪✳▼♦r❡♦✈❡r✱t❤❡♣r❡s✲ ❡♥❝❡♦❢♠❡♠♦r②❡✛❡❝ts ✐♥❡❧❡❝tr✐❝❞♦✉❜❧❡✲❧❛②❡r ❝❛♣❛❝✐t♦rs ✐s ♣r♦✈❡❞❡①♣❡r✐♠❡♥t❛❧❧②✐♥❬✹❪✳❚❤❡❢r❛❝t✐♦♥❛❧ ♦r❞❡r ❡❧❡♠❡♥ts ❝❛♥❜❡♣r♦❞✉❝❡❞✇✐t❤❞❡s✐r❡❞❝❤❛r❛❝t❡r✐st✐❝s✱❛s ❞❡♠♦♥str❛t❡❞✐♥❬✷✶✱✷✹❪✳◆♦t ♦♥❧②t❤❛t t❤❡❝❛♣❛❝✐t♦r ✐s ❣❡♥❡r❛❧✐③❡❞t♦✐♥❝❧✉❞❡♠❡♠♦r②❡✛❡❝ts✱❜✉t ❛❧s♦❞✐✛❡r❡♥t ♣❤❡♥♦♠❡♥❛✐♥✐♥❞✉❝t♦r ❝❛♥❜❡ ♠♦❞❡❧❡❞✉s✐♥❣❢r❛❝t✐♦♥❛❧❝❛❧❝✉❧✉s✱❛s ❞♦♥❡✐♥❬✷✷✱✸✺❪✳❋r❛❝t✐♦♥❛❧♦r❞❡r ❝❛♣❛❝✐t❛♥❝❡❛♥❞✐♥❞✉❝t❛♥❝❡❛r❡ ❛❧s♦❝♦♥s✐❞❡r❡❞✐♥❬✹✵❪✳❘❡✈✐❡✇♦❢❢r❛❝t✐♦♥❛❧♦r❞❡r ❡❧❡♠❡♥t✬s ❝❤❛r❛❝t❡r✐st✐❝s✱❛❧♦♥❣✇✐t❤t❤❡✐r ❞✐✛❡r❡♥t r❡❛❧✐③❛t✐♦♥s ❛♥❞t❤❡✐r ❛♣♣❧✐❝❛t✐♦♥✐♥♠♦❞❡❧✐♥❣✈❛r✐♦✉s ♣❤❡♥♦♠❡♥❛✐s ❣✐✈❡♥✐♥❬✸✻❪✳❈♦♥st✐t✉t✐✈❡♠♦❞❡❧s ♦❢❡❧❡❝tr✐❝❡❧❡♠❡♥ts ♠❛②❡✈❡♥❜❡❞❡r✐✈❡❞❢r♦♠♠♦❞❡❧✐♥❣t❤❡✐♥t❡r❛❝t✐♦♥♦❢t❤❡❡❧❡❝tr♦✲♠❛❣♥❡t✐❝✜❡❧❞✇✐t❤ ♠❛t❡r✐❛❧✱s❡❡❬✷✺✱✸✾❪✳ ❚❤❡❢r❛❝t✐♦♥❛❧♦r❞❡r ❡q✉❛t✐♦♥s ❣♦✈❡r♥✐♥❣tr❛♥s✐❡♥t r❡❣✐♠❡♦❢❡❧❡❝tr✐❝❝✐r❝✉✐ts ❞✐s♣❧❛②✐♥❣♠❡♠♦r②❡✛❡❝ts ❛r❡♦❜t❛✐♥❡❞✐♥❬✶✶✱✶✸❪❜②r❡♣❧❛❝✐♥❣t❤❡♦r❞✐♥❛r②t✐♠❡❞❡r✐✈❛t✐✈❡s ✇✐t❤t❤❡❢r❛❝t✐♦♥❛❧♦♥❡s ✐♥t❤❡❡q✉❛t✐♦♥s ❝♦rr❡s♣♦♥❞✐♥❣t♦t❤❡❝❧❛ss✐❝❛❧RLC ❛♥❞RC ❝✐r❝✉✐ts✳❯s✐♥❣❛♥❛❧②t✐❝❛❧t♦♦❧s ❛♥❞❜②❝♦♥s✐❞❡r✐♥❣t❤❡s❡r✐❡s ❝♦♥♥❡❝t✐♦♥♦❢r❡s✐st♦r ❛♥❞❢r❛❝t✐♦♥❛❧❝❛♣❛❝✐t♦r ❛s ❛❣❡♥❡r❛❧✐③❡❞❝❛♣❛❝✐t♦r✱t❤❡tr❛♥s✐❡♥t r❡❣✐♠❡❛♥❛❧②s✐s ♦❢t❤❡s❡r✐❡s RCα ❝✐r❝✉✐t ✐s ♣❡r❢♦r♠❡❞✐♥❬✶✺❪✱✇❤✐❧❡❜②❝♦♥s✐❞❡r✐♥❣t❤❡s❡r✐❡s ❝♦♥♥❡❝t✐♦♥♦❢r❡s✐st♦r ❛♥❞❢r❛❝t✐♦♥❛❧✐♥❞✉❝t♦r ❛s ❛❣❡♥❡r❛❧✐③❡❞✐♥❞✉❝t♦r✱t❤❡tr❛♥s✐❡♥t r❡❣✐♠❡❛♥❛❧②s✐s ♦❢t❤❡♣❛r❛❧❧❡❧RLβCα ✐s ♣❡r❢♦r♠❡❞✐♥❬✶✻✱✶✼❪✳❙✐♠♣❧❡RC ❛♥❞❛♥❡①❛♠♣❧❡♦❢❛♠♦r❡❝♦♠♣❧❡①❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣❢r❛❝t✐♦♥❛❧ ❝❛♣❛❝✐t♦r ❛♥❞✐♥❞✉❝t♦r ✐s ❝♦♥s✐❞❡r❡❞✐♥❬✺❪❢♦r t❤❡tr❛♥s✐❡♥t r❡❣✐♠❡✉s✐♥❣❛♥❛❧②t✐❝❛❧❛♣♣r♦❛❝❤✱✇❤✐❧❡✐♥ ❬✻✱✸✼✱✸✽❪♥✉♠❡r✐❝❛❧t♦♦❧s ❛r❡✉s❡❞t♦s♦❧✈❡❣♦✈❡r♥✐♥❣❡q✉❛t✐♦♥s ♦❢❢r❛❝t✐♦♥❛❧❧②❣❡♥❡r❛❧✐③❡❞❡❧❡❝tr✐❝❝✐r❝✉✐ts ✐♥t❤❡t✐♠❡❞♦♠❛✐♥✳ ❋r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢t❤❡❢r❛❝t✐♦♥❛❧RC✱RL✱❛♥❞LC ❝✐r❝✉✐ts ✐♥❝❧✉❞✐♥❣♣❛r❛♠❡t❡r ♦♣t✐♠✐③❛t✐♦♥ ♦❢RLβCα ❝✐r❝✉✐t ❛r❡✐♥✈❡st✐❣❛t❡❞✐♥❬✸✶✱✸✷❪❛♥❞✐♥❬✸✵❪✳❲✐❡♥❜r✐❞❣❡♦s❝✐❧❧❛t♦rs ❛♥❞r❡s♦♥❛♥❝❡♣❤❡♥♦♠❡♥❛ ✐♥❢r❛❝t✐♦♥❛❧♦r❞❡r ❝✐r❝✉✐ts ❛r❡❝♦♥s✐❞❡r❡❞✐♥❬✽✱✸✸❪❛♥❞✐♥❬✷✾✱✹✷❪✳ ❆❢t❡r ✐♥tr♦❞✉❝t♦r②r❡♠❛r❦s ❛♥❞❛❢t❡r ❢♦r♠✉❧❛t✐♥❣t✇♦t②♣❡s ♦❢❤❡r❡❞✐t❛r②❝♦♥st✐t✉t✐✈❡♠♦❞❡❧s ❢♦r ❝❛♣❛❝✐t♦r ❛♥❞✐♥❞✉❝t♦r✱t❤❡r♠♦❞②♥❛♠✐❝❝♦♥s✐❞❡r❛t✐♦♥s ❧❡❛❞✐♥❣t♦t❤❡♠♦❞❡❧❝❧❛ss✐✜❝❛t✐♦♥❛r❡♣❡r❢♦r♠❡❞ ✐♥❙❡❝t✐♦♥✷✱✇❤✐❧❡❞❡r✐✈❛t✐♦♥❛♥❞s♦❧✉t✐♦♥♦❢t❤❡❡q✉❛t✐♦♥s ❣♦✈❡r♥✐♥❣t❤❡tr❛♥s✐❡♥t r❡❣✐♠❡♦❢❢♦r❝❡❞s❡r✐❡s ❢r❛❝t✐♦♥❛❧❝✐r❝✉✐ts ✐s ♣r❡s❡♥t❡❞✐♥❙❡❝t✐♦♥✸✱❛❧♦♥❣✇✐t❤t❤❡♥✉♠❡r✐❝❛❧❡①❛♠♣❧❡s ✐❧❧✉str❛t✐♥❣t❤❡tr❛♥s✐❡♥t r❡s♣♦♥s❡s ✐♥❝❛s❡s ♦❢❝❛♣❛❝✐t♦r ♠♦❞❡❧s ❡①♣r❡ss✐♥❣❡✐t❤❡r ❝❤❛r❣❡✐♥t❡r♠s ♦❢✈♦❧t❛❣❡♠❡♠♦r②♦r ✈♦❧t❛❣❡ ✹ ✹ ✐♥t❡r♠s ♦❢❝❤❛r❣❡♠❡♠♦r②✳❚r❛♥s✐❡♥t r❡s♣♦♥s❡♦❢t❤❡❢♦r❝❡❞s❡r✐❡s ❢r❛❝t✐♦♥❛❧RL ❝✐r❝✉✐t ✐s ♣r♦✈❡❞t♦❜❡ ❣♦✈❡r♥❡❞❜②❡q✉❛t✐♦♥s ♦❢t❤❡s❛♠❡❢♦r♠❛s ✐♥t❤❡❝❛s❡♦❢RC ❝✐r❝✉✐t✳❙❡❝t✐♦♥✹❝♦♥t❛✐♥s t❤❡st❡❛❞②st❛t❡ r❡❣✐♠❡❛♥❛❧②s✐s ❛❧♦♥❣✇✐t❤t❤❡❝♦♠♣❛r✐s♦♥♦❢s♦❧✉t✐♦♥s ❢♦r tr❛♥s✐❡♥t ❛♥❞st❡❛❞②st❛t❡r❡❣✐♠❡s✱✇❤✐❧❡t❤❡ ❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✐❛♥❞❛r❣✉♠❡♥ts✱❛❧♦♥❣✇✐t❤t❤❡✐r ❛s②♠♣t♦t✐❝s✱ ❛r❡st✉❞✐❡❞✐♥❙❡❝t✐♦♥✺✳❋✐♥❛❧❧②✱❝♦♥❝❧✉❞✐♥❣r❡♠❛r❦s ❛r❡❣✐✈❡♥✐♥❙❡❝t✐♦♥✻✳ ✷ ❚❤❡r♠♦❞②♥❛♠✐❝❝♦♥s✐❞❡r❛t✐♦♥s r❡❣❛r❞✐♥❣❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s ■♥♦r❞❡r t♦❛♥❛❧②③❡❞✐ss✐♣❛t✐✈✐t②♣r♦♣❡rt✐❡s ♦❢❣❡♥❡r❛❧✐③❡❞❡❧❡❝tr✐❝❡❧❡♠❡♥ts✿♣❛ss✐✈❡❝❛♣❛❝✐t♦r ❛♥❞✐♥❞✉❝t♦r✱ ♠♦❞❡❧❡❞❜②t❤❡❝❤❛r❣❡✲✈♦❧t❛❣❡✭✶✮❛♥❞✢✉①✲❝✉rr❡♥t ✭✷✮❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s✱❛s ✇❡❧❧❛s ❛❝t✐✈❡❝❛♣❛❝✐t♦r ❛♥❞✐♥❞✉❝t♦r✱♠♦❞❡❧❡❞❜②t❤❡✈♦❧t❛❣❡✲❝❤❛r❣❡✭✸✮❛♥❞❝✉rr❡♥t✲✢✉①✭✹✮❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s✱t❤❡❡❧❡♠❡♥t ✐♥st❡❛❞②st❛t❡r❡❣✐♠❡✐s ❝♦♥s✐❞❡r❡❞❜②❛ss✉♠✐♥❣✐ts ✈♦❧t❛❣❡❛s t❤❡❤❛r♠♦♥✐❝❢✉♥❝t✐♦♥ ✉(t) = u0 ejωt ✭✾✮ ✭✾✮ ♦❢❛♠♣❧✐t✉❞❡u0 ❛♥❞❛♥❣✉❧❛r ❢r❡q✉❡♥❝②ω✱✐♠♣❧②✐♥❣t❤❛t ✐ts ❝✉rr❡♥t ✐s ❛❤❛r♠♦♥✐❝❢✉♥❝t✐♦♥♦❢t❤❡s❛♠❡ ❢r❡q✉❡♥❝②❛s ✈♦❧t❛❣❡✭✾✮✱❜✉t s❤✐❢t❡❞❜②♣❤❛s❡❛♥❣❧❡φi ❛♥❞♦❢❛♠♣❧✐t✉❞❡i0✱t❛❦✐♥❣t❤❡❢♦r♠✿ ✐(t) = i0 ej(ωt+φi), ✭✶✵✮ ✭✶✵✮ ❞✉❡t♦t❤❡❧✐♥❡❛r✐t②♦❢❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s✳ ❞✉❡t♦t❤❡❧✐♥❡❛r✐t②♦❢❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s✳ ❙✐♥❝❡t❤❡q✉❛♥t✐t✐❡s ❤❛✈✐♥❣♣❤②s✐❝❛❧♠❡❛♥✐♥❣✐♥✭✾✮❛♥❞✭✶✵✮❛r❡ ♥❝❡t❤❡q✉❛♥t✐t✐❡s ❤❛✈✐♥❣♣❤②s✐❝❛❧♠❡❛♥✐♥❣✐♥✭✾✮❛♥❞✭✶✵✮❛r❡ ❙✐♥❝❡t❤❡q✉❛♥t✐t✐❡s ❤❛✈✐♥❣♣❤②s✐❝❛❧♠❡❛♥✐♥❣✐♥✭✾✮❛♥❞✭✶✵✮❛r❡ u = Re ✉ ❛♥❞ i = Re ✐, u = Re ✉ ❛♥❞ i = Re ✐, r❡s♣❡❝t✐✈❡❧②✱t❤❡❡♥❡r❣②♦♥❣❡♥❡r❛❧✐③❡❞❡❧❡❝tr✐❝❡❧❡♠❡♥t ❞✉r✐♥❣❤❛r♠♦♥✐❝❢✉♥❝t✐♦♥✬s ♣❡r✐♦❞T r❡s♣❡❝t✐✈❡❧②✱t❤❡❡♥❡r❣②♦♥❣❡♥❡r❛❧✐③❡❞❡❧❡❝tr✐❝❡❧❡♠❡♥t ❞✉r✐♥❣❤❛r♠♦♥✐❝❢✉♥❝t✐♦♥✬s ♣❡r✐♦❞T W = Z (n+1)T nT u(t) i(t) dt = u0i0 Z (n+1)T nT cos(ωt) cos(ωt + φi) dt = 1 2u0i0T cos φi ✐s ❞✐ss✐♣❛t❡❞✐❢cos φi > 0 ❛♥❞❣❡♥❡r❛t❡❞✐❢cos φi < 0✳ ❚❤❡r❡❢♦r❡✱t❤❡❡❧❡♠❡♥t ❞✐ss✐♣❛t❡s ❡♥❡r❣②✱✐✳❡✳✱✐t ✐s ❝♦♥s✐❞❡r❡❞t♦❜❡♣❛ss✐✈❡✱✐❢✐ts ❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥ ✐♥t❤❡st❡❛❞②st❛t❡r❡❣✐♠❡✱✇✐t❤✈♦❧t❛❣❡❛♥❞❝✉rr❡♥t ❛ss✉♠❡❞✐♥t❤❡❢♦r♠s ❣✐✈❡♥❜②✭✾✮❛♥❞✭✶✵✮✱②✐❡❧❞s cos φi > 0 ❢♦r ❛❧❧❢r❡q✉❡♥❝✐❡s ω✱✇❤✐❧❡t❤❡❡❧❡♠❡♥t ❣❡♥❡r❛t❡s ❡♥❡r❣②✱✐✳❡✳✱✐t ✐s ❝♦♥s✐❞❡r❡❞t♦❜❡❛❝t✐✈❡✱✐❢ ✐ts ❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥✐♥t❤❡st❡❛❞②st❛t❡r❡❣✐♠❡✱✇✐t❤✈♦❧t❛❣❡❛♥❞❝✉rr❡♥t ❛ss✉♠❡❞✐♥t❤❡❢♦r♠s ❣✐✈❡♥ ❜②✭✾✮❛♥❞✭✶✵✮✱②✐❡❧❞s cos φi < 0 ❢♦r ❛❧❧❢r❡q✉❡♥❝✐❡s ω✳ ■♥t❤❡❝❛s❡♦❢♣❛ss✐✈❡❝❛♣❛❝✐t♦r✱s✐♥❝❡❡❧❡❝tr✐❝❝✉rr❡♥t ✐s i(t) = d dtq(t)✱t❤❡❝❤❛r❣❡✲✈♦❧t❛❣❡❝♦♥st✐t✉t✐✈❡ ✺ ❡q✉❛t✐♦♥✭✶✮❞✐✛❡r❡♥t✐❛t❡❞✇✐t❤r❡s♣❡❝t t♦t✐♠❡②✐❡❧❞s ✉❛t✐♦♥✭✶✮❞✐✛❡r❡♥t✐❛t❡❞✇✐t❤r❡s♣❡❝t t♦t✐♠❡②✐❡❧❞s i(t) = C d dtuC(t) + Cα 0Dα t uC(t), ✭✶✶✮ ✭✶✶✮ ✇❤❡r❡0Dα t ✱α ∈(0, 1)✱❞❡♥♦t❡s t❤❡♦♣❡r❛t♦r ♦❢❘✐❡♠❛♥♥✲▲✐♦✉✈✐❧❧❡❢r❛❝t✐♦♥❛❧❞✐✛❡r❡♥t✐❛t✐♦♥✱❞❡✜♥❡❞❛s ✇❤❡r❡0Dα t ✱α ∈(0, 1)✱❞❡♥♦t❡s t❤❡♦♣❡r❛t♦r ♦❢❘✐❡♠❛♥♥✲▲✐♦✉✈✐❧❧❡❢r❛❝t✐♦♥❛❧❞✐✛❡r❡♥t✐❛t✐♦♥✱❞❡✜♥❡❞❛s 0Dξ tf (t) = d dt 0I1−ξ t f (t) = d dt  t−ξ Γ (1 −ξ) ∗f (t)  ✇✐t❤ ξ ∈(0, 1), s♦t❤❛t✱❜②♣❧✉❣❣✐♥❣❝❛♣❛❝✐t♦r✬s ✈♦❧t❛❣❡❛♥❞❝✉rr❡♥t✱❛ss✉♠❡❞❛s ✭✾✮❛♥❞✭✶✵✮✱✐♥t♦✭✶✶✮❛♥❞❜②✉s✐♥❣ s♦t❤❛t✱❜②♣❧✉❣❣✐♥❣❝❛♣❛❝✐t♦r✬s ✈♦❧t❛❣❡❛♥❞❝✉rr❡♥t✱❛ss✉♠❡❞❛s ✭✾✮❛♥❞✭✶✵✮✱✐♥t♦✭✶✶✮❛♥❞❜②✉s✐♥❣ 0Dξ t ej(ωt+φ) = (jω)ξ ej(ωt+φ) = ωξ ej(ωt+φ+ ξπ 2 ) ❛s t →∞, ✭✶✷✮ ✭✶✷✮ s❡❡❬✶✵❪✱♦♥❡✜♥❞s t❤❛t s❡❡❬✶✵❪✱♦♥❡✜♥❞s t❤❛t sin φi = u0 i0  C ω + Cα ωα sin απ 2  > 0 ❛♥❞ cos φi = u0 i0 Cα ωα cos απ 2 > 0, ✇❤✐❧❡❢♦r t❤❡♣❛ss✐✈❡✐♥❞✉❝t♦r t❤❡✢✉①✲❝✉rr❡♥t ♠♦❞❡❧✭✷✮❞✐✛❡r❡♥t✐❛t❡❞✇✐t❤r❡s♣❡❝t t♦t✐♠❡②✐❡❧❞s ✇❤✐❧❡❢♦r t❤❡♣❛ss✐✈❡✐♥❞✉❝t♦r t❤❡✢✉①✲❝✉rr❡♥t ♠♦❞❡❧✭✷✮❞✐✛❡r❡♥t✐❛t❡❞✇✐t❤r❡s♣❡❝t t♦t✐♠❡②✐❡❧❞s uL(t) = L d dti(t) + Lβ 0Dβ t i(t), ✭✶✸✮ ✭✶✸✮ s✐♥❝❡uL(t) = d dtφ(t)✱s♦t❤❛t✱❜②♣❧✉❣❣✐♥❣✐♥❞✉❝t♦r✬s ✈♦❧t❛❣❡❛♥❞❝✉rr❡♥t✱❛ss✉♠❡❞❛s ✭✾✮❛♥❞✭✶✵✮✱✐♥t♦ ✭✶✸✮✱♦♥❡♦❜t❛✐♥s s✐♥❝❡uL(t) = d dtφ(t)✱s♦t❤❛t✱❜②♣❧✉❣❣✐♥❣✐♥❞✉❝t♦r✬s ✈♦❧t❛❣❡❛♥❞❝✉rr❡♥t✱❛ss✉♠❡❞❛s ✭✾✮❛♥❞✭✶✵✮✱✐♥t♦ ✭✶✸✮✱♦♥❡♦❜t❛✐♥s sin φi = −i0 u0  L ω + Lβ ωβ sin βπ 2  < 0 ❛♥❞ cos φi = i0 u0 Lβ ωβ cos βπ 2 > 0. ✷ ❚❤❡r♠♦❞②♥❛♠✐❝❝♦♥s✐❞❡r❛t✐♦♥s r❡❣❛r❞✐♥❣❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s ❖♥t❤❡♦t❤❡r ❤❛♥❞✱t❤❡✈♦❧t❛❣❡✲❝❤❛r❣❡❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥✭✸✮❞❡s❝r✐❜✐♥❣t❤❡❛❝t✐✈❡❝❛♣❛❝✐t♦r✱s✐♥❝❡ ❡❧❡❝tr✐❝❝❤❛r❣❡✐s q(t) = R t 0 i(t′)dt′ = 0I1 ti(t) ♣r♦✈✐❞❡❞t❤❛t q(0) = 0✱❜❡❝♦♠❡s uC(t) = 1 C 0I1 ti(t) + 1 Cµ 0I1+µ t i(t), ✭✶✹✮ ✭✶✹✮ ✇❤❡r❡t❤❡s❡♠✐✲❣r♦✉♣♣r♦♣❡rt②❢♦r ❢r❛❝t✐♦♥❛❧✐♥t❡❣r❛❧s✱✐✳❡✳✱0Iξ t 0Iζ t = 0Iζ t 0Iξ t = 0Iξ+ζ t ✱✐s ✉s❡❞✱s♦t❤❛t✱❜② ♣❧✉❣❣✐♥❣❝❛♣❛❝✐t♦r✬s ✈♦❧t❛❣❡❛♥❞❝✉rr❡♥t✱❛ss✉♠❡❞❛s ✭✾✮❛♥❞✭✶✵✮✱✐♥t♦✭✶✹✮❛♥❞❜②❡♠♣❧♦②✐♥❣ 0Iξ t ej(ωt+φ) = 1 (jω)ξ ej(ωt+φ) = 1 ωξ ej(ωt+φ−ξπ 2 ) ❛s t →∞, ✭✶✺✮ ✭✶✺✮ s❡❡❬✶✵❪✱♦♥❡✜♥❞s t❤❛t s❡❡❬✶✵❪✱♦♥❡✜♥❞s t❤❛t sin φi = i0 u0  1 C ω + 1 Cµ ω1+µ cos µπ 2  > 0 ❛♥❞ cos φi = −i0 u0 1 Cµ ω1+µ sin µπ 2 < 0, sin φi = i0 u0  1 C ω + 1 Cµ ω1+µ cos µπ 2  > 0 ❛♥❞ cos φi = −i0 u0 1 Cµ ω1+µ sin µπ 2 < 0, ✻ ❤✐❧❡❢♦r t❤❡❛❝t✐✈❡✐♥❞✉❝t♦r t❤❡❝✉rr❡♥t✲✢✉①♠♦❞❡❧✭✹✮✱r❡✇r✐tt❡♥❛s i(t) = 1 L 0I1 tuL(t) + 1 Lν 0I1+ν t uL(t), ✭✶✻✮ ✭✶✻✮ ✉s✐♥❣t❤❡♠❛❣♥❡t✐❝✢✉①t❛❦❡♥❛s φ(t) = R t 0 uL(t′)dt′ = 0I1 tuL(t) ♣r♦✈✐❞❡❞t❤❛t φ(0) = 0✱②✐❡❧❞s sin φi = −u0 i0  1 L ω + 1 Lν ω1+ν cos νπ 2  < 0 ❛♥❞ cos φi = −u0 i0 1 Lν ω1+ν sin νπ 2 < 0, t❤❛t ✐s ♦❜t❛✐♥❡❞❜②♣❧✉❣❣✐♥❣✐♥❞✉❝t♦r✬s ✈♦❧t❛❣❡❛♥❞❝✉rr❡♥t✱❛ss✉♠❡❞❛s ✭✾✮❛♥❞✭✶✵✮✱✐♥t♦✭✶✻✮✳ ❚❤❡r❡❢♦r❡✱❜♦t❤❝❛♣❛❝✐t♦r ❛♥❞✐♥❞✉❝t♦r✱♠♦❞❡❧❡❞❜②✭✶✮❛♥❞✭✷✮✱❞✐ss✐♣❛t❡❡♥❡r❣②❛♥❞t❤✉s t❤❡②❛r❡ ❝♦♥s✐❞❡r❡❞❛s ♣❛ss✐✈❡❡❧❡♠❡♥ts✱s✐♥❝❡cos φi > 0✱✇❤✐❧❡❜♦t❤❝❛♣❛❝✐t♦r ❛♥❞✐♥❞✉❝t♦r✱♠♦❞❡❧❡❞❜②✭✸✮❛♥❞ ✭✹✮✱❣❡♥❡r❛t❡❡♥❡r❣②❛♥❞t❤✉s t❤❡②❛r❡❝♦♥s✐❞❡r❡❞❛s ❛❝t✐✈❡❡❧❡♠❡♥ts✱s✐♥❝❡cos φi < 0✳❖♥t❤❡♦t❤❡r ❤❛♥❞✱t❤❡s✐❣♥♦❢sin φi ✐♥❞✐❝❛t❡s ✇❤❡t❤❡r t❤❡❡❧❡♠❡♥t ❤❛s ❝❛♣❛❝✐t✐✈❡♦r ✐♥❞✉❝t✐✈❡❝❤❛r❛❝t❡r✱s✐♥❝❡✐♥t❤❡ ❢♦r♠❡r ❝❛s❡sin φi > 0 ✐♠♣❧✐❡s t❤❛t ❝✉rr❡♥t ❧❡❛❞s t❤❡✈♦❧t❛❣❡✱✇❤✐❧❡✐♥t❤❡❧❛tt❡r ❝❛s❡sin φi < 0 ✐♠♣❧✐❡s t❤❛t ❝✉rr❡♥t ❧❛❣s t❤❡✈♦❧t❛❣❡✳ P❛ss✐✈❡❝❛♣❛❝✐t♦r✬s ❝♦♥st✐t✉t✐✈❡♠♦❞❡❧✭✶✶✮✐♥t❤❡❧✐♠✐t✐♥❣❝❛s❡s ♦❢❢r❛❝t✐♦♥❛❧❞✐✛❡r❡♥t✐❛t✐♦♥♦r❞❡r r❡❞✉❝❡s t♦t❤❡❝❧❛ss✐❝❛❧♠♦❞❡❧s✱s♦t❤❛t ✐❢α = 0 ♦♥❡♦❜t❛✐♥s t❤❡♠♦❞❡❧♦❢❝❧❛ss✐❝❛❧❝❛♣❛❝✐t♦r ❝♦♥♥❡❝t❡❞✐♥ ♣❛r❛❧❧❡❧✇✐t❤r❡s✐st♦r ♦❢❝♦♥❞✉❝t✐✈✐t②G ≡Cα [ S] ❛s i(t) = C d dtuC(t) + G uC(t), ✭✶✼✮ ✭✶✼✮ ♥❣❞✐ss✐♣❛t✐✈❡❡❧❡♠❡♥t ❛s ✇❡❧❧✱✇❤✐❧❡✐❢α = 1 ♦♥❡❤❛s i(t) = Ccl d dtuC(t), ✭✶✽✮ ✭✶✽✮ ❞❡s❝r✐❜✐♥❣t❤❡❝❧❛ss✐❝❛❧❝❛♣❛❝✐t♦r ♦❢❝❛♣❛❝✐t❛♥❝❡Ccl = C + Cα [ F]✱t❤❛t ♥❡✐t❤❡r ❞✐ss✐♣❛t❡s ♥♦r ❣❡♥❡r❛t❡s ❡♥❡r❣②✳❖♥t❤❡♦t❤❡r ❤❛♥❞✱❛❝t✐✈❡❝❛♣❛❝✐t♦r✬s ❝♦♥st✐t✉t✐✈❡♠♦❞❡❧✭✸✮✐♥t❤❡❧✐♠✐t✐♥❣❝❛s❡♦❢t❤❡❢r❛❝t✐♦♥❛❧ ✐♥t❡❣r❛t✐♦♥♦r❞❡r µ = 0 r❡❞✉❝❡s t♦ uC(t) = 1 Ccl q(t), ✭✶✾✮ ✭✶✾✮ ❞❡s❝r✐❜✐♥❣t❤❡❝❧❛ss✐❝❛❧❝❛♣❛❝✐t♦r ♦❢❝❛♣❛❝✐t❛♥❝❡Ccl =  1 C + 1 Cµ −1 [ F]✱✇❤✐❧❡✐♥t❤❡❝❛s❡µ = 1 ❝♦♥st✐✲ t✉t✐✈❡♠♦❞❡❧✭✸✮r❡❞✉❝❡s t♦❛❤❡r❡❞✐t❛r②♠♦❞❡❧♦❢✐♥t❡❣❡r ♦r❞❡r ❞❡s❝r✐❜✐♥❣t❤❡❝❧❛ss✐❝❛❧❝❛♣❛❝✐t♦r ♦❢❝❛♣❛❝✐t❛♥❝❡Ccl =  1 C + 1 Cµ −1 [ F]✱✇❤✐❧❡✐♥t❤❡❝❛s❡µ = 1 ❝♦♥st✐✲ t✉t✐✈❡♠♦❞❡❧✭✸✮r❡❞✉❝❡s t♦❛❤❡r❡❞✐t❛r②♠♦❞❡❧♦❢✐♥t❡❣❡r ♦r❞❡r uC(t) = 1 C q(t) + 1 Cα Z t 0 q(t′) dt′, ✭✷✵✮ ✭✷✵✮ ❝♦rr❡s♣♦♥❞✐♥❣t♦t❤❡s❡r✐❡s ❝♦♥♥❡❝t✐♦♥♦❢❝❧❛ss✐❝❛❧❝❛♣❛❝✐t♦r ❛♥❞❤❡r❡❞✐t❛r②t②♣❡❡❧❡♠❡♥t✳▼♦r❡♦✈❡r✱t❤❡ ❝♦♥st✐t✉t✐✈❡♠♦❞❡❧✭✷✵✮✱✇✐t❤❝❤❛r❣❡r❡✇r✐tt❡♥❛s q(t) = R t 0 i(t′)dt′ ✐❢q(0) = 0✱❞❡s❝r✐❜❡s t❤❡❝❛♣❛❝✐t✐✈❡ ❝♦rr❡s♣♦♥❞✐♥❣t♦t❤❡s❡r✐❡s ❝♦♥♥❡❝t✐♦♥♦❢❝❧❛ss✐❝❛❧❝❛♣❛❝✐t♦r ❛♥❞❤❡r❡❞✐t❛r②t②♣❡❡❧❡♠❡♥t✳▼♦r❡♦✈❡r✱t❤❡ ❝♦♥st✐t✉t✐✈❡♠♦❞❡❧✭✷✵✮✱✇✐t❤❝❤❛r❣❡r❡✇r✐tt❡♥❛s q(t) = R t 0 i(t′)dt′ ✐❢q(0) = 0✱❞❡s❝r✐❜❡s t❤❡❝❛♣❛❝✐t✐✈❡ ✼ t②♣❡❡❧❡♠❡♥t t❤❛t ❣❡♥❡r❛t❡s ❡♥❡r❣②✱s✐♥❝❡✐t ②✐❡❧❞s t②♣❡❡❧❡♠❡♥t t❤❛t ❣❡♥❡r❛t❡s ❡♥❡r❣②✱s✐♥❝❡✐t ②✐❡❧❞s t②♣❡❡❧❡♠❡♥t t❤❛t ❣❡♥❡r❛t❡s ❡♥❡r❣②✱s✐♥❝❡✐t ②✐❡❧❞s sin φi = i0 u0 1 C ω > 0 ❛♥❞ cos φi = −i0 u0 1 Cµ ω2 < 0 ✐♥t❤❡st❡❛❞②st❛t❡r❡❣✐♠❡✱✐✳❡✳✱✇❤❡♥✈♦❧t❛❣❡❛♥❞❝✉rr❡♥t ❛r❡❛ss✉♠❡❞❛s ❤❛r♠♦♥✐❝❢✉♥❝t✐♦♥s ✭✾✮❛♥❞ ✭✶✵✮✳ ✐♥t❤❡st❡❛❞②st❛t❡r❡❣✐♠❡✱✐✳❡✳✱✇❤❡♥✈♦❧t❛❣❡❛♥❞❝✉rr❡♥t ❛r❡❛ss✉♠❡❞❛s ❤❛r♠♦♥✐❝❢✉♥❝t✐♦♥s ✭✾✮❛♥❞ ✭✶✵✮✳ ❙✐♠✐❧❛r❧②✱t❤❡✈♦❧t❛❣❡✲❝✉rr❡♥t ❝♦♥st✐t✉t✐✈❡r❡❧❛t✐♦♥✭✶✸✮✱♠♦❞❡❧✐♥❣t❤❡♣❛ss✐✈❡✐♥❞✉❝t♦r✱✐♥t❤❡❧✐♠✐t✐♥❣ ❝❛s❡s ♦❢❢r❛❝t✐♦♥❛❧❞✐✛❡r❡♥t✐❛t✐♦♥♦r❞❡r β = 0 ❛♥❞β = 1 ❜❡❝♦♠❡s r❡s♣❡❝t✐✈❡❧② uL(t) = L d dti(t) + R i(t) ❛♥❞ uL(t) = Lcl d dti(t), uL(t) = L d dti(t) + R i(t) ❛♥❞ uL(t) = Lcl d dti(t), ❝♦rr❡s♣♦♥❞✐♥❣t♦t❤❡s❡r✐❡s ❝♦♥♥❡❝t✐♦♥♦❢t❤❡❝❧❛ss✐❝❛❧✐♥❞✉❝t♦r ❛♥❞r❡s✐st♦r ♦❢r❡s✐st❛♥❝❡R ≡Lβ[ Ω] ❛♥❞ t♦t❤❡❝❧❛ss✐❝❛❧✐♥❞✉❝t♦r ♦❢✐♥❞✉❝t❛♥❝❡Lcl = L + Lβ[ H]✱✇❤✐❧❡t❤❡❝✉rr❡♥t✲✢✉①♠♦❞❡❧✭✹✮❢♦r t❤❡❛❝t✐✈❡ ✐♥❞✉❝t♦r r❡❞✉❝❡s t♦ iL(t) = 1 Lcl φ(t) ❛♥❞ iL(t) = 1 L φ(t) + 1 Lν Z t 0 φ(t′) dt′ ✭✷✶✮ ✭✷✶✮ ✐♥t❤❡❧✐♠✐t✐♥❣❝❛s❡s ♦❢❢r❛❝t✐♦♥❛❧✐♥t❡❣r❛t✐♦♥♦r❞❡rs ν = 0 ❛♥❞ν = 1✱r❡s♣❡❝t✐✈❡❧②✱♠♦❞❡❧✐♥❣t❤❡❝❧❛ss✐❝❛❧ ✐♥❞✉❝t♦r ♦❢✐♥❞✉❝t❛♥❝❡Lcl =  1 L + 1 Lν −1 [ H] ❛♥❞✐♥❞✉❝t✐✈❡t②♣❡❡♥❡r❣②❣❡♥❡r❛t✐♥❣❡❧❡♠❡♥t✱t❤❛t ♠❛② ❜❡❝♦♥s✐❞❡r❡❞❛s t❤❡♣❛r❛❧❧❡❧❝♦♥♥❡❝t✐♦♥♦❢❝❧❛ss✐❝❛❧✐♥❞✉❝t♦r ❛♥❞❤❡r❡❞✐t❛r②t②♣❡❡❧❡♠❡♥t✱s✐♥❝❡✇❤❡♥ ✈♦❧t❛❣❡❛♥❞❝✉rr❡♥t ❛r❡❛ss✉♠❡❞❛s ❤❛r♠♦♥✐❝❢✉♥❝t✐♦♥s ✭✾✮❛♥❞✭✶✵✮✱♠♦❞❡❧✭✷✶✮2 ②✐❡❧❞s sin φi = −u0 i0 1 L ω < 0 ❛♥❞ cos φi = −u0 i0 1 Lν ω2 < 0, ✇❤❡r❡φ(t) = R t 0 uL(t′)dt′ ✇✐t❤φ(0) = 0 ✐s ✉s❡❞✳ ✸ ❚r❛♥s✐❡♥t r❡s♣♦♥s❡♦❢❢r❛❝t✐♦♥❛❧RC ❛♥❞RL ❝✐r❝✉✐ts ❈♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s ❢♦r ❣❡♥❡r❛❧✐③❡❞❝❛♣❛❝✐t♦r ❛♥❞✐♥❞✉❝t♦r ❛r❡✉s❡❞t♦♠♦❞❡❧❝♦rr❡s♣♦♥❞✐♥❣❡❧❡♠❡♥ts ✐♥RC ❛♥❞RL ❝✐r❝✉✐ts✱s♦t❤❛t ♦r❞✐♥❛r②❢r❛❝t✐♦♥❛❧❡q✉❛t✐♦♥s ❣♦✈❡r♥✐♥❣tr❛♥s✐❡♥t ♣r♦❝❡ss❡s ❛r❡♦❜t❛✐♥❡❞ ❜②❡♠♣❧♦②✐♥❣t❤❡s❡❝♦♥❞❑✐r❝❤❤♦✛✬s ❧❛✇✐♥❛❞❞✐t✐♦♥t♦t❤❡❝♦♥st✐t✉t✐✈❡r❡❧❛t✐♦♥s✳❋✉rt❤❡r✱❣♦✈❡r♥✐♥❣ ❡q✉❛t✐♦♥s ❛r❡s♦❧✈❡❞❛♥❞✉s❡❞✐♥♦r❞❡r t♦♣r♦❞✉❝❡✐❧❧✉str❛t✐✈❡♥✉♠❡r✐❝❛❧❡①❛♠♣❧❡s✳ ✸✳✶ ❉❡r✐✈❛t✐♦♥♦❢❣♦✈❡r♥✐♥❣❡q✉❛t✐♦♥s ❛♥❞t❤❡✐r s♦❧✉t✐♦♥s ❚r❛♥s✐❡♥t r❡s♣♦♥s❡♦❢RC ❝✐r❝✉✐t✱s✉❜❥❡❝t t♦t❤❡❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡E ❛♥❞❝♦♥s✐st✐♥❣♦❢r❡s✐st♦r ♦❢r❡s✐s✲ t❛♥❝❡R ❝♦♥♥❡❝t❡❞✐♥s❡r✐❡s ✇✐t❤❣❡♥❡r❛❧✐③❡❞❝❛♣❛❝✐t♦r✱✐s ❣♦✈❡r♥❡❞❜②t❤❡s❡❝♦♥❞❑✐r❝❤❤♦✛✬s ❧❛✇ E(t) = R i(t) + uC(t), ✭✷✷✮ ✭✷✷✮ E(t) = R i(t) + uC(t), E(t) = R i(t) + uC(t), ✽ ❝♦✉♣❧❡❞❡✐t❤❡r ✇✐t❤t❤❡❝✉rr❡♥t✲✈♦❧t❛❣❡♠♦❞❡❧✭✶✶✮✱❝♦rr❡s♣♦♥❞✐♥❣t♦t❤❡♣❛ss✐✈❡❝❛♣❛❝✐t♦r ❞❡s❝r✐❜❡❞❜② t❤❡❝❤❛r❣❡✲✈♦❧t❛❣❡r❡❧❛t✐♦♥✭✶✮✱♦r ✇✐t❤t❤❡✈♦❧t❛❣❡✲❝✉rr❡♥t r❡❧❛t✐♦♥✭✶✹✮✱❞❡s❝r✐❜✐♥❣t❤❡❛❝t✐✈❡❝❛♣❛❝✐t♦r ♠♦❞❡❧❡❞❜②t❤❡✈♦❧t❛❣❡✲❝❤❛r❣❡r❡❧❛t✐♦♥✭✸✮✳❊①♣r❡ss✐♥❣t❤❡s②st❡♠♦❢❡q✉❛t✐♦♥s ✭✶✶✮❛♥❞✭✷✷✮✐♥t❡r♠s ♦❢❝❛♣❛❝✐t♦r ✈♦❧t❛❣❡uC ✈s✳❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡E✱❛s ✇❡❧❧❛s s②st❡♠✭✶✹✮❛♥❞✭✷✷✮✐♥t❡r♠s ♦❢❝✉rr❡♥t i ✈s✳E✱t❤❡❡q✉❛t✐♦♥s ❣♦✈❡r♥✐♥❣RC ❝✐r❝✉✐ts✬r❡s♣♦♥s❡s ❛r❡ τ C d dtuC(t) + τ α 0Dα t uC(t) + uC(t) = E(t) ❛♥❞ ✭✷✸✮ i(t) + 1 τ C 0I1 ti(t) + 1 τ µ 0I1+µ t i(t) = 1 R E(t), ✭✷✹✮ ✭✷✸✮ ✭✷✹✮ ✇✐t❤τ C = RC [s]✱τ α = RCα [sα]✱❛♥❞τ µ = RCµ [s1+µ] ❜❡✐♥❣❝❧❛ss✐❝❛❧❛♥❞❢r❛❝t✐♦♥❛❧t✐♠❡✲❝♦♥st❛♥ts✳ ❚❤❡❣♦✈❡r♥✐♥❣❡q✉❛t✐♦♥✭✷✸✮❢♦✉♥❞✐ts ❛♣♣❧✐❝❛t✐♦♥✐♥♠♦❞❡❧✐♥❣❝❤❛r❣❡❛♥❞❞✐s❝❤❛r❣❡♣r♦❝❡ss❡s ♦❢s✉♣❡r✲ ❝❛♣❛❝✐t♦r✱st✉❞✐❡❞✐♥❬✹✶❪✳ ❚❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥❝♦♥♥❡❝t✐♥❣❝❛♣❛❝✐t♦r ✈♦❧t❛❣❡✇✐t❤❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡❛♥❞❝♦rr❡s♣♦♥❞✐♥❣t♦RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣t❤❡♣❛ss✐✈❡❝❛♣❛❝✐t♦r ✐s ♦❜t❛✐♥❡❞❢r♦♠t❤❡❣♦✈❡r♥✐♥❣❡q✉❛t✐♦♥✭✷✸✮✐♥t❤❡❢♦r♠ ˆg(1) C (s) = ˆuC(s) ˆE(s) = 1 τ C s + τ α sα + 1, ✭✷✺✮ ✭✷✺✮ ✇❤✐❧❡t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥r❡❧❛t✐♥❣❝✉rr❡♥t t♦❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡✐♥t❤❡❝❛s❡♦❢RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣ t❤❡❛❝t✐✈❡❝❛♣❛❝✐t♦r ✐s ♦❜t❛✐♥❡❞❢r♦♠t❤❡❣♦✈❡r♥✐♥❣❡q✉❛t✐♦♥✭✷✹✮❛s ˆg(2) i (s) = ˆi(s) ˆE(s) = 1 R s1+µ s1+µ + 1 τ C sµ + 1 τ µ = 1 R  1 −ˆg(2) C (s)  , ✇✐t❤ ✭✷✻✮ ˆg(2) C (s) = τ µ τ C sµ + 1 τ µs1+µ + τ µ τ C sµ + 1, ✭✷✼✮ ✭✷✻✮ ✭✷✼✮ ❜②❛♣♣❧②✐♥❣t❤❡▲❛♣❧❛❝❡tr❛♥s❢♦r♠✱❞❡✜♥❡❞❛s ❜②❛♣♣❧②✐♥❣t❤❡▲❛♣❧❛❝❡tr❛♥s❢♦r♠✱❞❡✜♥❡❞❛s ˆf(s) = L[f(t)](s) = Z ∞ 0 f(t) e−stdt, ❢♦r Res > 0, ❛♥❞✉s✐♥❣t❤❡▲❛♣❧❛❝❡tr❛♥s❢♦r♠s ♦❢❘✐❡♠❛♥♥✲▲✐♦✉✈✐❧❧❡❢r❛❝t✐♦♥❛❧❞❡r✐✈❛t✐✈❡❛♥❞✐♥t❡❣r❛❧ L h 0Dξ tf (t) i (s) = sξ ˆf(s) − h 0I1−ξ t f (t) i t=0 = sξ ˆf(s) ❛♥❞ L h 0Iξ tf (t) i (s) = 1 sξ ˆf(s), ❤♦❧❞✐♥❣❢♦r f ❜♦✉♥❞❡❞❛t ③❡r♦✱✇✐t❤t❤❡r❡♠❛✐♥✐♥❣tr❛♥s❢❡r ❢✉♥❝t✐♦♥s ˆg(1) i (s) = ˆi(s) ˆE(s) = 1 R  1 −ˆg(1) C (s)  ❛♥❞ ˆg(2) C (s) = ˆuC(s) ˆE(s) ✭✷✽✮ ✭✷✽✮ ❜❡✐♥❣♦❜t❛✐♥❡❞❢r♦♠t❤❡▲❛♣❧❛❝❡tr❛♥s❢♦r♠♦❢t❤❡s❡❝♦♥❞❑✐r❝❤❤♦✛✬s ❧❛✇✭✷✷✮✳ ✾ ❚❤❡✐♠♣✉❧s❡r❡s♣♦♥s❡s g(1) C ❛♥❞g(2) C ✱✐✳❡✳✱t❤❡❝❛♣❛❝✐t♦r ✈♦❧t❛❣❡s ❛s ❛❝♦♥s❡q✉❡♥❝❡s ♦❢❡❧❡❝tr♦♠♦t✐✈❡ ❢♦r❝❡❛ss✉♠❡❞❛s ❉✐r❛❝❞❡❧t❛❢✉♥❝t✐♦♥✱❛r❡♦❜t❛✐♥❡❞❜②✐♥✈❡rt✐♥❣t❤❡▲❛♣❧❛❝❡tr❛♥s❢♦r♠✐♥✭✷✺✮❛♥❞✭✷✼✮✱ s❡❡❛❧s♦✭✷✽✮2✱②✐❡❧❞✐♥❣ g(1) C (t) = 1 π Z ∞ 0 τ αρα sin(απ) |1 −τ Cρ + τ αραejαπ|2 e−ρtdρ ❛♥❞ ✭✷✾✮ g(2) C (t) = −1 π Z ∞ 0 τ µρ1+µ sin (µπ) 1 −τ µρµ  ρ − 1 τ C  ejµπ 2 e−ρtdρ + 2 Re   s1−µ 0  τ µ τ C sµ 0 + 1  (1 + µ) τ µs0 + µ τ µ τ C ejtIms0  e−|Res0|t, ✭✸✵✮ ✭✷✾✮ | ρ ρ | 1 π Z ∞ 0 τ µρ1+µ sin (µπ) 1 −τ µρµ  ρ − 1 τ C  ejµπ 2 e−ρtdρ + 2 Re   s1−µ 0  τ µ τ C sµ 0 + 1  (1 + µ) τ µs0 + µ τ µ τ C ejtIms0  e−|Res0|t, ✭✸✵✮ ✇✐t❤s0 ❜❡✐♥❣t❤❡♣♦❧❡♦❢ˆg(2) C ✱❣✐✈❡♥❜②✭✷✼✮✱❧②✐♥❣✐♥t❤❡✉♣♣❡r ❧❡❢t ❝♦♠♣❧❡①q✉❛rt❡r ♣❧❛♥❡❛♥❞♦❜t❛✐♥❡❞ ❛s ❛s♦❧✉t✐♦♥♦❢t❤❡❡q✉❛t✐♦♥ τ µs1+µ + τ µ τ C sµ + 1 = 0, τ µs1+µ + τ µ τ C sµ + 1 = 0, ❛s ♣r♦✈❡❞✐♥❆♣♣❡♥❞✐①❆✳✶✳❚❤❡✐♠♣✉❧s❡r❡s♣♦♥s❡s g(1) C ❛♥❞g(2) C ✱❣✐✈❡♥❜②✭✷✾✮❛♥❞✭✸✵✮✱❛r❡❝❛❧❝✉❧❛t❡❞ ❜②t❤❡❞❡✜♥✐t✐♦♥♦❢✐♥✈❡rs❡▲❛♣❧❛❝❡tr❛♥s❢♦r♠✳▼♦r❡♣r❡❝✐s❡❧②✱t❤❡✐♠♣✉❧s❡r❡s♣♦♥s❡g(1) C ✱❣✐✈❡♥❜②✭✷✾✮✱✐s ♥♦t ❝❛❧❝✉❧❛t❡❞✱s✐♥❝❡✐t ✐s ❛s♦❧✉t✐♦♥♦❢t❤❡♦r❞✐♥❛r②❢r❛❝t✐♦♥❛❧❞✐✛❡r❡♥t✐❛❧❡q✉❛t✐♦♥✭✷✸✮✱t❤❛t ✐s ✇❡❧❧✲❦♥♦✇♥ ❛s t❤❡❝♦♠♣♦s✐t❡❢r❛❝t✐♦♥❛❧r❡❧❛①❛t✐♦♥❡q✉❛t✐♦♥✱s❡❡❊q✳✭✹✳✶✮✐♥❬✶✷❪✱✇❤❡r❡✐t ✐s s♦❧✈❡❞❛♥❞❛♥❛❧②③❡❞❢♦r ❛s②♠♣t♦t✐❝❜❡❤❛✈✐♦r✱✇❤✐❧❡t❤❡✐♠♣✉❧s❡r❡s♣♦♥s❡g(2) C ✱❣✐✈❡♥❜②✭✸✵✮✱r❡♣r❡s❡♥t✐♥❣t❤❡s♦❧✉t✐♦♥❦❡r♥❡❧♦❢ t❤❡❢r❛❝t✐♦♥❛❧✐♥t❡❣r❛❧❡q✉❛t✐♦♥✭✷✹✮✱✐s ❝❛❧❝✉❧❛t❡❞✐♥❆♣♣❡♥❞✐①❆✳✷✳❖♥❝❡t❤❡✐♠♣✉❧s❡r❡s♣♦♥s❡s g(1) C ❛♥❞ g(2) C ❛r❡❦♥♦✇♥✱t❤❡r❡s♣♦♥s❡s g(1) i ❛♥❞g(2) i ❛r❡❡❛s✐❧②❝❛❧❝✉❧❛t❡❞❜②t❤❡✐♥✈❡rs❡▲❛♣❧❛❝❡tr❛♥s❢♦r♠♦❢✭✷✽✮1 ❛♥❞✭✷✻✮r❡s♣❡❝t✐✈❡❧②✱②✐❡❧❞✐♥❣ g(1,2) i (t) = 1 R  δ(t) −g(1,2) C (t)  , ✭✸✶✮ ✭✸✶✮ ✇❤❡r❡δ ✐s t❤❡❉✐r❛❝❞❡❧t❛❢✉♥❝t✐♦♥✳ ✇❤❡r❡δ ✐s t❤❡❉✐r❛❝❞❡❧t❛❢✉♥❝t✐♦♥✳ ✇❤❡r❡δ ✐s t❤❡❉✐r❛❝❞❡❧t❛❢✉♥❝t✐♦♥✳ ❆❧t❤♦✉❣❤♦r✐❣✐♥❛t✐♥❣❢r♦♠t❤❡❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s t❤❛t ❝♦♥♥❡❝t ♣❤②s✐❝❛❧q✉❛♥t✐t✐❡s ❜②t❛❦✐♥❣✐♥t♦ ❛❝❝♦✉♥t t❤❡✐♥st❛♥t❛♥❡♦✉s ❝♦♥tr✐❜✉t✐♦♥❛♥❞♣♦✇❡r t②♣❡❤❡r❡❞✐t❛r✐♥❡ss ♦❢t❤❡♣❤②s✐❝❛❧q✉❛♥t✐t②✱♥❛♠❡❧② ❝❤❛r❣❡✲✈♦❧t❛❣❡r❡❧❛t✐♦♥✭✶✮❛♥❞✈♦❧t❛❣❡✲❝❤❛r❣❡r❡❧❛t✐♦♥✭✸✮✱t❤❡✐♠♣✉❧s❡r❡s♣♦♥s❡s g(1) C ❛♥❞g(2) C ✱❣✐✈❡♥❜② ✭✷✾✮❛♥❞✭✸✵✮✱❤❛✈❡✉tt❡r❧②❞✐✛❡r❡♥t q✉❛❧✐t❛t✐✈❡♣r♦♣❡rt✐❡s✱t❤❛t ♠❛②❜❡t❤❡❝♦♥s❡q✉❡♥❝❡♦❢t❤❡❢❛❝t t❤❛t t❤❡❢♦r♠❡r ❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥❞❡s❝r✐❜❡s t❤❡❞✐ss✐♣❛t✐✈❡❡❧❡♠❡♥t ❝♦♥tr❛r②t♦t❤❡❧❛tt❡r ♦♥❡❞❡s❝r✐❜✐♥❣ t❤❡❣❡♥❡r❛t✐✈❡❡❧❡♠❡♥t✳❚❤❡✐♠♣✉❧s❡r❡s♣♦♥s❡g(1) C ✐s ❝♦♠♣❧❡t❡❧②♠♦♥♦t♦♥✐❝✱✐✳❡✳✱♣♦s✐t✐✈❡✱❞❡❝r❡❛s✐♥❣✱ ❝♦♥✈❡①❢✉♥❝t✐♦♥✱❞✉❡t♦♣♦s✐t✐✈✐t②♦❢t❤❡✐♥t❡❣r❛♥❞♠✉❧t✐♣❧②✐♥❣t❤❡❡①♣♦♥❡♥t✐❛❧❢✉♥❝t✐♦♥✐♥✭✷✾✮✱✇❤✐❧❡ t❤❡✐♠♣✉❧s❡r❡s♣♦♥s❡g(2) C ✐s ❛♥♦s❝✐❧❧❛t♦r②❢✉♥❝t✐♦♥❤❛✈✐♥❣❛♥❡①♣♦♥❡♥t✐❛❧❧②❞❡❝r❡❛s✐♥❣❛♠♣❧✐t✉❞❡✱❞✉❡ t♦t❤❡s❡❝♦♥❞t❡r♠✐♥✭✸✵✮✱✇✐t❤t❤❡✜rst t❡r♠❜❡✐♥❣♥❡❣❛t✐✈❡✱✐♥❝r❡❛s✐♥❣✱❝♦♥❝❛✈❡❢✉♥❝t✐♦♥✱❞✉❡t♦t❤❡ ❝♦♠♣❧❡t❡♠♦♥♦t♦♥✐❝✐t②♦❢t❤❡✐♥t❡❣r❛❧✳ ❱♦❧t❛❣❡uC ♦♥t❤❡❣❡♥❡r❛❧✐③❡❞❝❛♣❛❝✐t♦r ❛♥❞❝✉rr❡♥t i r✉♥♥✐♥❣t❤r♦✉❣❤RC ❝✐r❝✉✐t ✐♥t❤❡tr❛♥s✐❡♥t ✶✵ r❡❣✐♠❡❛r❡❡①♣r❡ss❡❞t❤♦r♦✉❣❤t❤❡❝♦♥✈♦❧✉t✐♦♥♦❢✐♠♣✉❧s❡r❡s♣♦♥s❡❛♥❞❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡❛s uC(t) = g(1,2) C (t) ∗E(t) ❛♥❞ i(t) = g(1,2) i (t) ∗E(t), ✭✸✷✮ ✭✸✷✮ ❜②✐♥✈❡rt✐♥❣t❤❡▲❛♣❧❛❝❡tr❛♥s❢♦r♠s ✐♥✭✷✺✮❛♥❞✭✷✽✮1 ✐♥t❤❡❝❛s❡♦❢♣❛ss✐✈❡❝❛♣❛❝✐t♦r ❛♥❞❜②t❤❡▲❛♣❧❛❝❡ tr❛♥s❢♦r♠✐♥✈❡rs✐♦♥✐♥✭✷✽✮2 ❛♥❞✭✷✻✮✐♥t❤❡❝❛s❡♦❢❛❝t✐✈❡❝❛♣❛❝✐t♦r✱✇✐t❤❝♦rr❡s♣♦♥❞✐♥❣✐♠♣✉❧s❡r❡s♣♦♥s❡s ❣✐✈❡♥❜②✭✷✾✮❛♥❞✭✸✵✮❢♦r ✈♦❧t❛❣❡s ❛♥❞❜②✭✸✶✮❢♦r ❝✉rr❡♥ts✳ ❈♦♥s✐❞❡r✐♥❣RL ❝✐r❝✉✐t✱t❤❛t ❝♦♥s✐sts ♦❢r❡s✐st♦r ❝♦♥♥❡❝t❡❞✐♥s❡r✐❡s ❡✐t❤❡r ✇✐t❤♣❛ss✐✈❡✐♥❞✉❝t♦r✱ ❝♦♥st✐t✉t✐✈❡❧②♠♦❞❡❧❡❞❜②✢✉①✲❝✉rr❡♥t r❡❧❛t✐♦♥✭✷✮✱♦r ✇✐t❤t❤❡❛❝t✐✈❡✐♥❞✉❝t♦r✱❞❡s❝r✐❜❡❞❜②t❤❡❝✉rr❡♥t✲ ✢✉①r❡❧❛t✐♦♥✭✹✮✱t❤❡tr❛♥s✐❡♥t r❡s♣♦♥s❡t♦❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡✐s ♦❜t❛✐♥❡❞❛s ❛s♦❧✉t✐♦♥♦❢t❤❡s❡❝♦♥❞ ❑✐r❝❤❤♦✛✬s ❧❛✇ E(t) = R i(t) + uL(t), ✭✸✸✮ ✭✸✸✮ ❝♦✉♣❧❡❞❡✐t❤❡r ✇✐t❤t❤❡✈♦❧t❛❣❡✲❝✉rr❡♥t ♠♦❞❡❧✭✶✸✮❞❡s❝r✐❜✐♥❣t❤❡♣❛ss✐✈❡✐♥❞✉❝t♦r✱♦r ✇✐t❤t❤❡❝✉rr❡♥t✲ ✈♦❧t❛❣❡r❡❧❛t✐♦♥✭✶✻✮♠♦❞❡❧✐♥❣t❤❡❛❝t✐✈❡✐♥❞✉❝t♦r✳ ❝♦✉♣❧❡❞❡✐t❤❡r ✇✐t❤t❤❡✈♦❧t❛❣❡✲❝✉rr❡♥t ♠♦❞❡❧✭✶✸✮❞❡s❝r✐❜✐♥❣t❤❡♣❛ss✐✈❡✐♥❞✉❝t♦r✱♦r ✇✐t❤t❤❡❝✉rr❡♥t✲ ✈♦❧t❛❣❡r❡❧❛t✐♦♥✭✶✻✮♠♦❞❡❧✐♥❣t❤❡❛❝t✐✈❡✐♥❞✉❝t♦r✳ ❊q✉❛t✐♦♥s ❣♦✈❡r♥✐♥❣t❤❡tr❛♥s✐❡♥t r❡❣✐♠❡✐♥RL ❝✐r❝✉✐t τ L d dti(t) + τ β 0Dβ t i(t) + i(t) = 1 R E(t) ❛♥❞ ✭✸✹✮ uL(t) + 1 τ L 0I1 tuL(t) + 1 τ ν 0I1+ν t uL(t) = E(t), ✭✸✺✮ ✭✸✹✮ ✭✸✺✮ ✇✐t❤τ L = L R [s]✱τ β = Lβ R [sβ]✱❛♥❞τ ν = Lν R [s1+ν] ❜❡✐♥❣❝❧❛ss✐❝❛❧❛♥❞❢r❛❝t✐♦♥❛❧t✐♠❡✲❝♦♥st❛♥ts✱❛r❡ r❡s♣❡❝t✐✈❡❧②♦❜t❛✐♥❡❞❜②r❡❞✉❝✐♥❣t❤❡s②st❡♠♦❢❡q✉❛t✐♦♥s ✭✶✸✮✱✭✸✸✮t♦❛s✐♥❣❧❡❡q✉❛t✐♦♥❡①♣r❡ss❡❞✐♥ t❡r♠s ♦❢❝✉rr❡♥t i ✐♥t❤❡❝❛s❡♦❢♣❛ss✐✈❡✐♥❞✉❝t♦r ❛♥❞❜②s♦❧✈✐♥❣t❤❡s②st❡♠♦❢❡q✉❛t✐♦♥s ✭✶✻✮✱✭✸✸✮✇✐t❤ r❡s♣❡❝t t♦✈♦❧t❛❣❡uL ✐♥t❤❡❝❛s❡♦❢❛❝t✐✈❡✐♥❞✉❝t♦r✳ ■❢RL ❝✐r❝✉✐t ❝♦♥t❛✐♥s ♣❛ss✐✈❡✐♥❞✉❝t♦r✱t❤❡♥✱❞✉❡t♦t❤❡❛♥❛❧♦❣♦✉s ❢♦r♠s ♦❢❣♦✈❡r♥✐♥❣❡q✉❛t✐♦♥s ✭✸✹✮ ❢♦r RL t♦✭✷✸✮❢♦r RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣♣❛ss✐✈❡❝❛♣❛❝✐t♦r✱t❤❡✐♠♣✉❧s❡r❡s♣♦♥s❡♦❢❝✉rr❡♥t ❝♦rr❡s♣♦♥❞✐♥❣ t♦✭✸✹✮❤❛s t❤❡s❛♠❡❢♦r♠❛s g(1) C ✱❣✐✈❡♥❜②✭✷✾✮✱✇❤✐❧❡✐❢RL ❝✐r❝✉✐t ❝♦♥t❛✐♥s ❛❝t✐✈❡✐♥❞✉❝t♦r✱t❤❡♥✱❞✉❡t♦ t❤❡❛♥❛❧♦❣♦✉s ❢♦r♠s ♦❢❣♦✈❡r♥✐♥❣❡q✉❛t✐♦♥s ✭✸✺✮❢♦r RL t♦✭✷✹✮❢♦r RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣❛❝t✐✈❡❝❛♣❛❝✐t♦r✱ t❤❡✐♠♣✉❧s❡r❡s♣♦♥s❡♦❢✐♥❞✉❝t♦r ✈♦❧t❛❣❡❝♦rr❡s♣♦♥❞✐♥❣t♦✭✸✺✮❤❛s t❤❡s❛♠❡❢♦r♠❛s g(2) i ✱❣✐✈❡♥❜②✭✸✶✮✳ ❆❧s♦✱❝✉rr❡♥t r✉♥♥✐♥❣t❤r♦✉❣❤RL ❝✐r❝✉✐t ❛♥❞✐♥❞✉❝t♦r ✈♦❧t❛❣❡❛r❡❡①♣r❡ss❡❞t❤♦r♦✉❣❤❝♦♥✈♦❧✉t✐♦♥s ♦❢t❤❡ ❢♦r♠❛♥❛❧♦❣♦✉s t♦t❤❡❝♦♥✈♦❧✉t✐♦♥s ❢♦r ❝❛♣❛❝✐t♦r ✈♦❧t❛❣❡uC ❛♥❞❝✉rr❡♥t i r✉♥♥✐♥❣t❤r♦✉❣❤RC ❝✐r❝✉✐t✱ ❣✐✈❡♥❜②✭✸✷✮✱r❡s♣❡❝t✐✈❡❧②✳ ✸✳✷ ◆✉♠❡r✐❝❛❧❡①❛♠♣❧❡s ❚✐♠❡❡✈♦❧✉t✐♦♥♦❢❝✉rr❡♥t ✐♥RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣t❤❡♣❛ss✐✈❡❝❛♣❛❝✐t♦r✱♠♦❞❡❧❡❞❜②t❤❡❝❤❛r❣❡✲✈♦❧t❛❣❡ ❝♦♥st✐t✉t✐✈❡r❡❧❛t✐♦♥✭✶✮✱❛s ❛r❡s♣♦♥s❡t♦t❤❡❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡t❛❦❡♥❛s t❤❡❍❡❛✈✐s✐❞❡st❡♣❢✉♥❝t✐♦♥✱ ✶✶ ✐✳❡✳✱❛s E(t) = E0 H(t)✱✇✐t❤E0 ❜❡✐♥❣t❤❡❝♦♥st❛♥t ✐♥t❡♥s✐t②♦❢t❤❡❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡✱✐s ❝❛❧❝✉❧❛t❡❞❜② ✭✸✷✮2 ❛♥❞♣r❡s❡♥t❡❞✐♥❋✐❣✉r❡✶✱❛❧♦♥❣✇✐t❤t❤❡r❡s♣♦♥s❡s ♦❢t❤❡❝❧❛ss✐❝❛❧RC ❝✐r❝✉✐ts✱s❡❡✭✶✼✮❛♥❞✭✶✽✮✱ t❤❛t ❛r❡❝❛❧❝✉❧❛t❡❞❜②✿ i(t) = E0 R τ α + e−τα+1 τC t τ α + 1 ❢♦r α = 0 ❛♥❞ i(t) = E0 R e− 1 τC +τα t ❢♦r α = 1. ✸✳✷ ◆✉♠❡r✐❝❛❧❡①❛♠♣❧❡s ❚❤❡t✐♠❡♣r♦✜❧❡s ♦❢❝✉rr❡♥t ❢♦r ❢r❛❝t✐♦♥❛❧♦r❞❡r RC ❝✐r❝✉✐t✱t❤❛t ❛r❡♠♦♥♦t♦♥✐❝❛❧❧②❞❡❝r❡❛s✐♥❣❢✉♥❝t✐♦♥s ♦❢t✐♠❡✱❢♦r ❜♦t❤s♠❛❧❧❛♥❞❧❛r❣❡t✐♠❡❧✐❡❜❡t✇❡❡♥t❤❡❝♦rr❡s♣♦♥❞✐♥❣t✐♠❡♣r♦✜❧❡s ❢♦r RC ❝✐r❝✉✐ts ❝♦♥t❛✐♥✐♥❣ ❝❧❛ss✐❝❛❧❡❧❡♠❡♥ts✱❛s ♦❜✈✐♦✉s ❢r♦♠❋✐❣✉r❡✶✳❚❤❡❞❡❝r❡❛s❡r❛t❡♦❢r❡s♣♦♥s❡s ❢♦r s♠❛❧❧t✐♠❡✐s t❤❡❣r❡❛t❡st ✐♥t❤❡❝❛s❡♦❢❝❧❛ss✐❝❛❧RC ❝✐r❝✉✐t ✇✐t❤α = 0✱✇❤✐❧❡r❡s♣♦♥s❡s✬❞❡❝r❡❛s❡r❛t❡❢♦r ❢r❛❝t✐♦♥❛❧RC ❝✐r❝✉✐ts ❞❡❝r❡❛s❡s ❛s t❤❡❢r❛❝t✐♦♥❛❧❞✐✛❡r❡♥t✐❛t✐♦♥♦r❞❡r α ✐♥❝r❡❛s❡s ❛♥❞✜♥❛❧❧②t❤❡❞❡❝r❡❛s❡r❛t❡✐s s♠❛❧❧❡st ✐♥t❤❡ ❝❛s❡♦❢❝❧❛ss✐❝❛❧RC ❝✐r❝✉✐t ✇✐t❤α = 1✱❛s ♦❜✈✐♦✉s ❢r♦♠❋✐❣✉r❡✶❛✳❚❤❡s✐t✉❛t✐♦♥✐s r❡✈❡rs❡❞❢♦r ❧❛r❣❡ t✐♠❡✱❛s ❞❡♣✐❝t❡❞✐♥❋✐❣✉r❡✶❜✱s✐♥❝❡t❤❡✐♥t❡❣❡r ♦r❞❡r r❡s♣♦♥s❡❢♦r α = 0 ✐s ❛❧♠♦st ❝♦♥st❛♥t✱❢r❛❝t✐♦♥❛❧ ♦r❞❡r r❡s♣♦♥s❡s ❞❡❝r❡❛s❡❛s t❤❡♣♦✇❡r t②♣❡❢✉♥❝t✐♦♥❛❝❝♦r❞✐♥❣t♦t❤❡❛s②♠♣t♦t✐❝s i(t) ∼E0 R      τ α t−α Γ(1−α) −τ 2 α t−2α Γ(1−2α)✱ ✐❢α ∈ 0, 1 2  ✱ τ α t−α Γ(1−α)✱ ✐❢α ∈ 1 2, 1  ✱ ❛s t →∞, s❡❡❬✶✷❪✱✇❤✐❧❡✐♥t❡❣❡r ♦r❞❡r r❡s♣♦♥s❡❢♦r α = 1 ❞❡❝r❡❛s❡s ❡①♣♦♥❡♥t✐❛❧❧②✳◆♦t❡t❤❛t ❛❧❧r❡s♣♦♥s❡s ❝♦rr❡✲ s♣♦♥❞✐♥❣t♦α ∈(0, 1] t❡♥❞t♦③❡r♦❢♦r ❧❛r❣❡t✐♠❡✱❡①❝❡♣t ❢♦r t❤❡r❡s♣♦♥s❡❝♦rr❡s♣♦♥❞✐♥❣t♦α = 0 t❤❛t t❡♥❞s t♦❛❝♦♥st❛♥t✿limt→∞i(t) = E0 R τ α τ α+1✳ s❡❡❬✶✷❪✱✇❤✐❧❡✐♥t❡❣❡r ♦r❞❡r r❡s♣♦♥s❡❢♦r α = 1 ❞❡❝r❡❛s❡s ❡①♣♦♥❡♥t✐❛❧❧②✳◆♦t❡t❤❛t ❛❧❧r❡s♣♦♥s❡s ❝♦rr❡✲ s♣♦♥❞✐♥❣t♦α ∈(0, 1] t❡♥❞t♦③❡r♦❢♦r ❧❛r❣❡t✐♠❡✱❡①❝❡♣t ❢♦r t❤❡r❡s♣♦♥s❡❝♦rr❡s♣♦♥❞✐♥❣t♦α = 0 t❤❛t t❡♥❞s t♦❛❝♦♥st❛♥t✿limt→∞i(t) = E0 R τ α τ α+1✳ α = 0 α = 0.25 α = 0.5 α = 0.75 α = 1 0 2 4 6 8 t 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 i(t) ✭❜✮Pr♦✜❧❡s ♦❢❝✉rr❡♥t ❢♦r ❧❛r❣❡t✐♠❡❛❧♦♥❣✇✐t❤❛s②♠♣t♦t✐❝ ❝✉r✈❡s ✭s♦❧✐❞❧✐♥❡✮✳ α = 1 α = 0.75 α = 0.5 α = 0.25 α = 0 0.0 0.2 0.4 0.6 0.8 t 0.0 0.2 0.4 0.6 0.8 1.0 i(t) ✭❛✮Pr♦✜❧❡s ♦❢❝✉rr❡♥t ❢♦r s♠❛❧❧t✐♠❡✳ α = 0 α = 0.25 α = 0.5 α = 0.75 α = 1 0 2 4 6 8 t 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 i(t) ✭❜✮Pr♦✜❧❡s ♦❢❝✉rr❡♥t ❢♦r ❧❛r❣❡t✐♠❡❛❧♦♥❣✇✐t❤❛s②♠♣t♦t✐❝ ❝✉r✈❡s ✭s♦❧✐❞❧✐♥❡✮✳ ❋✐❣✉r❡✶✿❈❛s❡♦❢♣❛ss✐✈❡❝❛♣❛❝✐t♦r ✕t✐♠❡♣r♦✜❧❡s ♦❢❝✉rr❡♥t ✐♥RC ❝✐r❝✉✐t ❛s ❛r❡s♣♦♥s❡t♦❡❧❡❝tr♦✲ ♠♦t✐✈❡❢♦r❝❡❛ss✉♠❡❞❛s ❍❡❛✈✐s✐❞❡✬s st❡♣❢✉♥❝t✐♦♥♦❢✐♥t❡♥s✐t②E0 = 1✱♦❜t❛✐♥❡❞❛♥❛❧②t✐❝❛❧❧②✭❧✐♥❡s✮❛♥❞ ♥✉♠❡r✐❝❛❧❧②✭❞♦ts✮❢♦r ♠♦❞❡❧♣❛r❛♠❡t❡rs✿R = 1✱τ C = 0.1✱❛♥❞τ α = 0.3✳ α = 0 α = 0.25 α = 0.5 α = 0.75 α = 1 0 2 4 6 8 t 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 i(t) ✭❜✮Pr♦✜❧❡s ♦❢❝✉rr❡♥t ❢♦r ❧❛r❣❡t✐♠❡❛❧♦♥❣✇✐t❤❛s②♠♣t♦t✐❝ ❝✉r✈❡s ✭s♦❧✐❞❧✐♥❡✮✳ α = 1 α = 0.75 α = 0.5 α = 0.25 α = 0 0.0 0.2 0.4 0.6 0.8 t 0.0 0.2 0.4 0.6 0.8 1.0 i(t) ✭❛✮Pr♦✜❧❡s ♦❢❝✉rr❡♥t ❢♦r s♠❛❧❧t✐♠❡✳ ✭❜✮Pr♦✜❧❡s ♦❢❝✉rr❡♥t ❢♦r ❧❛r❣❡t✐♠❡❛❧♦♥❣✇✐t❤❛s②♠♣t♦t✐❝ ❝✉r✈❡s ✭s♦❧✐❞❧✐♥❡✮✳ ❋✐❣✉r❡✶✿❈❛s❡♦❢♣❛ss✐✈❡❝❛♣❛❝✐t♦r ✕t✐♠❡♣r♦✜❧❡s ♦❢❝✉rr❡♥t ✐♥RC ❝✐r❝✉✐t ❛s ❛r❡s♣♦♥s❡t♦❡❧❡❝tr♦✲ ♠♦t✐✈❡❢♦r❝❡❛ss✉♠❡❞❛s ❍❡❛✈✐s✐❞❡✬s st❡♣❢✉♥❝t✐♦♥♦❢✐♥t❡♥s✐t②E0 = 1✱♦❜t❛✐♥❡❞❛♥❛❧②t✐❝❛❧❧②✭❧✐♥❡s✮❛♥❞ ♥✉♠❡r✐❝❛❧❧②✭❞♦ts✮❢♦r ♠♦❞❡❧♣❛r❛♠❡t❡rs✿R = 1✱τ C = 0.1✱❛♥❞τ α = 0.3✳ ❈✉rr❡♥t ✐♥RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣t❤❡❛❝t✐✈❡❝❛♣❛❝✐t♦r✱♠♦❞❡❧❡❞❜②✈♦❧t❛❣❡✲❝❤❛r❣❡❝♦♥st✐t✉t✐✈❡ ❈✉rr❡♥t ✐♥RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣t❤❡❛❝t✐✈❡❝❛♣❛❝✐t♦r✱♠♦❞❡❧❡❞❜②✈♦❧t❛❣❡✲❝❤❛r❣❡❝♦♥st✐t✉t✐✈❡r❡❧❛t✐♦♥ ✭✸✮✱❛s ❛r❡s♣♦♥s❡t♦t❤❡❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡t❛❦❡♥❛s t❤❡❍❡❛✈✐s✐❞❡st❡♣❢✉♥❝t✐♦♥✐s ❝❛❧❝✉❧❛t❡❞❜②✭✸✷✮2 ❛♥❞❝♦rr❡s♣♦♥❞✐♥❣t✐♠❡♣r♦✜❧❡s ❛r❡s❤♦✇♥✐♥❋✐❣✉r❡✷✱❛❧♦♥❣✇✐t❤t❤❡r❡s♣♦♥s❡s ♦❢t❤❡❝❧❛ss✐❝❛❧❛♥❞ ✶✷ ✐♥t❡❣❡r ♦r❞❡r ❤❡r❡❞✐t❛r②RC ❝✐r❝✉✐ts✱s❡❡✭✶✾✮❛♥❞✭✷✵✮✱t❤❛t ❛r❡❝❛❧❝✉❧❛t❡❞❜②✿ ✐♥t❡❣❡r ♦r❞❡r ❤❡r❡❞✐t❛r②RC ❝✐r❝✉✐ts✱s❡❡✭✶✾✮❛♥❞✭✷✵✮✱t❤❛t ❛r❡❝❛❧❝✉❧❛t❡❞❜②✿ i(t) = E0 R e −  1 τC + 1 τµ  t ❢♦r µ = 0 ❛♥❞ ✭✸✻✮ i(t) = E0 R                  e− t 2τC  cosh (λt) −sinh(λt) 2τ Cλ  , λ = r 1 2τ C 2 − 1 τ µ ✱ (1 −σt) e−σt, σ = 1 2τ C + r 1 2τ C 2 − 1 τ µ ✱ e− t 2τC  cos (ωt) −sin(ωt) 2τ Cω  , ω = r 1 τ µ −  1 2τ C 2 ✱ ❢♦r µ = 1. ✹ ❙t❡❛❞②st❛t❡r❡s♣♦♥s❡♦❢❢r❛❝t✐♦♥❛❧RC ❛♥❞RL ❝✐r❝✉✐ts ❚❤❡st❡❛❞②st❛t❡r❡❣✐♠❡♦❢❢♦r❝❡❞s❡r✐❡s ❢r❛❝t✐♦♥❛❧RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣❡✐t❤❡r ♣❛ss✐✈❡❝❛♣❛❝✐t♦r ♠♦❞❡❧❡❞ ❜②❝❤❛r❣❡✲✈♦❧t❛❣❡r❡❧❛t✐♦♥✭✶✮♦r ❛❝t✐✈❡❝❛♣❛❝✐t♦r ♠♦❞❡❧❡❞❜②✈♦❧t❛❣❡✲❝❤❛r❣❡❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥✭✸✮✐s ❝♦♥s✐❞❡r❡❞❜②❛ss✉♠✐♥❣t❤❡❤❛r♠♦♥✐❝❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡ E(t) = E0 ejωt ✭✸✽✮ ✭✸✽✮ ♦❢❛♠♣❧✐t✉❞❡E0 ❛♥❞❛♥❣✉❧❛r ❢r❡q✉❡♥❝②ω✱②✐❡❧❞✐♥❣t❤❡❝❛♣❛❝✐t♦r ✈♦❧t❛❣❡❛♥❞❝✉rr❡♥t ✐♥t❤❡❢♦r♠♦❢ ❤❛r♠♦♥✐❝❢✉♥❝t✐♦♥s ♦❢❛♠♣❧✐t✉❞❡E0 ❛♥❞❛♥❣✉❧❛r ❢r❡q✉❡♥❝②ω✱②✐❡❧❞✐♥❣t❤❡❝❛♣❛❝✐t♦r ✈♦❧t❛❣❡❛♥❞❝✉rr❡♥t ✐♥t❤❡❢♦r♠♦❢ ❤❛r♠♦♥✐❝❢✉♥❝t✐♦♥s uC(t) = uC0 ej(ωt+φC) ❛♥❞ i(t) = i0 ej(ωt+φi), ✭✸✾✮ ✭✸✾✮ ❛s ✇❡❧❧✱❤❛✈✐♥❣❛♠♣❧✐t✉❞❡s uC0 ❛♥❞i0 ❛♥❞♣❤❛s❡❛♥❣❧❡s φC ❛♥❞φi✱❞✉❡t♦❧✐♥❡❛r✐t②♦❢❢r❛❝t✐♦♥❛❧RC ❝✐r❝✉✐ts✬❣♦✈❡r♥✐♥❣❡q✉❛t✐♦♥s ✭✷✸✮❛♥❞✭✷✹✮✳◆♦t❡✱t❤❡q✉❛♥t✐t✐❡s ❤❛✈✐♥❣♣❤②s✐❝❛❧♠❡❛♥✐♥❣✐♥✭✸✽✮❛♥❞ ✭✸✾✮❛r❡Re E✱Re uC✱❛♥❞Re i✳ ■❢t✐♠❡✐s ❧❛r❣❡❡♥♦✉❣❤✱t❤❡❢r❛❝t✐♦♥❛❧RC ❝✐r❝✉✐ts ❡♥t❡r t❤❡st❡❛❞②st❛t❡r❡❣✐♠❡✱❞✉❡t♦t❤❡♣r❡✈❛❧❡♥❝❡ ♦❢❤❛r♠♦♥✐❝❢♦r❝✐♥❣♦✈❡r t❤❡✐♠♣✉❧s❡r❡s♣♦♥s❡s g(1) C ❛♥❞g(2) i ✱❣✐✈❡♥❜②✭✷✾✮❛♥❞✭✸✶✮✱s✐♥❝❡t❤❡②❞❡❝❛②t♦ ③❡r♦❛❝❝♦r❞✐♥❣t♦t❤❡✜♥❛❧✈❛❧✉❡❚❛✉❜❡r✐❛♥t❤❡♦r❡♠✿ lim t→∞g(1) C (t) = lim s→0  sˆg(1) C (s)  = 0 ❛♥❞ lim t→∞g(2) i (t) = lim s→0  sˆg(2) i (s)  = 0, ✇✐t❤ˆg(1) C ❛♥❞ˆg(2) i ❣✐✈❡♥❜②✭✷✺✮❛♥❞✭✷✻✮✱s♦t❤❛t t❤❡s♦❧✉t✐♦♥s ♦❢t❤❡❣♦✈❡r♥✐♥❣❡q✉❛t✐♦♥s ✭✷✸✮❛♥❞✭✷✹✮ ✐♥t❤❡tr❛♥s✐❡♥t r❡❣✐♠❡✱t❤❛t ❛r❡♦❜t❛✐♥❡❞❛s ❝♦♥✈♦❧✉t✐♦♥s ✇✐t❤t❤❡❤❛r♠♦♥✐❝❢♦r❝✐♥❣✭✸✽✮❛❝❝♦r❞✐♥❣t♦ ✭✸✷✮✱❢♦r ❧❛r❣❡t✐♠❡❜❡❝♦♠❡❤❛r♠♦♥✐❝❛s ✇❡❧❧✳ ✸✳✷ ◆✉♠❡r✐❝❛❧❡①❛♠♣❧❡s ✭✸✼✮ ✭✸✻✮ ✭✸✼✮ ❙✐♠✐❧❛r❧②❛s ✐♥t❤❡♣r❡✈✐♦✉s ❝❛s❡✱❛s ❞❡♣✐❝t❡❞✐♥❋✐❣✉r❡✷✱t❤❡t✐♠❡♣r♦✜❧❡s ♦❢❝✉rr❡♥t ❝♦rr❡s♣♦♥❞✐♥❣ t♦t❤❡❢r❛❝t✐♦♥❛❧♦r❞❡r ✈♦❧t❛❣❡✲❝❤❛r❣❡♠♦❞❡❧✭✸✮❧✐❡❜❡t✇❡❡♥t❤❡♣r♦✜❧❡s ❝♦rr❡s♣♦♥❞✐♥❣t♦t❤❡❝❧❛ss✐❝❛❧ ❛♥❞✐♥t❡❣❡r ♦r❞❡r ❤❡r❡❞✐t❛r②RC ❝✐r❝✉✐ts✱♦❜t❛✐♥❡❞❛❝❝♦r❞✐♥❣t♦✭✸✻✮❛♥❞✭✸✼✮r❡s♣❡❝t✐✈❡❧②✳❈❧❛ss✐❝❛❧ RC ❝✐r❝✉✐t✱❛❝❝♦r❞✐♥❣t♦✭✸✻✮✱❤❛s ❛♠♦♥♦t♦♥✐❝❡①♣♦♥❡♥t✐❛❧❧②❞❡❝r❡❛s✐♥❣r❡s♣♦♥s❡❢♦r ❛♥②✈❛❧✉❡s ♦❢t❤❡ ♠♦❞❡❧♣❛r❛♠❡t❡rs✱✇❤✐❧❡✐♥t❤❡❝❛s❡♦❢✐♥t❡❣❡r ♦r❞❡r ❤❡r❡❞✐t❛r②RC ❝✐r❝✉✐t ♠♦❞❡❧♣❛r❛♠❡t❡rs ❞❡t❡r♠✐♥❡ ✇❤❡t❤❡r t❤❡r❡s♣♦♥s❡✐s ❛♣❡r✐♦❞✐❝♦r ♦s❝✐❧❧❛t♦r②✱s♦t❤❛t t❤❡r❡s♣♦♥s❡❢r♦♠❋✐❣✉r❡✷❛✱♦❜t❛✐♥❡❞❜②✭✸✼✮2✱ ✐s ❛♣❡r✐♦❞✐❝❜✉t ❛❧s♦♥♦♥✲♠♦♥♦t♦♥✐❝✱✇❤✐❧❡t❤❡r❡s♣♦♥s❡❢r♦♠❋✐❣✉r❡✷❜✱♦❜t❛✐♥❡❞❜②✭✸✼✮3✱❤❛s ❞❛♠♣❡❞ ♦s❝✐❧❧❛t♦r②❝❤❛r❛❝t❡r✳❚❤❡r❡s♣♦♥s❡♦❢❢r❛❝t✐♦♥❛❧RC ❝✐r❝✉✐t ❞✐s♣❧❛②s t❤❡❞❛♠♣❡❞♦s❝✐❧❧❛t♦r②❜❡❤❛✈✐♦r✱s❡❡ ❝✉r✈❡s ❢♦r µ = 0.5 ❛♥❞µ = 0.75 ❢r♦♠❋✐❣✉r❡✷❜✱t❤❛t ❝❛♥❜❡❛tt❡♥✉❛t❡❞t♦s✉❝❤❛♥❡①t❡♥t✱s♦t❤❛t ♦♥❧② ♦♥❡♠✐♥✐♠✉♠r❡♠❛✐♥s✱s❡❡❛❧❧❝✉r✈❡s ❢r♦♠❋✐❣✉r❡✷❛❛♥❞❝✉r✈❡❢♦r µ = 0.25 ❢r♦♠❋✐❣✉r❡✷❜✳ μ = 0 μ = 1 1 2 3 4 t 0.0 0.2 0.4 0.6 0.8 1.0 i(t) μ = 1 μ = 0 0.0 0.2 0.4 0.6 0.8 t 0.0 0.2 0.4 0.6 0.8 1.0 i(t) ✭❛✮❚✐♠❡♣r♦✜❧❡s✱❞❡♣✐❝t❡❞❜②❞♦tt❡❞✱❞❛s❤❡❞✱❛♥❞❞♦t✲ ❞❛s❤❡❞❧✐♥❡❢♦r µ = 0.25, 0.5, 0.75✱r❡s♣❡❝t✐✈❡❧②✱❛♥❞❢♦r ♠♦❞❡❧♣❛r❛♠❡t❡rs✿R = 1✱τ C = 0.5✱❛♥❞τ µ = 1✳ μ = 0 μ = 0.25 μ = 0.5 μ = 0.75 μ = 1 0.5 1.0 1.5 2.0 2.5 t -0.5 0.0 0.5 1.0 i(t) ✭❜✮❚✐♠❡♣r♦✜❧❡s ❢♦r ♠♦❞❡❧♣❛r❛♠❡t❡rs✿R = 1✱τ C = 1✱ ❛♥❞τ µ = 0.1✳ μ = 0 μ = 0.25 μ = 0.5 μ = 0.75 μ = 1 0.5 1.0 1.5 2.0 2.5 t -0.5 0.0 0.5 1.0 i(t) ✭❜✮❚✐♠❡♣r♦✜❧❡s ❢♦r ♠♦❞❡❧♣❛r❛♠❡t❡rs✿R = 1✱τ C = 1✱ ❛♥❞τ µ = 0.1✳ ✭❛✮❚✐♠❡♣r♦✜❧❡s✱❞❡♣✐❝t❡❞❜②❞♦tt❡❞✱❞❛s❤❡❞✱❛♥❞❞♦t✲ ❞❛s❤❡❞❧✐♥❡❢♦r µ = 0.25, 0.5, 0.75✱r❡s♣❡❝t✐✈❡❧②✱❛♥❞❢♦r ♠♦❞❡❧♣❛r❛♠❡t❡rs✿R = 1✱τ C = 0.5✱❛♥❞τ µ = 1✳ ✭❜✮❚✐♠❡♣r♦✜❧❡s ❢♦r ♠♦❞❡❧♣❛r❛♠❡t❡rs✿R = 1✱τ C = 1✱ ❛♥❞τ µ = 0.1✳ ✭❜✮❚✐♠❡♣r♦✜❧❡s ❢♦r ♠♦❞❡❧♣❛r❛♠❡t❡rs✿R = 1✱τ C = 1✱ ❛♥❞τ µ = 0.1✳ ✭❛✮❚✐♠❡♣r♦✜❧❡s✱❞❡♣✐❝t❡❞❜②❞♦tt❡❞✱❞❛s❤❡❞✱❛♥❞❞♦t✲ ❞❛s❤❡❞❧✐♥❡❢♦r µ = 0.25, 0.5, 0.75✱r❡s♣❡❝t✐✈❡❧②✱❛♥❞❢♦r ♠♦❞❡❧♣❛r❛♠❡t❡rs✿R = 1✱τ C = 0.5✱❛♥❞τ µ = 1✳ ✭❜✮❚✐♠❡♣r♦✜❧❡s ❢♦r ♠♦❞❡❧♣❛r❛♠❡t❡rs✿R = 1✱τ C = 1✱ ❛♥❞τ µ = 0.1✳ ❋✐❣✉r❡✷✿❈❛s❡♦❢❛❝t✐✈❡❝❛♣❛❝✐t♦r ✕t✐♠❡♣r♦✜❧❡s ♦❢❝✉rr❡♥t ✐♥RC ❝✐r❝✉✐t ❛s ❛r❡s♣♦♥s❡t♦❡❧❡❝tr♦♠♦✲ t✐✈❡❢♦r❝❡❛ss✉♠❡❞❛s ❍❡❛✈✐s✐❞❡✬s st❡♣❢✉♥❝t✐♦♥♦❢✐♥t❡♥s✐t②E0 = 1✱♦❜t❛✐♥❡❞❛♥❛❧②t✐❝❛❧❧②✭❧✐♥❡s✮❛♥❞ ♥✉♠❡r✐❝❛❧❧②✭❞♦ts✮✳ ❋✐❣✉r❡✷✿❈❛s❡♦❢❛❝t✐✈❡❝❛♣❛❝✐t♦r ✕t✐♠❡♣r♦✜❧❡s ♦❢❝✉rr❡♥t ✐♥RC ❝✐r❝✉✐t ❛s ❛r❡s♣♦♥s❡t♦❡❧❡❝tr♦♠♦✲ t✐✈❡❢♦r❝❡❛ss✉♠❡❞❛s ❍❡❛✈✐s✐❞❡✬s st❡♣❢✉♥❝t✐♦♥♦❢✐♥t❡♥s✐t②E0 = 1✱♦❜t❛✐♥❡❞❛♥❛❧②t✐❝❛❧❧②✭❧✐♥❡s✮❛♥❞ ♥✉♠❡r✐❝❛❧❧②✭❞♦ts✮✳ ❆s ❡✈✐❞❡♥t ❢r♦♠❋✐❣✉r❡s ✶❛♥❞✷✱t❤❡r❡✐s ❛♣❡r❢❡❝t ❛❣r❡❡♠❡♥t ❜❡t✇❡❡♥❝✉r✈❡s ♦❜t❛✐♥❡❞t❤r♦✉❣❤ ❛♥❛❧②t✐❝❛❧❡①♣r❡ss✐♦♥s ❛♥❞t❤♦s❡❝❛❧❝✉❧❛t❡❞❜②t❤❡✜①❡❞❚❛❧❜♦t ♥✉♠❡r✐❝❛❧▲❛♣❧❛❝❡✐♥✈❡rs✐♦♥▼❛t❤❡♠❛t✐❝❛ ❢✉♥❝t✐♦♥✱❞❡✈❡❧♦♣❡❞❜②❏✳❆❜❛t❡❛♥❞P✳P✳❱❛❧❦ó ❛❝❝♦r❞✐♥❣t♦❬✶❪❛♥❞❛✈❛✐❧❛❜❧❡❛t✿ ❤tt♣✿✴✴❧✐❜r❛r②✳✇♦❧❢r❛♠✳❝♦♠✴✐♥❢♦❝❡♥t❡r✴▼❛t❤❙♦✉r❝❡✴✹✼✸✽✴✳ ✶✸ ✹✳✶ ❉❡r✐✈❛t✐♦♥♦❢❛♠♣❧✐t✉❞❡s ❛♥❞♣❤❛s❡❛♥❣❧❡s ♦❢st❡❛❞②st❛t❡r❡s♣♦♥s❡s P❧✉❣❣✐♥❣t❤❡❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡✭✸✽✮❛♥❞❝❛♣❛❝✐t♦r ✈♦❧t❛❣❡✭✸✾✮1 ✐♥t♦❡q✉❛t✐♦♥✭✷✸✮❣♦✈❡r♥✐♥❣t❤❡r❡s♣♦♥s❡ ♦❢RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣♣❛ss✐✈❡❝❛♣❛❝✐t♦r✱♦♥❡✜♥❞s sin φ(1) C = −uC0 E0  τ C ω + τ α ωα sin απ 2  , cos φ(1) C = uC0 E0  τ α ωα cos απ 2 + 1  , ❛❝❝♦r❞✐♥❣t♦t❤❡❢♦r♠✉❧❛❢♦r ❢r❛❝t✐♦♥❛❧❞❡r✐✈❛t✐✈❡♦❢❤❛r♠♦♥✐❝❢✉♥❝t✐♦♥✭✶✷✮✱s♦t❤❛t ♣❛ss✐✈❡❝❛♣❛❝✐t♦r✬s ✈♦❧t❛❣❡❛♠♣❧✐t✉❞❡❛♥❞♣❤❛s❡❛♥❣❧❡❛r❡✿ ✭✹✵✮ u(1) C0 = E0 q τ 2 C ω2 + 2τ Cτ α ω1+α sin απ 2 + τ 2α ω2α + 2τ α ωα cos απ 2 + 1 , ✭✹✵✮ ✶✹ φ(1) C = −arctan τ C ω + τ α ωα sin απ 2 τ α ωα cos απ 2 + 1 , ✭✹✶✮ φ(1) C = −arctan τ C ω + τ α ωα sin απ 2 τ α ωα cos απ 2 + 1 , ✭✹✶✮ ✭✹✶✮ ✇❤✐❧❡t❤❡❡q✉❛t✐♦♥✭✷✹✮❣♦✈❡r♥✐♥❣t❤❡r❡s♣♦♥s❡♦❢RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣❛❝t✐✈❡❝❛♣❛❝✐t♦r✱✇✐t❤t❤❡❡❧❡❝✲ tr♦♠♦t✐✈❡❢♦r❝❡❛♥❞❝✉rr❡♥t ❛ss✉♠❡❞❛s ✭✸✽✮❛♥❞✭✸✾✮2✱②✐❡❧❞s ✇❤✐❧❡t❤❡❡q✉❛t✐♦♥✭✷✹✮❣♦✈❡r♥✐♥❣t❤❡r❡s♣♦♥s❡♦❢RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣❛❝t✐✈❡❝❛♣❛❝✐t♦r✱✇✐t❤t❤❡❡❧❡❝✲ tr♦♠♦t✐✈❡❢♦r❝❡❛♥❞❝✉rr❡♥t ❛ss✉♠❡❞❛s ✭✸✽✮❛♥❞✭✸✾✮2✱②✐❡❧❞s sin φ(2) i = −R i0 E0  1 τ C ω + 1 τ µ ω1+µ cos µπ 2  , cos φ(2) i = R i0 E0  1 − 1 τ µ ω1+µ sin µπ 2  , t❤❛t s♦❧✈❡❞✇✐t❤r❡s♣❡❝t t♦t❤❡❝✉rr❡♥t ❛♠♣❧✐t✉❞❡❛♥❞♣❤❛s❡❛♥❣❧❡❣✐✈❡s t❤❛t s♦❧✈❡❞✇✐t❤r❡s♣❡❝t t♦t❤❡❝✉rr❡♥t ❛♠♣❧✐t✉❞❡❛♥❞♣❤❛s❡❛♥❣❧❡❣✐✈❡s i(2) 0 = E0 R 1 q 1 −2 1 τ µ ω1+µ sin µπ 2 + 1 τ 2 C ω2 + 2 1 τ Cτ µ ω2+µ cos µπ 2 + 1 τ 2 µ ω2+2µ , ✭✹✷✮ φ(2) i = arctan 1 τ C ω + 1 τ µ ω1+µ cos µπ 2 1 − 1 τ µ ω1+µ sin µπ 2 , ✭✹✸✮ ✭✹✷✮ ✭✹✸✮ ❡t❤❡❢♦r♠✉❧❛❢♦r ❢r❛❝t✐♦♥❛❧✐♥t❡❣r❛❧♦❢❤❛r♠♦♥✐❝❢✉♥❝t✐♦♥✭✶✺✮✐s ✉s❡❞✳ ✇❤❡r❡t❤❡❢♦r♠✉❧❛❢♦r ❢r❛❝t✐♦♥❛❧✐♥t❡❣r❛❧♦❢❤❛r♠♦♥✐❝❢✉♥❝t✐♦♥✭✶✺✮✐s ✉s❡❞✳ ■♥♦r❞❡r t♦♦❜t❛✐♥❝✉rr❡♥t ✐♥t❤❡st❡❛❞②st❛t❡r❡❣✐♠❡♦❢RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣♣❛ss✐✈❡❝❛♣❛❝✐t♦r ✐♥t❤❡ ❢♦r♠❣✐✈❡♥❜②✭✸✾✮2✱♦♥❡r❡✇r✐t❡s ❡q✉❛t✐♦♥✭✶✶✮❛s i(t) = 1 R  τ C d dtuC(t) + τ α 0Dα t uC(t)  , t❤❛t ❛❧♦♥❣✇✐t❤♣❛ss✐✈❡❝❛♣❛❝✐t♦r✬s ✈♦❧t❛❣❡✭✸✾✮1 ②✐❡❧❞s t❤❛t ❛❧♦♥❣✇✐t❤♣❛ss✐✈❡❝❛♣❛❝✐t♦r✬s ✈♦❧t❛❣❡✭✸✾✮1 ②✐❡❧❞s t❤❛t ❛❧♦♥❣✇✐t❤♣❛ss✐✈❡❝❛♣❛❝✐t♦r✬s ✈♦❧t❛❣❡✭✸✾✮1 ②✐❡❧❞s t❤❛t ❛❧♦♥❣✇✐t❤♣❛ss✐✈❡❝❛♣❛❝✐t♦r✬s ✈♦❧t❛❣❡✭✸✾✮1 ②✐❡❧❞s sin(φ(1) i −φ(1) C ) = u(1) C0 R i(1) 0  τ C ω + τ α ωα sin απ 2  , cos(φ(1) i −φ(1) C ) = u(1) C0 R i(1) 0 τ α ωα cos απ 2 , ✇❤❡r❡✭✶✷✮✐s ✉s❡❞✱✐♠♣❧②✐♥❣t❤❡❝✉rr❡♥t ❛♠♣❧✐t✉❞❡❛♥❞♣❤❛s❡❛♥❣❧❡✐♥t❤❡❢♦❧❧♦✇✐♥❣❢♦r♠s i(1) 0 = u(1) C0 R r τ 2 C ω2 + 2τ Cτ α ω1+α sin απ 2 + τ 2α ω2α, ✭✹✹✮ φ(1) i = φ(1) C + arctan τ C τ α ω1−α 1 cos απ 2 + tan απ 2  , ✭✹✺✮ ✭✹✹✮ ✭✹✺✮ ✇✐t❤♣❛ss✐✈❡❝❛♣❛❝✐t♦r✬s ✈♦❧t❛❣❡❛♠♣❧✐t✉❞❡❛♥❞♣❤❛s❡❛♥❣❧❡❣✐✈❡♥❜②✭✹✵✮❛♥❞✭✹✶✮✳❖♥t❤❡♦t❤❡r ❤❛♥❞✱ ❛❝t✐✈❡❝❛♣❛❝✐t♦r✬s ✈♦❧t❛❣❡✐♥t❤❡st❡❛❞②st❛t❡r❡❣✐♠❡♦❢RC ❝✐r❝✉✐t ✐♥t❤❡❢♦r♠❣✐✈❡♥❜②✭✸✾✮1 ✐s ♦❜t❛✐♥❡❞ ❢r♦♠✐ts ❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥✭✶✹✮✱r❡✇r✐tt❡♥❛s ✇✐t❤♣❛ss✐✈❡❝❛♣❛❝✐t♦r✬s ✈♦❧t❛❣❡❛♠♣❧✐t✉❞❡❛♥❞♣❤❛s❡❛♥❣❧❡❣✐✈❡♥❜②✭✹✵✮❛♥❞✭✹✶✮✳❖♥t❤❡♦t❤❡r ❤❛♥❞✱ ❛❝t✐✈❡❝❛♣❛❝✐t♦r✬s ✈♦❧t❛❣❡✐♥t❤❡st❡❛❞②st❛t❡r❡❣✐♠❡♦❢RC ❝✐r❝✉✐t ✐♥t❤❡❢♦r♠❣✐✈❡♥❜②✭✸✾✮1 ✐s ♦❜t❛✐♥❡❞ ❢r♦♠✐ts ❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥✭✶✹✮✱r❡✇r✐tt❡♥❛s uC(t) = R  1 τ C 0I1 ti(t) + 1 τ µ 0I1+µ t i(t)  , ✶✺ t❤❛t ❛❧♦♥❣✇✐t❤t❤❡❝✉rr❡♥t ✭✸✾✮2 ②✐❡❧❞s t❤❛t ❛❧♦♥❣✇✐t❤t❤❡❝✉rr❡♥t ✭✸✾✮2 ②✐❡❧❞s sin(φ(2) C −φ(2) i ) = −R i(2) 0 u(2) C0  1 τ C ω + 1 τ µ ω1+µ cos µπ 2  , cos(φ(2) C −φ(2) i ) = −R i(2) 0 u(2) C0 1 τ µ ω1+µ sin µπ 2 , ✇❤❡r❡✭✶✷✮✐s ✉s❡❞✱✐♠♣❧②✐♥❣❛❝t✐✈❡❝❛♣❛❝✐t♦r✬s ✈♦❧t❛❣❡❛♠♣❧✐t✉❞❡❛♥❞♣❤❛s❡❛♥❣❧❡✐♥t❤❡❢♦❧❧♦✇✐♥❣❢♦r♠s u(2) C0 = R i(2) 0 s 1 τ 2 C ω2 + 2 1 τ Cτ µ ω2+µ cos µπ 2 + 1 τ 2µ ω2+2µ , ✭✹✻✮ φ(2) C = φ(2) i + arctan 1 τ C ω 1 τ µ ω1+µ sin µπ 2 + cot µπ 2 ! ✹✳✶ ❉❡r✐✈❛t✐♦♥♦❢❛♠♣❧✐t✉❞❡s ❛♥❞♣❤❛s❡❛♥❣❧❡s ♦❢st❡❛❞②st❛t❡r❡s♣♦♥s❡s , ✭✹✼✮ ✭✹✻✮ ✭✹✼✮ ✇✐t❤❝✉rr❡♥t ❛♠♣❧✐t✉❞❡❛♥❞♣❤❛s❡❛♥❣❧❡❣✐✈❡♥❜②✭✹✷✮❛♥❞✭✹✸✮✳ ✇✐t❤❝✉rr❡♥t ❛♠♣❧✐t✉❞❡❛♥❞♣❤❛s❡❛♥❣❧❡❣✐✈❡♥❜②✭✹✷✮❛♥❞✭✹✸✮✳ ✇✐t❤❝✉rr❡♥t ❛♠♣❧✐t✉❞❡❛♥❞♣❤❛s❡❛♥❣❧❡❣✐✈❡♥❜②✭✹✷✮❛♥❞✭✹✸✮✳ ■❢t❤❡♣❛ss✐✈❡✐♥❞✉❝t♦r ✐♥t❤❡s❡r✐❡s ❢r❛❝t✐♦♥❛❧RL ❝✐r❝✉✐t ❢♦r❝❡❞❜②t❤❡❤❛r♠♦♥✐❝❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡ ✭✸✽✮✐s ♠♦❞❡❧❡❞❜②t❤❡✢✉①✲❝✉rr❡♥t ❝♦♥st✐t✉t✐✈❡r❡❧❛t✐♦♥✭✷✮✱t❤❡♥t❤❡❝✉rr❡♥t ✐s ❣✐✈❡♥❜②✭✸✾✮2✱✇✐t❤ ❛♠♣❧✐t✉❞❡❛♥❞♣❤❛s❡❛♥❣❧❡❤❛✈✐♥❣t❤❡s❛♠❡❢♦r♠❛s t❤❡♣❛ss✐✈❡❝❛♣❛❝✐t♦r ✈♦❧t❛❣❡❛♠♣❧✐t✉❞❡u(1) C0 ❛♥❞ ♣❤❛s❡❛♥❣❧❡φ(1) C ✱❣✐✈❡♥❜②✭✹✵✮❛♥❞✭✹✶✮✱❞✉❡t♦t❤❡s❛♠❡❢♦r♠s ♦❢❣♦✈❡r♥✐♥❣❡q✉❛t✐♦♥s ✭✸✹✮❢♦r RL ❝✐r❝✉✐t ❛♥❞✭✷✸✮❢♦r RC ❝✐r❝✉✐t✱✇❤✐❧❡✱❜②t❤❡❛♥❛❧♦❣②♦❢❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s ✭✶✸✮❛♥❞✭✶✶✮✱♣❛ss✐✈❡✐♥❞✉❝t♦r✬s ✈♦❧t❛❣❡✐s ❛❤❛r♠♦♥✐❝❢✉♥❝t✐♦♥♦❢❛♠♣❧✐t✉❞❡❛♥❞♣❤❛s❡❛♥❣❧❡❤❛✈✐♥❣t❤❡s❛♠❡❢♦r♠s ❛s t❤❡❛♠♣❧✐t✉❞❡ i(1) 0 ❛♥❞♣❤❛s❡❛♥❣❧❡φ(1) i ✱❣✐✈❡♥❜②✭✹✹✮❛♥❞✭✹✺✮✳❆❧s♦✱✐♥t❤❡❝❛s❡♦❢❛❝t✐✈❡✐♥❞✉❝t♦r ♠♦❞❡❧❡❞❜②t❤❡ ❝✉rr❡♥t✲✢✉①❝♦♥st✐t✉t✐✈❡r❡❧❛t✐♦♥✭✹✮✱❞✉❡t♦t❤❡❛♥❛❧♦❣②♦❢❣♦✈❡r♥✐♥❣❡q✉❛t✐♦♥s ✭✸✺✮❢♦r RL ❛♥❞✭✷✹✮ ❢♦r RC ❝✐r❝✉✐t✱❛❝t✐✈❡✐♥❞✉❝t♦r✬s ✈♦❧t❛❣❡✐s ❛❤❛r♠♦♥✐❝❢✉♥❝t✐♦♥❤❛✈✐♥❣❛♠♣❧✐t✉❞❡❛♥❞♣❤❛s❡❛♥❣❧❡♦❢ t❤❡s❛♠❡❢♦r♠❛s t❤❡❝✉rr❡♥t ❛♠♣❧✐t✉❞❡i(2) 0 ❛♥❞♣❤❛s❡❛♥❣❧❡φ(2) i ✱❣✐✈❡♥❜②✭✹✷✮❛♥❞✭✹✸✮✱✇❤✐❧❡✱❜②t❤❡ ❛♥❛❧♦❣②♦❢❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s ✭✶✻✮❛♥❞✭✶✹✮✱t❤❡❝✉rr❡♥t ✐s ❛❤❛r♠♦♥✐❝❢✉♥❝t✐♦♥✇✐t❤❛♠♣❧✐t✉❞❡❛♥❞ ♣❤❛s❡❛♥❣❧❡♦❢t❤❡s❛♠❡❢♦r♠❛s ❢♦r t❤❡❛❝t✐✈❡❝❛♣❛❝✐t♦r ✈♦❧t❛❣❡❛♠♣❧✐t✉❞❡u(2) C0 ❛♥❞♣❤❛s❡❛♥❣❧❡φ(2) C ✱ ❣✐✈❡♥❜②✭✹✻✮❛♥❞✭✹✼✮✳ ✹✳✷ ◆✉♠❡r✐❝❛❧❡①❛♠♣❧❡s ❆ss✉♠✐♥❣t❤❡❤❛r♠♦♥✐❝❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝✐♥❣❛s E(t) = E0 cos(ωt) t❤❡tr❛♥s✐t✐♦♥❢r♦♠tr❛♥s✐❡♥t t♦st❡❛❞②st❛t❡r❡❣✐♠❡✐s ✐❧❧✉str❛t❡❞✐♥❋✐❣✉r❡✸❜②s❤♦✇✐♥❣t❤❡t✐♠❡♣r♦✜❧❡s ♦❢ ❝✉rr❡♥t ✐♥t❤❡❢r❛❝t✐♦♥❛❧RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣♣❛ss✐✈❡❝❛♣❛❝✐t♦r✱♠♦❞❡❧❡❞❜②❝❤❛r❣❡✲✈♦❧t❛❣❡❝♦♥st✐t✐t✉t✐✈❡ ❡q✉❛t✐♦♥✭✶✮✱❛s ✇❡❧❧❛s ✐♥❋✐❣✉r❡✹✐♥t❤❡❝❛s❡♦❢❛❝t✐✈❡❝❛♣❛❝✐t♦r ♠♦❞❡❧❡❞❜②✈♦❧t❛❣❡✲❝❤❛r❣❡❝♦♥st✐t✐t✉t✐✈❡ ✶✻ ❡q✉❛t✐♦♥✭✸✮✳❚❤❡r❡s♣♦♥s❡✐♥tr❛♥s✐❡♥t r❡❣✐♠❡✐s ❝❛❧❝✉❧❛t❡❞❛❝❝♦r❞✐♥❣t♦✭✸✷✮❛s ❡q✉❛t✐♦♥✭✸✮✳❚❤❡r❡s♣♦♥s❡✐♥tr❛♥s✐❡♥t r❡❣✐♠❡✐s ❝❛❧❝✉❧❛t❡❞❛❝❝♦r❞✐♥❣t♦✭✸✷✮❛s i(t) = E0 g(1,2) i (t) ∗cos(ωt)  = E0 R cos(ωt) −E0 R Z t 0 g(1,2) C (t −t′) cos(ωt′) dt′, ✭✹✽✮ ✭✹✽✮ s✐♥❝❡t❤❡✐♠♣✉❧s❡r❡s♣♦♥s❡s g(1,2) i t❛❦❡t❤❡❢♦r♠✭✸✶✮✱✇❤❡r❡g(1,2) C ❛r❡❣✐✈❡♥❜②✭✷✾✮❛♥❞✭✸✵✮✱✇❤✐❧❡t❤❡ st❡❛❞②st❛t❡r❡s♣♦♥s❡ i(t) = i(1,2) 0 cos(ωt + φ(1,2) i ), ✭✹✾✮ ✭✹✾✮ ✇✐t❤t❤❡❝✉rr❡♥t ❛♠♣❧✐t✉❞❡s i(1,2) 0 ❣✐✈❡♥❜②✭✹✹✮❛♥❞✭✹✷✮❛♥❞♣❤❛s❡❛♥❣❧❡s φ(1,2) i ❣✐✈❡♥❜②✭✹✺✮❛♥❞✭✹✸✮ ✐s ♦❜t❛✐♥❡❞❛s t❤❡r❡❛❧♣❛rt ♦❢✭✸✾✮2✳ ❆s ♦❜✈✐♦✉s ❢r♦♠❋✐❣✉r❡✸✱❢r❛❝t✐♦♥❛❧RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣♣❛ss✐✈❡❝❛♣❛❝✐t♦r ❡♥t❡rs t❤❡st❡❛❞②st❛t❡ r❡❣✐♠❡q✉✐t❡r❛♣✐❞❧②r❡❣❛r❞❧❡ss ♦❢t❤❡✈❛❧✉❡♦❢❢r❛❝t✐♦♥❛❧❞✐✛❡r❡♥t✐❛t✐♦♥♦r❞❡r α✱s✐♥❝❡❝✉r✈❡s ❝♦rr❡s♣♦♥❞✲ ✐♥❣t♦t❤❡tr❛♥s✐❡♥t ❛♥❞st❡❛❞②st❛t❡r❡❣✐♠❡♦✈❡r❧❛♣❡✈❡♥❢♦r s♠❛❧❧t✐♠❡✱♣r❡s✉♠❛❜❧②❞✉❡t♦t❤❡❝♦♠♣❧❡t❡ ♠♦♥♦t♦♥✐❝✐t②♦❢✐♠♣✉❧s❡r❡s♣♦♥s❡g(1) C ✳❚❤❡❣♦♦❞❛❣r❡❡♠❡♥t ❜❡t✇❡❡♥t❤❡❝✉r✈❡s ♦❜t❛✐♥❡❞t❤r♦✉❣❤❛♥❛✲ ❧②t✐❝❛❧❡①♣r❡ss✐♦♥❢♦r tr❛♥s✐❡♥t r❡❣✐♠❡❛♥❞❜②♥✉♠❡r✐❝❛❧▲❛♣❧❛❝❡tr❛♥s❢♦r♠✐♥✈❡rs✐♦♥♣r♦❝❡❞✉r❡✐s ❡✈✐❞❡♥t ❛s ✇❡❧❧✳ ❍❛✈✐♥❣t❤❡❛♥❣✉❧❛r ❢r❡q✉❡♥❝②✜①❡❞✱t❤❡t✐♠❡♣r♦✜❧❡s ♦❢❝✉rr❡♥t ✐♥RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣t❤❡❛❝t✐✈❡ ❝❛♣❛❝✐t♦r✱❛s ✇❡❧❧❛s t❤❡❝✉rr❡♥t ❛♠♣❧✐t✉❞❡❛♥❞♣❤❛s❡❛♥❣❧❡❛r❡❞❡♣✐❝t❡❞✐♥❋✐❣✉r❡✹t❛❦✐♥❣❞✐✛❡r❡♥t ✈❛❧✉❡s ♦❢t❤❡❢r❛❝t✐♦♥❛❧✐♥t❡❣r❛t✐♦♥♦r❞❡r µ✳❙✐♠✐❧❛r❧②❛s ✐♥t❤❡❝❛s❡♦❢RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣t❤❡♣❛ss✐✈❡ ❝❛♣❛❝✐t♦r✱t❤❡t✐♠❡♣r♦✜❧❡s ❝♦rr❡s♣♦♥❞✐♥❣t♦tr❛♥s✐❡♥t ❛♥❞st❡❛❞②st❛t❡r❡s♣♦♥s❡s s❤♦✇♥✐♥❋✐❣✉r❡✹❛ st❛rt ♦✈❡r❧❛♣♣✐♥❣❡✈❡♥❢♦r s♠❛❧❧t✐♠❡✐♥❝❛s❡♦❢❧♦✇✈❛❧✉❡s ♦❢t❤❡♣❛r❛♠❡t❡r µ✱✇❤✐❧❡✇✐t❤✐ts ✐♥❝r❡❛s❡t❤❡ t✐♠❡♥❡❡❞❡❞t♦r❡❛❝❤t❤❡st❡❛❞②st❛t❡✐♥❝r❡❛s❡s ❛s ✇❡❧❧✳❆❣❛✐♥♦♥❡♥♦t✐❝❡s t❤❡❣♦♦❞❛❣r❡❡♠❡♥t ❜❡t✇❡❡♥ t❤❡❝✉r✈❡s ♦❜t❛✐♥❡❞❜②❛♥❛❧②t✐❝❛❧❛♥❞♥✉♠❡r✐❝❛❧❛♣♣r♦❛❝❤✳❚❤❡❝✉rr❡♥t ❛♠♣❧✐t✉❞❡✱❞❡♣✐❝t❡❞✐♥❋✐❣✉r❡✹❜✱ ✐s ❢♦✉♥❞t♦✐♥❝r❡❛s❡♠♦♥♦t♦♥✐❝❛❧❧②✇✐t❤t❤❡✐♥❝r❡❛s❡♦❢♣❛r❛♠❡t❡r µ✱✇❤✐❧❡✱❛s ❝❛♥❜❡s❡❡♥❢r♦♠❋✐❣✉r❡ ✹❝✱t❤❡♣❤❛s❡❛♥❣❧❡✐♥❝r❡❛s❡s ✉♣t♦t❤❡♠❛①✐♠✉♠✱❛tt❛✐♥❡❞❛t (µ, φi) = (0.75286, 0.579321π)✱❧②✐♥❣✐♥t❤❡ ✐♥t❡r✈❛❧[0.299579, 0.979149] ♦❢♣❛r❛♠❡t❡r µ ✇✐t❤t❤❡✈❛❧✉❡s ♦❢♣❤❛s❡❛♥❣❧❡φi ❣r❡❛t❡r t❤❛♥π 2 ✱♠❡❛♥✐♥❣ t❤❛t t❤❡r❡s✐st♦r ❛♥❞❛❝t✐✈❡❝❛♣❛❝✐t♦r ❝♦♥s✐❞❡r❡❞❛s ❛s✐♥❣❧❡❡❧❡♠❡♥t ✐♥t❤✐s ✐♥t❡r✈❛❧♦❢♣❛r❛♠❡t❡r µ ❜❡❤❛✈❡ ❛s ❛❣❡♥❡r❛t✐✈❡❡❧❡♠❡♥t✱s✐♥❝❡cos φi < 0✳ ✺ ❋r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢❢r❛❝t✐♦♥❛❧RC ❛♥❞RL ❝✐r❝✉✐t ■♥♦r❞❡r t♦❛♥❛❧②③❡t❤❡❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣❡✐t❤❡r ♣❛ss✐✈❡♦r ❛❝t✐✈❡❝❛♣❛❝✐t♦r✱ ♦♥❡❝♦♥s✐❞❡rs tr❛♥s❢❡r ❢✉♥❝t✐♦♥s ˆg(1,2) R = R ˆg(1,2) i , ✭✺✵✮ ✭✺✵✮ ✇❤❡r❡ˆg(1,2) i ❛r❡❣✐✈❡♥❜②✭✷✻✮❛♥❞✭✷✽✮✱✇✐t❤ˆg(1,2) i ❝♦rr❡s♣♦♥❞✐♥❣t♦t❤❡❝✉rr❡♥t r✉♥♥✐♥❣t❤r♦✉❣❤t❤❡ RC ❝✐r❝✉✐t ❛♥❞ˆg(1,2) R ❝♦rr❡s♣♦♥❞✐♥❣t♦t❤❡✈♦❧t❛❣❡♦♥r❡s✐st♦r uR✱s♦t❤❛t t❤❡♠♦❞✉❧✉s ❛♥❞❛r❣✉♠❡♥t ♦❢ ✶✼ α = 0.05 α = 0.5 α = 0.95 5 10 15 20 t -0.2 0.0 0.2 0.4 0.6 0.8 1.0 i(t) α = 0.05 α = 0.95 2 4 6 8 10 t -0.2 0.0 0.2 0.4 0.6 0.8 1.0 i(t) ❋✐❣✉r❡✸✿❚✐♠❡♣r♦✜❧❡s ♦❢❝✉rr❡♥t ✐♥RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣♣❛ss✐✈❡❝❛♣❛❝✐t♦r ❛s ❛r❡s♣♦♥s❡t♦❡❧❡❝tr♦♠♦t✐✈❡ ❢♦r❝❡❛ss✉♠❡❞❛s ❛❝♦s✐♥❡❢✉♥❝t✐♦♥♦❢❛♠♣❧✐t✉❞❡E0 = 1 ❛♥❞❛♥❣✉❧❛r ❢r❡q✉❡♥❝②ω = 0.3✱♦❜t❛✐♥❡❞❜②❛♥❛✲ ❧②t✐❝❛❧❡①♣r❡ss✐♦♥✭✹✽✮✭❧✐♥❡✮❛♥❞♥✉♠❡r✐❝❛❧❧②✭❞♦ts✮✐♥t❤❡tr❛♥s✐❡♥t r❡❣✐♠❡❛♥❞❜②❛♥❛❧②t✐❝❛❧❡①♣r❡ss✐♦♥ ✭✹✾✮✭t❤✐♥❧✐♥❡✮✐♥t❤❡st❡❛❞②st❛t❡r❡❣✐♠❡❢♦r ♠♦❞❡❧♣❛r❛♠❡t❡rs✿R = 1✱τ C = 0.2✱❛♥❞τ α = 0.5✳ α = 0.05 α = 0.5 α = 0.95 5 10 15 20 t -0.2 0.0 0.2 0.4 0.6 0.8 1.0 i(t) α = 0.05 α = 0.95 2 4 6 8 10 t -0.2 0.0 0.2 0.4 0.6 0.8 1.0 i(t) ❋✐❣✉r❡✸✿❚✐♠❡♣r♦✜❧❡s ♦❢❝✉rr❡♥t ✐♥RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣♣❛ss✐✈❡❝❛♣❛❝✐t♦r ❛s ❛r❡s♣♦♥s❡t♦❡❧❡❝tr♦♠♦t✐✈❡ ❢♦r❝❡❛ss✉♠❡❞❛s ❛❝♦s✐♥❡❢✉♥❝t✐♦♥♦❢❛♠♣❧✐t✉❞❡E0 = 1 ❛♥❞❛♥❣✉❧❛r ❢r❡q✉❡♥❝②ω = 0.3✱♦❜t❛✐♥❡❞❜②❛♥❛✲ ❧②t✐❝❛❧❡①♣r❡ss✐♦♥✭✹✽✮✭❧✐♥❡✮❛♥❞♥✉♠❡r✐❝❛❧❧②✭❞♦ts✮✐♥t❤❡tr❛♥s✐❡♥t r❡❣✐♠❡❛♥❞❜②❛♥❛❧②t✐❝❛❧❡①♣r❡ss✐♦♥ ✭✹✾✮✭t❤✐♥❧✐♥❡✮✐♥t❤❡st❡❛❞②st❛t❡r❡❣✐♠❡❢♦r ♠♦❞❡❧♣❛r❛♠❡t❡rs✿R = 1✱τ C = 0.2✱❛♥❞τ α = 0.5✳ μ = 0.05 μ = 0.5 μ = 0.95 1 2 3 4 5 6 7 t -2 -1 1 2 i(t) ✭❛✮❚✐♠❡♣r♦✜❧❡s ♦❢❝✉rr❡♥t ♦❜t❛✐♥❡❞❜②❛♥❛❧②t✐❝❛❧❡①♣r❡ss✐♦♥✭✹✽✮✭❧✐♥❡✮❛♥❞ ♥✉♠❡r✐❝❛❧❧②✭❞♦ts✮✐♥t❤❡tr❛♥s✐❡♥t r❡❣✐♠❡❛♥❞❜②❛♥❛❧②t✐❝❛❧❡①♣r❡ss✐♦♥✭✹✾✮ ✭t❤✐♥❧✐♥❡✮✐♥t❤❡st❡❛❞②st❛t❡r❡❣✐♠❡✳ 0.2 0.4 0.6 0.8 1.0 μ 0.5 1.0 1.5 2.0 2.5 3.0 i0 ✭❜✮❆♠♣❧✐t✉❞❡♦❢❝✉rr❡♥t ❞❡♣❡♥❞✐♥❣♦♥♣❛r❛♠❡t❡r µ✳ 0.0 0.2 0.4 0.6 0.8 1.0 μ 2 π 5 π 2 3 π 5 ϕi ✭❝✮P❤❛s❡❛♥❣❧❡♦❢❝✉rr❡♥t ❞❡♣❡♥❞✐♥❣♦♥♣❛r❛♠❡t❡r µ✳ ❋✐❣✉r❡✹✿❈✉rr❡♥t ✐♥RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣❛❝t✐✈❡❝❛♣❛❝✐t♦r ❛s ❛r❡s♣♦♥s❡t♦❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡❛ss✉♠❡❞ ❛s ❛❝♦s✐♥❡❢✉♥❝t✐♦♥♦❢❛♠♣❧✐t✉❞❡E0 = 1 ❛♥❞❛♥❣✉❧❛r ❢r❡q✉❡♥❝②ω = 3.2✱♦❜t❛✐♥❡❞❢♦r ♠♦❞❡❧♣❛r❛♠❡t❡rs✿ R = 1✱τ C = 1✱❛♥❞τ µ = 0.1✳ μ = 0.05 μ = 0.5 μ = 0.95 1 2 3 4 5 6 7 t -2 -1 1 2 i(t) ✭❛✮❚✐♠❡♣r♦✜❧❡s ♦❢❝✉rr❡♥t ♦❜t❛✐♥❡❞❜②❛♥❛❧②t✐❝❛❧❡①♣r❡ss✐♦♥✭✹✽✮✭❧✐♥❡✮❛♥❞ ♥✉♠❡r✐❝❛❧❧②✭❞♦ts✮✐♥t❤❡tr❛♥s✐❡♥t r❡❣✐♠❡❛♥❞❜②❛♥❛❧②t✐❝❛❧❡①♣r❡ss✐♦♥✭✹✾✮ ✭t❤✐♥❧✐♥❡✮✐♥t❤❡st❡❛❞②st❛t❡r❡❣✐♠❡✳ ✭❛✮❚✐♠❡♣r♦✜❧❡s ♦❢❝✉rr❡♥t ♦❜t❛✐♥❡❞❜②❛♥❛❧②t✐❝❛❧❡①♣r❡ss✐♦♥✭✹✽✮✭❧✐♥❡✮❛♥❞ ♥✉♠❡r✐❝❛❧❧②✭❞♦ts✮✐♥t❤❡tr❛♥s✐❡♥t r❡❣✐♠❡❛♥❞❜②❛♥❛❧②t✐❝❛❧❡①♣r❡ss✐♦♥✭✹✾✮ ✭t❤✐♥❧✐♥❡✮✐♥t❤❡st❡❛❞②st❛t❡r❡❣✐♠❡✳ 0.0 0.2 0.4 0.6 0.8 1.0 μ 2 π 5 π 2 3 π 5 ϕi ✭❝✮P❤❛s❡❛♥❣❧❡♦❢❝✉rr❡♥t ❞❡♣❡♥❞✐♥❣♦♥♣❛r❛♠❡t❡r µ✳ 0.2 0.4 0.6 0.8 1.0 μ 0.5 1.0 1.5 2.0 2.5 3.0 i0 ✭❜✮❆♠♣❧✐t✉❞❡♦❢❝✉rr❡♥t ❞❡♣❡♥❞✐♥❣♦♥♣❛r❛♠❡t❡r µ✳ 0.0 0.2 0.4 0.6 0.8 1.0 μ 2 π 5 π 2 3 π 5 ϕi ✭❝✮P❤❛s❡❛♥❣❧❡♦❢❝✉rr❡♥t ❞❡♣❡♥❞✐♥❣♦♥♣❛r❛♠❡t❡r µ✳ ❋✐❣✉r❡✹✿❈✉rr❡♥t ✐♥RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣❛❝t✐✈❡❝❛♣❛❝✐t♦r ❛s ❛r❡s♣♦♥s❡t♦❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡❛ss✉♠❡❞ ❛s ❛❝♦s✐♥❡❢✉♥❝t✐♦♥♦❢❛♠♣❧✐t✉❞❡E0 = 1 ❛♥❞❛♥❣✉❧❛r ❢r❡q✉❡♥❝②ω = 3.2✱♦❜t❛✐♥❡❞❢♦r ♠♦❞❡❧♣❛r❛♠❡t❡rs✿ R = 1✱τ C = 1✱❛♥❞τ µ = 0.1✳ 0.2 0.4 0.6 0.8 1.0 μ 0.5 1.0 1.5 2.0 2.5 3.0 ✭❜✮❆♠♣❧✐t✉❞❡♦❢❝✉rr❡♥t ❞❡♣❡♥❞✐♥❣♦♥♣❛r❛♠❡t❡r µ✳ ❋✐❣✉r❡✹✿❈✉rr❡♥t ✐♥RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣❛❝t✐✈❡❝❛♣❛❝✐t♦r ❛s ❛r❡s♣♦♥s❡t♦❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡❛ss✉♠❡❞ ❛s ❛❝♦s✐♥❡❢✉♥❝t✐♦♥♦❢❛♠♣❧✐t✉❞❡E0 = 1 ❛♥❞❛♥❣✉❧❛r ❢r❡q✉❡♥❝②ω = 3.2✱♦❜t❛✐♥❡❞❢♦r ♠♦❞❡❧♣❛r❛♠❡t❡rs✿ R = 1✱τ C = 1✱❛♥❞τ µ = 0.1✳ ❋✐❣✉r❡✹✿❈✉rr❡♥t ✐♥RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣❛❝t✐✈❡❝❛♣❛❝✐t♦r ❛s ❛r❡s♣♦♥s❡t♦❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡❛ss✉♠❡❞ ❛s ❛❝♦s✐♥❡❢✉♥❝t✐♦♥♦❢❛♠♣❧✐t✉❞❡E0 = 1 ❛♥❞❛♥❣✉❧❛r ❢r❡q✉❡♥❝②ω = 3.2✱♦❜t❛✐♥❡❞❢♦r ♠♦❞❡❧♣❛r❛♠❡t❡rs✿ R = 1✱τ C = 1✱❛♥❞τ µ = 0.1✳ ✶✽ tr❛♥s❢❡r ❢✉♥❝t✐♦♥s ✭✺✵✮❛r❡ tr❛♥s❢❡r ❢✉♥❝t✐♦♥s ✭✺✵✮❛r❡ ˆg(1,2) R (ω) = u(1,2) R0 (ω) E0 = R i(1,2) 0 (ω) E0 ❛♥❞ arg ˆg(1,2) R (ω) = arg ˆg(1,2) i (ω) = φ(1,2) i (ω) , ✭✺✶✮ ✭✺✶✮ ✇❤❡r❡t❤❡❝✉rr❡♥t ❛♠♣❧✐t✉❞❡i(1) 0 ❛♥❞♣❤❛s❡❛♥❣❧❡φ(1) i ❛r❡♦❜t❛✐♥❡❞❛s ✭✹✹✮❛♥❞✭✹✺✮✐♥t❤❡st❡❛❞②st❛t❡ r❡❣✐♠❡♦❢RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣t❤❡♣❛ss✐✈❡❝❛♣❛❝✐t♦r✱s♦t❤❛t ✭✺✶✮r❡❛❞s ˆg(1) R (ω) dB = 20 log ˆg(1) R (ω) = 10 log  1 − 2τ α ωα cos απ 2 + 1 τ 2 C ω2 + 2τ Cτ α ω1+α sin απ 2 + τ 2α ω2α + 2τ α ωα cos απ 2 + 1  ❛♥❞ ✭✺✷✮ arg ˆg(1) R (ω) = −arctan τ C ω + τ α ωα sin απ 2 τ α ωα cos απ 2 + 1 + arctan τ C τ α ω1−α 1 cos απ 2 + tan απ 2  = arctan τ C ω + τ α ωα sin απ 2 τ 2 C ω2 + 2τ Cτ α ω1+α sin απ 2 + τ 2α ω2α + τ α ωα cos απ 2 , ✭✺✸✮ ✭✺✷✮ ✭✺✸✮ ✇❤✐❧❡t❤❡❝✉rr❡♥t ❛♠♣❧✐t✉❞❡i(2) 0 ❛♥❞♣❤❛s❡❛♥❣❧❡φ(2) i ❛r❡♦❜t❛✐♥❡❞❛s ✭✹✷✮❛♥❞✭✹✸✮✐♥t❤❡st❡❛❞②st❛t❡ r❡❣✐♠❡♦❢RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣t❤❡❛❝t✐✈❡❝❛♣❛❝✐t♦r✱✐♠♣❧②✐♥❣❜②✭✺✶✮ ✇❤✐❧❡t❤❡❝✉rr❡♥t ❛♠♣❧✐t✉❞❡i(2) 0 ❛♥❞♣❤❛s❡❛♥❣❧❡φ(2) i ❛r❡♦❜t❛✐♥❡❞❛s ✭✹✷✮❛♥❞✭✹✸✮✐♥t❤❡st❡❛❞②st❛t❡ r❡❣✐♠❡♦❢RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣t❤❡❛❝t✐✈❡❝❛♣❛❝✐t♦r✱✐♠♣❧②✐♥❣❜②✭✺✶✮ ˆg(2) R (ω) dB = 20 log ˆg(2) R (ω) = −10 log  1 −2 1 τ µ ω1+µ sin µπ 2 + 1 τ 2 C ω2 + 2 1 τ Cτ µ ω2+µ cos µπ 2 + 1 τ 2µ ω2+2µ  ❛♥❞ ✭✺✹✮ arg ˆg(2) R (ω) = arctan 1 τ C ω + 1 τ µ ω1+µ cos µπ 2 1 − 1 τ µ ω1+µ sin µπ 2 = arctan τ µωµ + τ C cos µπ 2 τ Cτ µω1+µ −τ C sin µπ 2 . ✺✳✶ ❆s②♠♣t♦t✐❝❛♥❛❧②s✐s ❆s②♠♣t♦t✐❝❡①♣❛♥s✐♦♥s ♦❢t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s✱♦❜t❛✐♥❡❞❢r♦♠✭✺✷✮✐♥t❤❡❝❛s❡♦❢RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣t❤❡♣❛ss✐✈❡❝❛♣❛❝✐t♦r✱❢♦r ❧♦✇❛♥❞❤✐❣❤❢r❡q✉❡♥❝✐❡s r❡❛❞s ˆg(1) R (ω) dB ∼10 log      τ 2 α ω2α −2τ 3 α ω3α cos απ 2 ✱ ✐❢α ∈ 0, 1 2  ✱ τ 2 α ω2α + 2τ ατ C ω1+α sin απ 2 ✱ ✐❢α ∈ 1 2, 1  ✱ ❛s ω →0, ✭✺✻✮ ˆg(1) R (ω) dB ∼10 log                                  1 − 1 τ 2 C ω2 2τ α ωα cos απ 2 + 1  ✱ ✐❢α ∈ 0, 1 2  ✱ 1 − 1 τ 2 C ω2 2τ α ωα cos απ 2 −2 τ 2 α τ C ω2α−1 sin(απ) + 1  ✱ ✐❢α ∈ 1 2, 2 3  ✱ 1 − 1 τ 2 C ω2 2τ α ωα cos απ 2 −2 τ 2 α τ C ω2α−1 sin(απ) +2 τ 3 α τ 2 C ω3α−2 cos απ 2 4 sin2 απ 2 −1  + 1  ✱ ✐❢α ∈ 2 3, 1  ✱ ❛s ω →∞, ∼10 log      τ 2 α ω2α −2τ 3 α ω3α cos απ 2 ✱ ✐❢α ∈ 0, 1 2  ✱ τ 2 α ω2α + 2τ ατ C ω1+α sin απ 2 ✱ ✐❢α ∈ 1 2, 1  ✱ ❛s ω →0, ✭✺✻✮ ✭✺✻✮ ✐❢α ∈ 0, 1 2  ✱ ✐❢α ∈ 2 3, 1  ✱ ✭✺✼✮ r❡s♣❡❝t✐✈❡❧②✱✇❤✐❧❡t❤❡❛s②♠♣t♦t✐❝❡①♣❛♥s✐♦♥s ♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t✱♦❜t❛✐♥❡❞❢r♦♠✭✺✸✮✱✐s s♣❡❝t✐✈❡❧②✱✇❤✐❧❡t❤❡❛s②♠♣t♦t✐❝❡①♣❛♥s✐♦♥s ♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t✱♦❜t❛✐♥❡❞❢r♦♠✭✺✸✮✱✐s arg ˆg(1) R (ω) ∼arctan                      tan απ 2  1 −τ α ωα 1 cos απ 2 + τ 2 α ω2α 1 cos2 απ 2  ✱ ✐❢α ∈ 0, 1 3  ✱ tan απ 2  1 −τ α ωα 1 cos απ 2 + τ C τ α ω1−α 1 sin απ 2 +τ 2 α ω2α 1 cos2 απ 2  ✱ ✐❢α ∈ 1 3, 1 2  ✱ tan απ 2  1 + τ C τ α ω1−α 1 sin απ 2 −τ α ωα 1 cos απ 2  ✱ ✐❢α ∈ 1 2, 1  ✱ ❛s ω →0, ✭✺✽✮ arg ˆg(1) R (ω) ∼arctan  1 τ C ω  1 −τ α τ C 1 ω1−α sin απ 2  ❛s ω →∞. ✺ ❋r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢❢r❛❝t✐♦♥❛❧RC ❛♥❞RL ❝✐r❝✉✐t ✭✺✺✮ ✭✺✹✮ ✭✺✺✮ ❚❤❡❡①♣r❡ss✐♦♥s ✭✺✷✮✲✭✺✺✮❢♦r tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✐❛♥❞❛r❣✉♠❡♥ts ❛r❡❛❧s♦♦❜t❛✐♥❡❞❜②s✉❜st✐t✉t✐♥❣ s = jω ✐♥t♦tr❛♥s❢❡r ❢✉♥❝t✐♦♥s ˆg(1,2) i (s)✱❣✐✈❡♥❜②✭✷✻✮❛♥❞✭✷✽✮✱❛♥❞s✉❜s❡q✉❡♥t❧②✜♥❞✐♥❣t❤❡✐r ♠♦❞✉❧✉s ❛♥❞❛r❣✉♠❡♥t✳ ❈♦♥s✐❞❡r✐♥❣t❤❡❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢RL ❝✐r❝✉✐t✱❞✉❡t♦t❤❡❛♥❛❧♦❣✐❡s ❜❡t✇❡❡♥♣❤②s✐❝❛❧q✉❛♥✲ t✐t✐❡s ♦❢RL ❛♥❞RC ❝✐r❝✉✐ts ❞✐s❝✉ss❡❞✐♥❙❡❝t✐♦♥✹✳✶✱♦♥❡❤❛s t❤❛t t❤❡❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢t❤❡ ✐♥❞✉❝t♦r ✈♦❧t❛❣❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(1,2) L ❤❛✈❡t❤❡s❛♠❡❢♦r♠❛s t❤❡❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢t❤❡tr❛♥s✲ ❢❡r ❢✉♥❝t✐♦♥ˆg(1,2) R ✱❣✐✈❡♥❜②✭✺✵✮✱❛♥❞❛❧s♦t❤❛t t❤❡❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥ ❝♦rr❡s♣♦♥❞✐♥❣t♦❝✉rr❡♥t ✐♥RL ❝✐r❝✉✐t ❤❛✈❡t❤❡s❛♠❡❢♦r♠❛s t❤❡❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥❝♦rr❡s♣♦♥❞✐♥❣t♦t❤❡❝❛♣❛❝✐t♦r ✈♦❧t❛❣❡✐♥RC ❝✐r❝✉✐t✳ ✶✾ ✶✾ ✺✳✶ ❆s②♠♣t♦t✐❝❛♥❛❧②s✐s ✺✳✶ ❆s②♠♣t♦t✐❝❛♥❛❧②s✐s ✭✺✾✮ arg ˆg(1) R (ω) ∼arctan  1 τ C ω  1 −τ α τ C 1 ω1−α sin απ 2  ❛s ω →∞. ✭✺✾✮ ✭✺✾✮ ❈❧❡❛r❧②✱❜②r❡t❛✐♥✐♥❣♦♥❧②t❤❡❧❡❛❞✐♥❣t❡r♠s ✐♥t❤❡♣r❡✈✐♦✉s ❛s②♠♣t♦t✐❝❡①♣❛♥s✐♦♥s✱♦♥❡❤❛s ˆg(1) R (ω) dB ∼20 α log ω + 20 log τ α ❛♥❞arg ˆg(1) R (ω) ∼απ 2 ❛s ω →0, ✭✻✵✮ ˆg(1) R (ω) dB ∼0 ❛♥❞arg ˆg(1) R (ω) ∼arctan 1 τ C ω ∼ 1 τ C ω ❛s ω →∞, ✭✻✵✮ s✐♥❝❡arctan x ∼x ❢♦r x ≪1✱✐♠♣❧②✐♥❣t❤❛t ❢♦r ❧♦✇❢r❡q✉❡♥❝✐❡s t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ✐s ❛ ❧✐♥❡❛r ❢✉♥❝t✐♦♥♦❢log ω ❤❛✈✐♥❣s❧♦♣❡♣r♦♣♦rt✐♦♥❛❧t♦t❤❡❢r❛❝t✐♦♥❛❧❞✐✛❡r❡♥t✐❛t✐♦♥♦r❞❡r α ❛♥❞✐♥t❡r❝❡♣t ♣r♦♣♦rt✐♦♥❛❧t♦t❤❡❢r❛❝t✐♦♥❛❧t✐♠❡❝♦♥st❛♥t τ α✱✇❤✐❧❡t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t ✐s ♣r♦♣♦rt✐♦♥❛❧ t♦♣❛r❛♠❡t❡r α✱❛♥❞✐♥t❤❡❝❛s❡♦❢❤✐❣❤❢r❡q✉❡♥❝✐❡s✱t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t t❡♥❞s t♦③❡r♦❛s ❛ ❤②♣❡r❜♦❧✐❝❢✉♥❝t✐♦♥✇✐t❤t❤❡❝♦❡✣❝✐❡♥t ✐♥✈❡rs❡❧②♣r♦♣♦rt✐♦♥❛❧t♦t❤❡t✐♠❡❝♦♥st❛♥t τ C✳ s✐♥❝❡arctan x ∼x ❢♦r x ≪1✱✐♠♣❧②✐♥❣t❤❛t ❢♦r ❧♦✇❢r❡q✉❡♥❝✐❡s t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ✐s ❛ ❧✐♥❡❛r ❢✉♥❝t✐♦♥♦❢log ω ❤❛✈✐♥❣s❧♦♣❡♣r♦♣♦rt✐♦♥❛❧t♦t❤❡❢r❛❝t✐♦♥❛❧❞✐✛❡r❡♥t✐❛t✐♦♥♦r❞❡r α ❛♥❞✐♥t❡r❝❡♣t ♣r♦♣♦rt✐♦♥❛❧t♦t❤❡❢r❛❝t✐♦♥❛❧t✐♠❡❝♦♥st❛♥t τ α✱✇❤✐❧❡t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t ✐s ♣r♦♣♦rt✐♦♥❛❧ t♦♣❛r❛♠❡t❡r α✱❛♥❞✐♥t❤❡❝❛s❡♦❢❤✐❣❤❢r❡q✉❡♥❝✐❡s✱t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t t❡♥❞s t♦③❡r♦❛s ❛ ❤②♣❡r❜♦❧✐❝❢✉♥❝t✐♦♥✇✐t❤t❤❡❝♦❡✣❝✐❡♥t ✐♥✈❡rs❡❧②♣r♦♣♦rt✐♦♥❛❧t♦t❤❡t✐♠❡❝♦♥st❛♥t τ C✳ ❆s②♠♣t♦t✐❝❜❡❤❛✈✐♦r ♦❢t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ❛♥❞❛r❣✉♠❡♥t✱♦❜t❛✐♥❡❞❢r♦♠✭✺✹✮❛♥❞✭✺✺✮✐♥ ✷✵ t❤❡❝❛s❡♦❢RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣t❤❡❛❝t✐✈❡❝❛♣❛❝✐t♦r✱✐s ❞❡s❝r✐❜❡❞❜② t❤❡❝❛s❡♦❢RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣t❤❡❛❝t✐✈❡❝❛♣❛❝✐t♦r✱✐s ❞❡s❝r✐❜❡❞❜② t❤❡❝❛s❡♦❢RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣t❤❡❛❝t✐✈❡❝❛♣❛❝✐t♦r✱✐s ❞❡s❝r✐❜❡❞❜② ˆg(2) R (ω) dB ∼−10 log      1 τ 2µ ω2+2µ + 2 1 τ Cτ µ ω2+µ cos µπ 2 ✱ ❛s ω →0✱ 1 −2 1 τ µ ω1+µ sin µπ 2 ✱ ❛s ω →∞✱ ✭✻✶✮ arg ˆg(2) R (ω) ∼arctan      tan (1+µ)π 2  1 + τ µ τ C ωµ 1 cos µπ 2  ✱ ❛s ω →0✱ 1 τ C ω  1 + τ C τ µ 1 ωµ cos µπ 2  ✱ ❛s ω →∞✱ ✭✻✷✮ ✭✻✶✮ ✭✻✷✮ ❛♥❞❛❣❛✐♥❜②r❡t❛✐♥✐♥❣♦♥❧②t❤❡❧❡❛❞✐♥❣t❡r♠s ✐♥t❤❡♣r❡✈✐♦✉s ❛s②♠♣t♦t✐❝❡①♣❛♥s✐♦♥s✱♦♥❡❤❛s ˆg(2) R (ω) dB ∼20 (1 + µ) log ω + 20 log τ µ ❛♥❞arg ˆg(2) R (ω) ∼(1 + µ)π 2 ❛s ω →0, ✭✻✸✮ ˆg(2) R (ω) dB ∼0 ❛♥❞arg ˆg(2) R (ω) ∼arctan 1 τ C ω ∼ 1 τ C ω ❛s ω →∞, ✭✻✹✮ ✭✻✸✮ ✭✻✹✮ s✐♥❝❡arctan x ∼x ❢♦r x ≪1✱❛❣❛✐♥✐♠♣❧②✐♥❣t❤❛t ❢♦r ❧♦✇❢r❡q✉❡♥❝✐❡s t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ✐s ❛ ❧✐♥❡❛r ❢✉♥❝t✐♦♥♦❢log ω ❤❛✈✐♥❣s❧♦♣❡♣r♦♣♦rt✐♦♥❛❧t♦t❤❡❢r❛❝t✐♦♥❛❧✐♥t❡❣r❛t✐♦♥♦r❞❡r 1 + µ ❛♥❞✐♥t❡r❝❡♣t ♣r♦♣♦rt✐♦♥❛❧t♦t❤❡❢r❛❝t✐♦♥❛❧t✐♠❡❝♦♥st❛♥t τ µ✱✇❤✐❧❡t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t ✐s ♣r♦♣♦rt✐♦♥❛❧t♦ ♣❛r❛♠❡t❡r µ✱❛♥❞✐♥t❤❡❝❛s❡♦❢❤✐❣❤❢r❡q✉❡♥❝✐❡s✱❛s ❜❡❢♦r❡✱t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t t❡♥❞s t♦③❡r♦ ❛s ❛❤②♣❡r❜♦❧✐❝❢✉♥❝t✐♦♥✇✐t❤t❤❡❝♦❡✣❝✐❡♥t ✐♥✈❡rs❡❧②♣r♦♣♦rt✐♦♥❛❧t♦t❤❡t✐♠❡❝♦♥st❛♥t τ C✳ ❚❤❡r❡❢♦r❡✱r❡❣❛r❞❧❡ss ♦❢t❤❡❢❛❝t ✇❡❛t❤❡r RC ❝✐r❝✉✐t ❝♦♥t❛✐♥s ♣❛ss✐✈❡♦r ❛❝t✐✈❡❝❛♣❛❝✐t♦r✱♠♦❞❡❧♣❛r❛♠✲ ❡t❡rs ❝❛♥❡❛s✐❧②❜❡❡st✐♠❛t❡❞❢r♦♠t❤❡❛s②♠♣t♦t✐❝❡①♣r❡ss✐♦♥s ♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✐❛♥❞❛r❣✉♠❡♥ts✳ ❉❡r✐✈❛t✐♦♥♦❢❛s②♠♣t♦t✐❝❢♦r♠✉❧❛❡✭✺✻✮✲✭✺✾✮✱❝♦rr❡s♣♦♥❞✐♥❣t♦tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(1) R ✱❛♥❞✭✻✶✮❛♥❞✭✻✷✮✱ ❝♦rr❡s♣♦♥❞✐♥❣t♦tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(2) R ✱✐s ♣❡r❢♦r♠❡❞✐♥❆♣♣❡♥❞✐①❇✳ ✺✳✷ ◆✉♠❡r✐❝❛❧❡①❛♠♣❧❡s ❋r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ❝♦rr❡s♣♦♥❞✐♥❣t♦RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣t❤❡♣❛ss✐✈❡❝❛♣❛❝✐t♦r✱✐✳❡✳✱❇♦❞❡♣❧♦ts✱ ❛r❡♣r❡s❡♥t❡❞✐♥❋✐❣✉r❡✺✱t♦❣❡t❤❡r ✇✐t❤t❤❡✐r ❛s②♠♣t♦t✐❝s✳❆s ❡①♣❡❝t❡❞❢r♦♠t❤❡❢♦r♠♦❢t❤❡tr❛♥s❢❡r ❢✉♥❝✲ t✐♦♥ˆg(1) R ✱✐ts ♠♦❞✉❧✉s ❤❛s ❛③❡r♦♦❢♥♦♥✲✐♥t❡❣❡r ♦r❞❡r ❛t t❤❡♦r✐❣✐♥✱s✐♥❝❡✐t ❞❡❝r❡❛s❡s ❧✐♥❡❛r❧②t♦♥❡❣❛t✐✈❡ ✐♥✜♥✐t②❛s t❤❡❢r❡q✉❡♥❝②t❡♥❞s t♦③❡r♦✱❛s ♣r❡❞✐❝t❡❞❜②t❤❡❛s②♠♣t♦t✐❝❡①♣❛♥s✐♦♥✭✻✵✮1 ❛♥❞❛s ♦❜s❡r✈❡❞ ❢r♦♠❋✐❣✉r❡✺❛✳❋✉rt❤❡r✱t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ♠♦♥♦t♦♥✐❝❛❧❧②✐♥❝r❡❛s❡s ❛♥❞✱✐♥❛❝❝♦r❞❛♥❝❡✇✐t❤ ✐ts ❛s②♠♣t♦t✐❝s ✭✺✼✮✱t❡♥❞s t♦③❡r♦❢♦r ❤✐❣❤❢r❡q✉❡♥❝✐❡s r❡❣❛r❞❧❡ss ♦❢t❤❡✈❛❧✉❡♦❢❢r❛❝t✐♦♥❛❧❞✐✛❡r❡♥t✐❛t✐♦♥ ♦r❞❡r α✱❛s ♦❜✈✐♦✉s ❢r♦♠❋✐❣✉r❡✺❜✳❆❧t❤♦✉❣❤✐t ✐s ♥♦t t❤❡❝❛s❡✱s✉❝❤❜❡❤❛✈✐♦r ♦❢❝❤❛r❛❝t❡r✐st✐❝s ❢♦r ❤✐❣❤ ❢r❡q✉❡♥❝✐❡s ♠✐❣❤t ❜❡✐♥t❡r♣r❡t❡❞❛s ✐❢t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥❤❛s ❛r❡❛❧♣♦❧❡♦❢t❤❡s❛♠❡♦r❞❡r ❛s ✐ts ③❡r♦ ❛t t❤❡♦r✐❣✐♥✳❖♥t❤❡♦t❤❡r ❤❛♥❞✱t❤❡❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t ❝❤❛♥❣❡ ❢r♦♠♥♦♥✲♠♦♥♦t♦♥✐❝❢✉♥❝t✐♦♥✱✇❤✐❝❤❛tt❛✐♥s ❛♠❛①✐♠✉♠✱t♦❛♠♦♥♦t♦♥✐❝❛❧❧②❞❡❝r❡❛s✐♥❣❢✉♥❝t✐♦♥❛s t❤❡ ♣❛r❛♠❡t❡r α ✐♥❝r❡❛s❡s✱s❡❡❋✐❣✉r❡✺❝✳❚❤❡❧♦✇❢r❡q✉❡♥❝②❛s②♠♣t♦t✐❝s ♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t ✭✻✵✮2 s❤♦✇s t❤❛t t❤❡❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ❤❛✈❡❛❝♦♥st❛♥t ✈❛❧✉❡❞❡♣❡♥❞✐♥❣♦♥t❤❡♣❛r❛♠❡t❡r α ❝♦♥✜r♠✐♥❣ ✷✶ t❤❡❝♦♥❝❧✉s✐♦♥❞❡r✐✈❡❞❢r♦♠t❤❡❛s②♠♣t♦t✐❝s ♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s✳❋✐❣✉r❡✺❞s❤♦✇s t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t✱❛❧♦♥❣✇✐t❤✐ts ❛s②♠♣t♦t✐❝s✱t❡♥❞✐♥❣t♦③❡r♦❢♦r ❤✐❣❤❢r❡q✉❡♥❝✐❡s r❡❣❛r❞❧❡ss ♦❢t❤❡ ♣❛r❛♠❡t❡r α✱❛❣❛✐♥♠✐s❧❡❛❞✐♥❣t♦t❤❡❝♦♥❝❧✉s✐♦♥❛❜♦✉t t❤❡♣♦❧❡s ♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥✳ α = 0.2 α = 0.4 α = 0.6 α = 0.8 0.01 0.10 1 10 100 -40 -30 -20 -10 0 ω |g R (1)(ω) dB α = 0.2 α = 0.4 α = 0.6 α = 0.8 0.01 0.10 1 10 100 -40 -30 -20 -10 0 ω |g R (1)(ω) dB ✭❛✮❚r❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ✭s♦❧✐❞❧✐♥❡✮❛♥❞✐ts ❧♦✇❢r❡✲ q✉❡♥❝②❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮✳ α = 0.2 α = 0.4 α = 0.6 α = 0.8 5000 1 × 104 5 × 104 1 × 105 -0.0004 -0.0003 -0.0002 -0.0001 0.0000 ω |g R (1)(ω) dB ✭❜✮❊♥❧❛r❣❡❞❞❡t❛✐❧♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ✭s♦❧✐❞❧✐♥❡✮ ❛♥❞✐ts ❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮❢♦r ❤✐❣❤❢r❡q✉❡♥❝✐❡s✳ α = 0.1 α = 0.2 α = 0.3 α = 0.5 α = 0.7 0.01 0.10 1 10 100 1000 0.0 0.2 0.4 0.6 0.8 1.0 ω arg g R (1)(ω) ✭❝✮❚r❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t ✭s♦❧✐❞❧✐♥❡✮❛♥❞✐ts ❧♦✇❢r❡✲ q✉❡♥❝②❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮✳ α = 0.1 α = 0.3 α = 0.5 α = 0.7 20 50 100 200 0.00 0.05 0.10 0.15 0.20 ω arg g R (1)(ω) ✭❞✮❊♥❧❛r❣❡❞❞❡t❛✐❧♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t ✭s♦❧✐❞ ❧✐♥❡✮❛♥❞✐ts ❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮❢♦r ❤✐❣❤❢r❡q✉❡♥❝✐❡s✳ ❋✐❣✉r❡✺✿❈❛s❡♦❢♣❛ss✐✈❡❝❛♣❛❝✐t♦r ✕❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(1) R ♦❜t❛✐♥❡❞❢♦r ♠♦❞❡❧ ♣❛r❛♠❡t❡rs✿τ C = 0.2 ❛♥❞τ α = 0.5✳ α = 0.2 α = 0.4 α = 0.6 α = 0.8 5000 1 × 104 5 × 104 1 × 105 -0.0004 -0.0003 -0.0002 -0.0001 0.0000 ω |g R (1)(ω) dB ✭❜✮❊♥❧❛r❣❡❞❞❡t❛✐❧♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ✭s♦❧✐❞❧✐♥❡✮ ❛♥❞✐ts ❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮❢♦r ❤✐❣❤❢r❡q✉❡♥❝✐❡s✳ ✭❛✮❚r❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ✭s♦❧✐❞❧✐♥❡✮❛♥❞✐ts ❧♦✇❢r❡✲ q✉❡♥❝②❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮✳ α = 0.1 α = 0.3 α = 0.5 α = 0.7 20 50 100 200 0.00 0.05 0.10 0.15 0.20 ω arg g R (1)(ω) α = 0.1 α = 0.2 α = 0.3 α = 0.5 α = 0.7 0.01 0.10 1 10 100 1000 0.0 0.2 0.4 0.6 0.8 1.0 ω arg g R (1)(ω) 100 ✭❞✮❊♥❧❛r❣❡❞❞❡t❛✐❧♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t ✭s♦❧✐❞ ❧✐♥❡✮❛♥❞✐ts ❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮❢♦r ❤✐❣❤❢r❡q✉❡♥❝✐❡s✳ ✭❝✮❚r❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t ✭s♦❧✐❞❧✐♥❡✮❛♥❞✐ts ❧♦✇❢r❡✲ q✉❡♥❝②❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮✳ ❋✐❣✉r❡✺✿❈❛s❡♦❢♣❛ss✐✈❡❝❛♣❛❝✐t♦r ✕❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(1) R ♦❜t❛✐♥❡❞❢♦r ♠♦❞❡❧ ♣❛r❛♠❡t❡rs✿τ C = 0.2 ❛♥❞τ α = 0.5✳ ❋✐❣✉r❡✺✿❈❛s❡♦❢♣❛ss✐✈❡❝❛♣❛❝✐t♦r ✕❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(1) R ♦❜t❛✐♥❡❞❢♦r ♠♦❞❡❧ ♣❛r❛♠❡t❡rs✿τ C = 0.2 ❛♥❞τ α = 0.5✳ ■♥t❤❡❝❛s❡♦❢RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣t❤❡❛❝t✐✈❡❝❛♣❛❝✐t♦r✱t❤❡❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s✱✐♥❝❧✉❞✐♥❣ t❤❡✐r ❛s②♠♣t♦t✐❝s✱❛r❡s❤♦✇♥✐♥❋✐❣✉r❡✻✳❈♦♥tr❛r②t♦t❤❡♠♦❞✉❧✉s ♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(1) R ✱t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ˆg(2) R dB✱❛s ♦❜✈✐♦✉s ❢♦r♠❋✐❣✉r❡✻❛✱❜❡❤❛✈❡s ♥♦♥✲♠♦♥♦t♦♥✐❝❛❧❧②❛♥❞❛tt❛✐♥s ❛♠❛①✐✲ ♠✉♠❛s ❛❝♦♥s❡q✉❡♥❝❡♦❢t❤❡❢❛❝t t❤❛t tr❛♥s❢❡r ❢✉♥❝t✐♦♥❤❛s ❛♣❛✐r ♦❢❝♦♠♣❧❡①❝♦♥❥✉❣❛t❡❞♣♦❧❡s✳❋♦r ❧♦✇❢r❡q✉❡♥❝✐❡s✱t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ✐♥❝r❡❛s❡s ❧✐♥❡❛r❧②❢r♦♠t❤❡♥❡❣❛t✐✈❡✐♥✜♥✐t②✱s❡❡❛❧s♦t❤❡ ❛s②♠♣t♦t✐❝❡①♣❛♥s✐♦♥✭✻✸✮1✱✐♠♣❧②✐♥❣t❤❛t t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(2) R ❤❛s ❛③❡r♦♦❢♥♦♥✲✐♥t❡❣❡r ♦r❞❡r ❛t t❤❡♦r✐❣✐♥✱❛s ❡①♣❡❝t❡❞❢r♦♠✐ts ❢♦r♠✳❆♥❛❧♦❣♦✉s❧②t♦t❤❡❝❛s❡♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ˆg(1) R dB✱t❤❡ ❝❤❛r❛❝t❡r✐st✐❝s ♦❢ ˆg(2) R dB ❛❧s♦t❡♥❞t♦③❡r♦❢♦r ❤✐❣❤❢r❡q✉❡♥❝✐❡s ✐♥❛❝❝♦r❞❛♥❝❡✇✐t❤t❤❡✐r ❛s②♠♣t♦t✐❝s ✭✻✶✮ r❡❣❛r❞❧❡ss ♦❢t❤❡✈❛❧✉❡♦❢❢r❛❝t✐♦♥❛❧✐♥t❡❣r❛t✐♦♥♦r❞❡r µ✱❛s ✐t ❝❛♥❜❡s❡❡♥❢r♦♠❋✐❣✉r❡✻❜✳❚❤❡❢r❡q✉❡♥❝② ❝❤❛r❛❝t❡r✐st✐❝s ♦❢t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t ♠♦♥♦t♦♥✐❝❛❧❧②❞❡❝r❡❛s❡❢r♦♠❛❝♦♥st❛♥t ✈❛❧✉❡❞❡♣❡♥❞✐♥❣ ♦♥t❤❡♣❛r❛♠❡t❡r µ✱❛s ♣r❡❞✐❝t❡❞❜②❛s②♠♣t♦t✐❝❡①♣❛♥s✐♦♥✭✻✸✮2✱t♦❛③❡r♦✈❛❧✉❡❛❝❝♦r❞✐♥❣t♦❛s②♠♣t♦t✐❝ ❢♦r♠✉❧❛✭✻✹✮2✱s❡❡❋✐❣✉r❡s ✻❝❛♥❞✻❞✳ ■t ✐s ❝❧❡❛r ❢r♦♠t❤❡❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✐t❤❛t RC ❝✐r❝✉✐t r❡❣❛r❞❧❡ss ✷✷ μ = 0.8 μ = 0.6 μ = 0.4 μ = 0.2 0.5 1 5 10 -25 -20 -15 -10 -5 0 5 ω |g R (2)(ω) dB ✭❛✮❚r❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ✭s♦❧✐❞❧✐♥❡✮❛♥❞✐ts ❧♦✇❢r❡✲ q✉❡♥❝②❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮✳ μ = 0.8 μ = 0.6 μ = 0.4 μ = 0.2 40 60 80 100 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 ω |g R (2)(ω) dB ✭❜✮❊♥❧❛r❣❡❞❞❡t❛✐❧♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ✭s♦❧✐❞❧✐♥❡✮ ❛♥❞✐ts ❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮❢♦r ❤✐❣❤❢r❡q✉❡♥❝✐❡s✳ μ = 0.8 μ = 0.6 μ = 0.4 μ = 0.2 0.1 0.5 1 5 10 50 0.0 0.5 1.0 1.5 2.0 2.5 ω arg g R (2)(ω) ✭❝✮❚r❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t ✭s♦❧✐❞❧✐♥❡✮❛♥❞✐ts ❧♦✇❢r❡✲ q✉❡♥❝②❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮✳ μ = 0.2 μ = 0.4 μ = 0.6 μ = 0.8 2 5 10 20 50 0.0 0.1 0.2 0.3 0.4 0.5 0.6 ω arg g R (2)(ω) ✭❞✮❊♥❧❛r❣❡❞❞❡t❛✐❧♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t ✭s♦❧✐❞ ❧✐♥❡✮❛♥❞✐ts ❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮❢♦r ❤✐❣❤❢r❡q✉❡♥❝✐❡s✳ μ = 0.8 μ = 0.6 μ = 0.4 μ = 0.2 40 60 80 100 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 ω |g R (2)(ω) dB μ = 0.8 μ = 0.6 μ = 0.4 μ = 0.2 0.5 1 5 10 -25 -20 -15 -10 -5 0 5 ω |g R (2)(ω) dB ✭❛✮❚r❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ✭s♦❧✐❞❧✐♥❡✮❛♥❞✐ts ❧♦✇❢r❡✲ q✉❡♥❝②❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮✳ ✭❜✮❊♥❧❛r❣❡❞❞❡t❛✐❧♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ✭s♦❧✐❞❧✐♥❡✮ ❛♥❞✐ts ❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮❢♦r ❤✐❣❤❢r❡q✉❡♥❝✐❡s✳ ✭❜✮❊♥❧❛r❣❡❞❞❡t❛✐❧♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ✭s♦❧✐❞❧✐♥❡✮ ❛♥❞✐ts ❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮❢♦r ❤✐❣❤❢r❡q✉❡♥❝✐❡s✳ μ = 0.2 μ = 0.4 μ = 0.6 μ = 0.8 2 5 10 20 50 0.0 0.1 0.2 0.3 0.4 0.5 0.6 ω arg g R (2)(ω) μ = 0.8 μ = 0.6 μ = 0.4 μ = 0.2 0.1 0.5 1 5 10 50 0.0 0.5 1.0 1.5 2.0 2.5 ω arg g R (2)(ω) ✭❞✮❊♥❧❛r❣❡❞❞❡t❛✐❧♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t ✭s♦❧✐❞ ❧✐♥❡✮❛♥❞✐ts ❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮❢♦r ❤✐❣❤❢r❡q✉❡♥❝✐❡s✳ ✭❝✮❚r❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t ✭s♦❧✐❞❧✐♥❡✮❛♥❞✐ts ❧♦✇❢r❡✲ q✉❡♥❝②❛s②♠♣t♦t✐❝s ✭❞❛s❤❡❞❧✐♥❡✮✳ ❋✐❣✉r❡✻✿❈❛s❡♦❢❛❝t✐✈❡❝❛♣❛❝✐t♦r ✕❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(2) R ♦❜t❛✐♥❡❞❢♦r ♠♦❞❡❧ ♣❛r❛♠❡t❡rs✿τ C = 5 ❛♥❞τ µ = 0.5✳ ❋✐❣✉r❡✻✿❈❛s❡♦❢❛❝t✐✈❡❝❛♣❛❝✐t♦r ✕❢r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(2) R ♦❜t❛✐♥❡❞❢♦r ♠♦❞❡❧ ♣❛r❛♠❡t❡rs✿τ C = 5 ❛♥❞τ µ = 0.5✳ ✇❤❡t❤❡r ✐t ❝♦♥t❛✐♥s ♣❛ss✐✈❡♦r ❛❝t✐✈❡❝❛♣❛❝✐t♦r ❜❡❤❛✈❡s ❛s t❤❡❤✐❣❤♣❛ss ✜❧t❡r✱s❡❡❋✐❣✉r❡s ✺❛❛♥❞✻❛✳ ❤❡t❤❡r ✐t ❝♦♥t❛✐♥s ♣❛ss✐✈❡♦r ❛❝t✐✈❡❝❛♣❛❝✐t♦r ❜❡❤❛✈❡s ❛s t❤❡❤✐❣❤♣❛ss ✜❧t❡r✱s❡❡❋✐❣✉r❡s ✺❛❛♥❞✻ ✻ ❈♦♥❝❧✉s✐♦♥ ❈❧❛ss✐❝❛❧❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s✱❞❡s❝r✐❜✐♥❣❜❡❤❛✈✐♦r ♦❢❝❛♣❛❝✐t♦r ❛♥❞✐♥❞✉❝t♦r ❛s ❜❛s✐❝❡❧❡♠❡♥ts ♦❢❡❧❡❝tr✐❝ ❝✐r❝✉✐ts✱❛r❡❣❡♥❡r❛❧✐③❡❞❛♥❞♣r♦♣♦s❡❞✐♥t❤❡❢♦r♠t❤❛t ✐♥❝❧✉❞❡❡❧❡♠❡♥t✬s ✐♥st❛♥t❛♥❡♦✉s ❛♥❞❤❡r❡❞✐t❛r② r❡s♣♦♥s❡✱✇✐t❤t❤❡❤❡r❡❞✐t❛r✐♥❡ss ♠♦❞❡❧❡❞❜②t❤❡❧♦♥❣♠❡♠♦r②❦❡r♥❡❧♦❢♣♦✇❡r t②♣❡✱✐✳❡✳✱❜②t❤❡❢r❛❝t✐♦♥❛❧ ✐♥t❡❣r❛❧✱②✐❡❧❞✐♥❣t✇♦t②♣❡s ♦❢❝♦♥st✐t✉t✐✈❡❡q✉❛t✐♦♥s ❞❡♣❡♥❞✐♥❣♦♥t❤❡♣❤②s✐❝❛❧q✉❛♥t✐t✐❡s ✇❤♦s❡♠❡♠♦r② ✐s ❝♦♥s✐❞❡r❡❞✳❚❤❡r♠♦❞②♥❛♠✐❝❛❧❝♦♥s✐❞❡r❛t✐♦♥s ✐♠♣❧②t❤❛t ❝❤❛r❣❡✲✈♦❧t❛❣❡✭✶✮❛♥❞✢✉①✲❝✉rr❡♥t ✭✷✮❝♦♥✲ st✐t✉t✐✈❡r❡❧❛t✐♦♥s ❞❡s❝r✐❜❡♣❛ss✐✈❡❝❛♣❛❝✐t♦r ❛♥❞✐♥❞✉❝t♦r✱✇❤✐❧❡✈♦❧t❛❣❡✲❝❤❛r❣❡✭✸✮❛♥❞❝✉rr❡♥t✲✢✉①✭✹✮ ♠♦❞❡❧s ❞❡s❝r✐❜❡❛❝t✐✈❡❝❛♣❛❝✐t♦r ❛♥❞✐♥❞✉❝t♦r✳❆❧s♦✱❡q✉✐✈❛❧❡♥t ♠♦❞❡❧s ♦❢❣❡♥❡r❛❧✐③❡❞❡❧❡❝tr✐❝❡❧❡♠❡♥t ❝❛♥❜❡♦❜t❛✐♥❡❞❜②t❤❡s✐♠✉❧t❛♥❡♦✉s ❝❤❛♥❣❡♦❢✐ts ♠❡♠♦r②❦❡r♥❡❧❛♥❞t♦♣♦❧♦❣②✳ ❈❤❛r❣❡✲✈♦❧t❛❣❡❛♥❞✈♦❧t❛❣❡✲❝❤❛r❣❡❝♦♥st✐t✉t✐✈❡♠♦❞❡❧s ✭✶✮❛♥❞✭✸✮❛r❡❢✉rt❤❡r ✉s❡❞✐♥❞❡r✐✈✐♥❣t❤❡ ❡q✉❛t✐♦♥s ✭✷✸✮❛♥❞✭✷✹✮✱❣♦✈❡r♥✐♥❣tr❛♥s✐❡♥t r❡❣✐♠❡✐♥t❤❡s❡r✐❡s RC ❝✐r❝✉✐t s✉❜❥❡❝t t♦❡❧❡❝tr♦♠♦t✐✈❡❢♦r❝❡✱ t❤❛t ②✐❡❧❞❡❞❞✐✛❡r❡♥t q✉❛❧✐t❛t✐✈❡❜❡❤❛✈✐♦r ♦❢t❤❡❣❡♥❡r❛❧✐③❡❞❝❛♣❛❝✐t♦rs✬✐♠♣✉❧s❡r❡s♣♦♥s❡s✱♦❜t❛✐♥❡❞❛s ✭✷✾✮❛♥❞✭✸✵✮❜②t❤❡▲❛♣❧❛❝❡tr❛♥s❢♦r♠♠❡t❤♦❞✳◆❛♠❡❧②✱t❤❡✐♠♣✉❧s❡r❡s♣♦♥s❡❝♦rr❡s♣♦♥❞✐♥❣t♦t❤❡ ✷✸ ♣❛ss✐✈❡❝❛♣❛❝✐t♦r✱♠♦❞❡❧❡❞❜②t❤❡❝❤❛r❣❡✲✈♦❧t❛❣❡r❡❧❛t✐♦♥✭✶✮✱✐s ❛♣♦s✐t✐✈❡♠♦♥♦t♦♥✐❝❛❧❧②❞❡❝r❡❛s✐♥❣ ❝♦♥✈❡①❢✉♥❝t✐♦♥✱✇❤✐❧❡t❤❡✐♠♣✉❧s❡r❡s♣♦♥s❡❝♦rr❡s♣♦♥❞✐♥❣t♦t❤❡❛❝t✐✈❡❝❛♣❛❝✐t♦r✱♠♦❞❡❧❡❞❜②✈♦❧t❛❣❡✲ ❝❤❛r❣❡r❡❧❛t✐♦♥✭✸✮✱✐s ❛❞❛♠♣❡❞♦s❝✐❧❧❛t♦r②❢✉♥❝t✐♦♥✱✇✐t❤t❤❡♣♦ss✐❜✐❧✐t②♦❢s✉❝❤❛♥❡①t❡♥s✐✈❡❞❛♠♣✐♥❣ t❤❛t t❤❡r❡❛r❡♥♦✈✐s✐❜❧❡♦s❝✐❧❧❛t✐♦♥s✳◆✉♠❡r✐❝❛❧❡①❛♠♣❧❡s ❢♦r t❤❡❍❡❛✈✐s✐❞❡❢✉♥❝t✐♦♥t②♣❡❡❧❡❝tr♦♠♦t✐✈❡ ❢♦r❝❡✐❧❧✉str❛t❡❞♠❡♥t✐♦♥❡❞❞✐✛❡r❡♥❝❡s ✐♥t❤❡q✉❛❧✐t❛t✐✈❡❜❡❤❛✈✐♦r ♦❢r❡s♣♦♥s❡s✳ ❈✉rr❡♥t ✐♥t❤❡❢r❛❝t✐♦♥❛❧RC ❝✐r❝✉✐t✱♦❜t❛✐♥❡❞❛s ❛tr❛♥s✐❡♥t r❡❣✐♠❡r❡s♣♦♥s❡t♦t❤❡❤❛r♠♦♥✐❝❡❧❡❝✲ tr♦♠♦t✐✈❡❢♦r❝❡❛❝❝♦r❞✐♥❣t♦✭✹✽✮✱✐s ❝❤❡❝❦❡❞❛❣❛✐♥st t❤❡s♦❧✉t✐♦♥❛❜✐♥✐t✐♦♦❜t❛✐♥❡❞❢♦r t❤❡st❡❛❞②st❛t❡ r❡❣✐♠❡❛❝❝♦r❞✐♥❣t♦✭✹✾✮✱✇✐t❤t❤❡♥✉♠❡r✐❝❛❧❡①❛♠♣❧❡s ✐❧❧✉str❛t✐♥❣♣❡r❢❡❝t ❛❣r❡❡♠❡♥t ❜❡t✇❡❡♥t❤❡s❡t✇♦ t②♣❡s ♦❢s♦❧✉t✐♦♥s✳▼♦r❡♦✈❡r✱t❤❡❢r❛❝t✐♦♥❛❧RC ❝✐r❝✉✐t ❝♦♥t❛✐♥✐♥❣❛❝t✐✈❡❝❛♣❛❝✐t♦r ♣r♦✈❡❞t♦❜❡❤❛✈❡✐♥ t❤❡st❡❛❞②st❛t❡r❡❣✐♠❡❛s ❛♥❡❧❡♠❡♥t t❤❛t ❡✐t❤❡r ❝♦♥s✉♠❡s ♦r ♣r♦❞✉❝❡s ❡❧❡❝tr✐❝❡♥❡r❣②❞❡♣❡♥❞✐♥❣♦♥t❤❡ ❢r❛❝t✐♦♥❛❧✐♥t❡❣r❛t✐♦♥♦r❞❡r ✐♥t❤❡❝♦♥st✐t✉t✐✈❡r❡❧❛t✐♦♥♦❢t❤❡❣❡♥❡r❛❧✐③❡❞❝❛♣❛❝✐t♦r✳ ❋r❡q✉❡♥❝②❝❤❛r❛❝t❡r✐st✐❝s ❛♥❛❧②s✐s ♦❢RC ❝✐r❝✉✐ts✬tr❛♥s❢❡r ❢✉♥❝t✐♦♥s s✉♣♣♦rt❡❞t❤❡❝♦♥❝❧✉s✐♦♥s ❛❜♦✉t t❤❡♦r❞❡r ❛♥❞♥❛t✉r❡♦❢t❤❡✐r ♣♦❧❡s ❛♥❞③❡r♦s ❞r❛✇♥❢r♦♠t❤❡❢♦r♠♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥s ❛♥❞❢r♦♠t❤❡ r❡s♣♦♥s❡s ✐♥tr❛♥s✐❡♥t r❡❣✐♠❡❛♥❞❛❧s♦♣r♦✈❡❞t❤❛t RC ❝✐r❝✉✐t ❜❡❤❛✈❡s ❛s t❤❡❤✐❣❤♣❛ss ✜❧t❡r r❡❣❛r❞❧❡ss ♦❢t❤❡t②♣❡♦❢❣❡♥❡r❛❧✐③❡❞❝❛♣❛❝✐t♦r✳❚❤❡❛s②♠♣t♦t✐❝s ♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ❛♥❞❛r❣✉♠❡♥t ✐♥ ♥✉♠❡r✐❝❛❧❡①❛♠♣❧❡s s❤♦✇❡❞❣♦♦❞❛❣r❡❡♠❡♥t ✇✐t❤t❤❡❝❤❛r❛❝t❡r✐st✐❝s ♣r♦✈✐❞✐♥❣t❤❡♣♦ss✐❜✐❧✐t②t♦❡st✐♠❛t❡ ♠♦❞❡❧♣❛r❛♠❡t❡rs ♦❢RC ❝✐r❝✉✐t✳ ❆ ❈❛❧❝✉❧❛t✐♦♥♦❢✐♠♣✉❧s❡r❡s♣♦♥s❡g(2) C ❙t❛rt✐♥❣❢♦r♠t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(2) C ✱❣✐✈❡♥❜②✭✷✼✮✱t❤❡✐♠♣✉❧s❡r❡s♣♦♥s❡g(2) C ✐s ♦❜t❛✐♥❡❞✐♥t❤❡❢♦r♠ ✭✸✵✮❜②❛♣♣❧②✐♥❣t❤❡▲❛♣❧❛❝❡✐♥✈❡rs✐♦♥❢♦r♠✉❧❛ g(2) C (t) = L−1[ˆg(2) C (s)](t) = 1 2πj Z ΓBr ˆg(2) C (s) est ds ✭✻✺✮ ✭✻✺✮ ❛♥❞✉s✐♥❣t❤❡❈❛✉❝❤②r❡s✐❞✉❡s t❤❡♦r❡♠✱❝❧❛✐♠✐♥❣t❤❛t ❛♥❞✉s✐♥❣t❤❡❈❛✉❝❤②r❡s✐❞✉❡s t❤❡♦r❡♠✱❝❧❛✐♠✐♥❣t❤❛t I Γ f(z)dz = 2πj X k Res(f(z), zk), ✭✻✻✮ ✭✻✻✮ ✐❢❢✉♥❝t✐♦♥f ❤❛s ♣♦❧❡s zk ✐♥t❤❡❞♦♠❛✐♥❡♥❝✐r❝❧❡❞❜②t❤❡❝♦♥t♦✉r Γ✱✇❤✐❝❤✐s ❝❤♦s❡♥t♦❝♦♥t❛✐♥t❤❡ ❇r♦♠✇✐❝❤♣❛t❤ΓBr✳ ❇r♦♠✇✐❝❤♣❛t❤ΓBr✳ ✷✹ ❆✳✶ ◆❛t✉r❡♦❢♣♦❧❡s ♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(2) C ❚❤❡❡①✐st❡♥❝❡♦❢♣♦❧❡s ♦❢t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(2) C ✐♥t❤❡✜rst ❘✐❡♠❛♥♥s❤❡❡t ✐s ❞❡t❡r♠✐♥❡❞❜②t❤❡ ♦❝❝✉rr❡♥❝❡♦❢③❡r♦s ♦❢t❤❡❞❡♥♦♠✐♥❛t♦r ♦❢ˆg(2) C ✱r❡✇r✐tt❡♥❛s ψ(s) = as1+µ + bsµ + 1, ✇✐t❤ a = τ µ ❛♥❞b = τ µ τ C . ✭✻✼✮ ✭✻✼✮ ■♥♦r❞❡r t♦✜♥❞③❡r♦s ♦❢t❤❡❢✉♥❝t✐♦♥ψ✱❣✐✈❡♥❜②✭✻✼✮✱✐ts r❡❛❧❛♥❞✐♠❛❣✐♥❛r②♣❛rts ❛r❡s❡♣❛r❛t❡❞❛s Reψ(ρ, ϕ) = aρ1+µ cos((1 + µ)ϕ) + bρµ cos(µϕ) + 1, ✭✻✽✮ Imψ(ρ, ϕ) = aρ1+µ sin((1 + µ)ϕ) + bρµ sin(µϕ), ✭✻✾✮ ✭✻✽✮ ✭✻✾✮ ❜②s✉❜st✐t✉t✐♥❣s = ρejϕ ✐♥t♦✭✻✼✮✳Pr♦♣❡rt✐❡s ♦❢t❤❡r❡❛❧❛♥❞✐♠❛❣✐♥❛r②♣❛rts ♦❢❢✉♥❝t✐♦♥ψ Reψ(ρ, −ϕ) = Reψ(ρ, ϕ) ❛♥❞ Imψ(ρ, −ϕ) = −Imψ(ρ, ϕ) ✐♠♣❧②t❤❛t ✐t ✐s s②♠♠❡tr✐❝✇✐t❤r❡s♣❡❝t t♦t❤❡r❡❛❧❛①✐s✱s♦t❤❛t ✐❢ψ ❤❛s ❛③❡r♦s0 ✐♥t❤❡✉♣♣❡r ❝♦♠♣❧❡① ❤❛❧❢✲♣❧❛♥❡✱t❤❡♥✐t ❛❧s♦❤❛s ✐ts ❝♦♠♣❧❡①❝♦♥❥✉❣❛t❡¯s0 ❛s ❛③❡r♦✱t❤✉s ✐t ✐s s✉✣❝✐❡♥t t♦s❡❡❦❢♦r ③❡r♦s ✐♥ t❤❡✉♣♣❡r ❝♦♠♣❧❡①❤❛❧❢✲♣❧❛♥❡♦♥❧②✳▼♦r❡♦✈❡r✱❢✉♥❝t✐♦♥ψ ❞♦❡s ♥♦t ❤❛✈❡③❡r♦s ✐♥t❤❡✉♣♣❡r r✐❣❤t ❝♦♠♣❧❡① q✉❛rt❡r✲♣❧❛♥❡✭❛♥❞t❤❡r❡❢♦r❡✐♥t❤❡❧♦✇❡r r✐❣❤t ❝♦♠♣❧❡①q✉❛rt❡r✲♣❧❛♥❡❛s ✇❡❧❧✮✱s✐♥❝❡❢♦r ϕ ∈ 0, π 2  ✱❜② ✭✻✾✮✱♦♥❡❤❛s Imψ(ρ, ϕ) > 0✱✇❤✐❧❡✐❢ϕ = 0✱t❤❡♥Imψ(ρ, ϕ) = 0✱❜✉t ♦♥❡❤❛s Reψ(ρ, ϕ) > 0✱❜②✭✻✽✮✳ ❚❤✉s✱✐❢ψ ❤❛s ③❡r♦s✱t❤❡②❧✐❡✐♥t❤❡✉♣♣❡r ❧❡❢t ❝♦♠♣❧❡①q✉❛rt❡r✲♣❧❛♥❡❛♥❞t❤❡✐r ❝♦♠♣❧❡①❝♦♥❥✉❣❛t❡s ✐♥ t❤❡❧♦✇❡r ❧❡❢t ❝♦♠♣❧❡①q✉❛rt❡r✲♣❧❛♥❡✳ ❚❤❡❡q✉❛t✐♦♥Imψ(ρ, ϕ) = 0 s♦❧✈❡❞✇✐t❤r❡s♣❡❝t t♦ρ > 0 ②✐❡❧❞s ρ = −b a sin(µϕ) sin((1 + µ)ϕ) = b a sin(µϕ) | sin((1 + µ)ϕ)| ❢♦r ϕ ∈  π 1 + µ, π  , ❛♥❞✇❤❡♥s✉❝❤♦❜t❛✐♥❡❞ρ ✐s s✉❜st✐t✉t❡❞✐♥t♦❡q✉❛t✐♦♥Reψ(ρ, ϕ) = 0 ♦♥❡♦❜t❛✐♥s ❛♥❞✇❤❡♥s✉❝❤♦❜t❛✐♥❡❞ρ ✐s s✉❜st✐t✉t❡❞✐♥t♦❡q✉❛t✐♦♥Reψ(ρ, ϕ) = 0 ♦♥❡♦❜t❛✐♥s  a sin(µϕ) µ = sin ϕ  b sin((1 + µ)ϕ) 1+µ . ✭✼✵✮ ✭✼✵✮ ■t ✐s ✉♥❝❧❡❛r ✇❤❡t❤❡r ♦r ♥♦t t❤❡❡q✉❛t✐♦♥✭✼✵✮❤❛s ❛s♦❧✉t✐♦♥ϕ ∈ π 1+µ, π  ❛♥❞t❤❡r❡❢♦r❡t❤❡❡①✐st❡♥❝❡ ♦❢③❡r♦s ♦❢❢✉♥❝t✐♦♥ψ✱❣✐✈❡♥❜②✭✻✼✮✱✐♥t❤❡✉♣♣❡r ❧❡❢t ❝♦♠♣❧❡①q✉❛rt❡r✲♣❧❛♥❡✐s ✐♥✈❡st✐❣❛t❡❞❜②✉s✐♥❣t❤❡ ❛r❣✉♠❡♥t ♣r✐♥❝✐♣❧❡✇✐t❤t❤❡❝♦♥t♦✉r γ ❛s ✐♥❋✐❣✉r❡✼✱s✐♥❝❡s = 0 ✐s ✐ts ♦♥❧②❜r❛♥❝❤✐♥❣♣♦✐♥t✳❘❡❝❛❧❧✱ t❤❡❛r❣✉♠❡♥t ♣r✐♥❝✐♣❧❡✐s st❛t✐♥❣t❤❛t ✐❢t❤❡✐♥❞❡♣❡♥❞❡♥t ✈❛r✐❛❜❧❡z ❝❤❛♥❣❡s ❛❧♦♥❣t❤❡❝❧♦s❡❞❝♦♥t♦✉r γ ✐♥t❤❡❝♦♠♣❧❡①♣❧❛♥❡✱t❤❡♥t❤❡♥✉♠❜❡r ♦❢③❡r♦s N ♦❢❢✉♥❝t✐♦♥f(z) ✐♥t❤❡❞♦♠❛✐♥❜♦✉♥❞❡❞❜②❝♦♥t♦✉r γ ✐s ❞❡t❡r♠✐♥❡❞❜②t❤❡❝❤❛♥❣❡♦❢❛r❣✉♠❡♥t✿∆arg f(z) = 2πN✱❛ss✉♠✐♥❣t❤❛t ❢✉♥❝t✐♦♥f ❞♦❡s ♥♦t ❤❛✈❡ ✷✺ ♣♦❧❡s ✐♥t❤❡♠❡♥t✐♦♥❡❞❞♦♠❛✐♥✳ Re s Im s R r γ2 γ1 γ3 γ4 ❋✐❣✉r❡✼✿❈♦♥t♦✉r γ = γ1 ∪γ2 ∪γ3 ∪γ4. ❋✐❣✉r❡✼✿❈♦♥t♦✉r γ = γ1 ∪γ2 ∪γ3 ∪γ4. ❋✐❣✉r❡✼✿❈♦♥t♦✉r γ = γ1 ∪γ2 ∪γ3 ∪γ4. ❆✳✶ ◆❛t✉r❡♦❢♣♦❧❡s ♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(2) C ❆❧♦♥❣t❤❡❝♦♥t♦✉r γ1✱♣❛r❛♠❡t❡r✐③❡❞❜②s = ρej π 2 ✱✇✐t❤ρ ∈(0, ∞)✱t❤❡✐♠❛❣✐♥❛r②♣❛rt ♦❢❢✉♥❝t✐♦♥ψ✱ ❜②✭✻✾✮✱✐s Imψ  ρ, π 2  = aρ1+µ cos µπ 2 + bρµ sin µπ 2 > 0, s✐♥❝❡µ ∈(0, 1)✱✇❤✐❧❡✐♥t❤❡❧✐♠✐t✐♥❣❝❛s❡s✱♦♥❡❤❛s Reψ  ρ, π 2  ∼1 ❛♥❞ Imψ  ρ, π 2  ∼bρµ sin µπ 2 →0 ❛s ρ →0, Reψ  ρ, π 2  ∼−aρ1+µ sin µπ 2 →−∞ ❛♥❞ Imψ  ρ, π 2  ∼aρ1+µ cos µπ 2 →∞ ❛ ❆❧♦♥❣t❤❡❝♦♥t♦✉r γ2✱♣❛r❛♠❡t❡r✐③❡❞❜②s = Rejϕ✱✇✐t❤ϕ ∈  π 2 , π  ❛s R →∞✱❛❝❝♦r❞✐♥❣t♦✭✻✽✮❛♥❞ ✭✻✾✮✱♦♥❡❤❛s Reψ(R, ϕ) ∼aR1+µ cos((1 + µ)ϕ) ❛♥❞ Imψ(R, ϕ) ∼aR1+µ sin((1 + µ)ϕ) ✐♠♣❧②✐♥❣ |ψ| ∼aR1+µ →∞, ✇❤✐❧❡❢♦r ϕ = π 2 ❛♥❞ϕ = π ✐t ❤♦❧❞s ✇❤✐❧❡❢♦r ϕ = π 2 ❛♥❞ϕ = π ✐t ❤♦❧❞s Reψ  R, π 2  ∼−aR1+µ sin µπ 2 →−∞ ❛♥❞ Imψ  R, π 2  ∼aR1+µ cos µπ 2 →∞, Reψ (R, π) ∼−aR1+µ cos(µπ) →      −∞, ✐❢µ < 1 2 ∞, ✐❢µ > 1 2 ❛♥❞ Imψ (R, π) ∼−aR1+µ sin(µπ) →−∞. ❆❧♦♥❣t❤❡❝♦♥t♦✉r γ3✱♣❛r❛♠❡t❡r✐③❡❞❜②s = ρejπ✱✇✐t❤ρ ∈(0, ∞)✱t❤❡r❡❛❧❛♥❞✐♠❛❣✐♥❛r②♣❛rts ♦❢ ❢✉♥❝t✐♦♥ψ✱❛❝❝♦r❞✐♥❣t♦✭✻✽✮❛♥❞✭✻✾✮✱r❡❛❞ Reψ(ρ, ϕ) = −aρ1+µ cos(µπ) + bρµ cos(µπ) + 1 = ρµ cos(µπ)(b −aρ) + 1, ✭✼✶✮ ✭✼✶✮ ✷✻ Imψ(ρ, ϕ) = −aρ1+µ sin(µπ) + bρµ sin(µπ) = ρµ sin(µπ)(b −aρ). ✭✼✷✮ Imψ(ρ, ϕ) = −aρ1+µ sin(µπ) + bρµ sin(µπ) = ρµ sin(µπ)(b −aρ). ✭✼✷✮ ✭✼✷✮ ❚❤❡❛s②♠♣t♦t✐❝❜❡❤❛✈✐♦r ❚❤❡❛s②♠♣t♦t✐❝❜❡❤❛✈✐♦r ❚❤❡❛s②♠♣t♦t✐❝❜❡❤❛✈✐♦r ❚❤❡❛s②♠♣t♦t✐❝❜❡❤❛✈✐♦r Reψ (ρ, π) ∼1 ❛♥❞ Imψ (ρ, π) ∼bρµ sin(µπ) →0+ ❛s ρ →0, Reψ (ρ, π) ∼1 ❛♥❞ Imψ (ρ, π) ∼bρµ sin(µπ) →0+ ❛s ρ →0, Reψ (ρ, π) ∼−aρ1+µ cos(µπ) →      −∞, ✐❢µ < 1 2 ∞, ✐❢µ > 1 2 ❛♥❞ Imψ (ρ, π) ∼−aρ1+µ sin(µπ) →−∞. Reψ (ρ, π) ∼−aρ1+µ cos(µπ) →      −∞, ✐❢µ < 1 2 ∞, ✐❢µ > 1 2 ❛♥❞ Imψ (ρ, π) ∼−aρ1+µ sin(µπ) →−∞ ♦❢Reψ ❛♥❞Imψ✱❣✐✈❡♥❜②✭✼✶✮❛♥❞✭✼✷✮✱✐♠♣❧✐❡s t❤❛t ✐♠❛❣✐♥❛r②♣❛rt ♦❢❢✉♥❝t✐♦♥ψ ❛❧♦♥❣γ3 ❝❤❛♥❣❡s ✐ts s✐❣♥❢r♦♠♥❡❣❛t✐✈❡t♦♣♦s✐t✐✈❡❛♥❞t❤❡r❡❢♦r❡ ♦❢Reψ ❛♥❞Imψ✱❣✐✈❡♥❜②✭✼✶✮❛♥❞✭✼✷✮✱✐♠♣❧✐❡s t❤❛t ✐♠❛❣✐♥❛r②♣❛rt ♦❢❢✉♥❝t✐♦♥ψ ❛❧♦♥❣γ3 ❝❤❛♥❣❡s ✐ts s✐❣♥❢r♦♠♥❡❣❛t✐✈❡t♦♣♦s✐t✐✈❡❛♥❞t❤❡r❡❢♦r❡ Imψ(ρ∗, π) = 0 ✐♠♣❧✐❡s ρ∗= b a ❛♥❞ Re Ψ (ρ∗, π) = 1. ❆❧♦♥❣t❤❡❝♦♥t♦✉r γ4✱♣❛r❛♠❡t❡r✐③❡❞❜②s = rejϕ✱✇✐t❤ϕ ∈  π 2 , π  ❛s r →0✱❛❝❝♦r❞✐♥❣t♦✭✻✽✮❛♥❞ ✭✻✾✮✱♦♥❡❤❛s Reψ(r, ϕ) ∼1 ❛♥❞ Imψ(r, ϕ) ∼brµ sin(µπ) →0+. Reψ(r, ϕ) ∼1 ❛♥❞ Imψ(r, ϕ) ∼brµ sin(µπ) →0+. ❆✳✶ ◆❛t✉r❡♦❢♣♦❧❡s ♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(2) C ■♥❝♦♥❝❧✉s✐♦♥✱♦♥❡✜♥❞s t❤❛t ∆arg ψ(s) = 2π✱s♦❜②t❤❡❛r❣✉♠❡♥t ♣r✐♥❝✐♣❧❡❢✉♥❝t✐♦♥ψ ❤❛s ❛s✐♥❣❧❡ ③❡r♦✐♥t❤❡✉♣♣❡r ❧❡❢t ❝♦♠♣❧❡①q✉❛rt❡r✲♣❧❛♥❡❛♥❞t❤❡r❡❢♦r❡❤❛s ❛♣❛✐r ♦❢❝♦♠♣❧❡①❝♦♥❥✉❣❛t❡❞③❡r♦s ✇✐t❤ ♥❡❣❛t✐✈❡r❡❛❧♣❛rt ✐♥t❤❡✜rst ❘✐❡♠❛♥♥s❤❡❡t✳ ❆✳✷ ▲❛♣❧❛❝❡tr❛♥s❢♦r♠✐♥✈❡rs✐♦♥♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(2) C ❚❤❡✐♠♣✉❧s❡r❡s♣♦♥s❡g(2) C ✐s ♦❜t❛✐♥❡❞❢r♦♠t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(2) C ✱❣✐✈❡♥❜②✭✷✼✮✱❜②t❤❡▲❛♣❧❛❝❡ ✐♥✈❡rs✐♦♥❢♦r♠✉❧❛✭✻✺✮✉s✐♥❣t❤❡❈❛✉❝❤②r❡s✐❞✉❡s t❤❡♦r❡♠✭✻✻✮✱✐✳❡✳✱ I Γ ˆg(2) C (s) est ds = 2πj  Res  ˆg(2) C (s) est, s0  + Res  ˆg(2) C (s) est, ¯s0  , ✭✼✸✮ ✭✼✸✮ ✇❤❡r❡t❤❡✐♥t❡❣r❛t✐♦♥♣❛t❤Γ ✐s ❝❤♦s❡♥❛s ✐♥❋✐❣✉r❡✽✱s✐♥❝❡❢✉♥❝t✐♦♥ˆg(2) C ❤❛s s = 0 ❛s t❤❡❜r❛♥❝❤✐♥❣♣♦✐♥t ❛♥❞❛♣❛✐r ♦❢❝♦♠♣❧❡①❝♦♥❥✉❣❛t❡❞♣♦❧❡s s0 ❛♥❞¯s0 ❤❛✈✐♥❣♥❡❣❛t✐✈❡r❡❛❧♣❛rt✱❛s ♣r♦✈❡❞✐♥❙❡❝t✐♦♥❆✳✶✳ ■♥t❡❣r❛t✐♦♥❛❧♦♥❣❝♦♥t♦✉rs Γ3 ❛♥❞Γ5✱♣❛r❛♠❡t❡r✐③❡❞❜②s = ρejπ ❛♥❞s = ρe−jπ✱✇✐t❤ρ ∈(0, ∞)✱ r❡s♣❡❝t✐✈❡❧②②✐❡❧❞s ✇❤❡r❡t❤❡✐♥t❡❣r❛t✐♦♥♣❛t❤Γ ✐s ❝❤♦s❡♥❛s ✐♥❋✐❣✉r❡✽✱s✐♥❝❡❢✉♥❝t✐♦♥ˆg(2) C ❤❛s s = 0 ❛s t❤❡❜r❛♥❝❤✐♥❣♣♦✐♥t ❛♥❞❛♣❛✐r ♦❢❝♦♠♣❧❡①❝♦♥❥✉❣❛t❡❞♣♦❧❡s s0 ❛♥❞¯s0 ❤❛✈✐♥❣♥❡❣❛t✐✈❡r❡❛❧♣❛rt✱❛s ♣r♦✈❡❞✐♥❙❡❝t✐♦♥❆✳✶✳ ■♥t❡❣r❛t✐♦♥❛❧♦♥❣❝♦♥t♦✉rs Γ3 ❛♥❞Γ5✱♣❛r❛♠❡t❡r✐③❡❞❜②s = ρejπ ❛♥❞s = ρe−jπ✱✇✐t❤ρ ∈(0, ∞)✱ r❡s♣❡❝t✐✈❡❧②②✐❡❧❞s IΓ3 = lim R→∞ r→0 Z Γ3 ˆg(2) C (s)estds = Z 0 ∞ τ µ τ C ρµejµπ + 1 ψ(ρejπ) eρtejπejπdρ = Z ∞ 0 τ µ τ C ρµejµπ + 1 ¯ψ(ρejπ) |ψ(ρejπ)|2 e−ρtdρ, ✭✼✹✮ IΓ5 = lim R→∞ r→0 Z Γ5 ˆg(2) C (s)estds = Z ∞ 0 τ µ τ C ρµe−jµπ + 1 ψ(ρe−jπ) eρte−jπe−jπdρ ✭✼✹✮ IΓ5 = lim R→∞ r→0 Z Γ5 ˆg(2) C (s)estds = Z ∞ 0 τ µ τ C ρµe−jµπ + 1 ψ(ρe−jπ) eρte−jπe−jπdρ ✷✼ Γ2 Γ6 Γ7 Γ0 Γ1 Γ3 Γ4 Γ5 Re s Im s R r p0 ❋✐❣✉r❡✽✿❈♦♥t♦✉r Γ = Γ0 ∪Γ1 ∪Γ2 ∪Γ3 ∪Γ4 ∪Γ5 ∪Γ6 ∪Γ7✳ Γ2 Γ6 Γ7 Γ0 Γ1 Γ3 Γ4 Γ5 Re s Im s R r p0 ❋✐❣✉r❡✽✿❈♦♥t♦✉r Γ = Γ0 ∪Γ1 ∪Γ2 ∪Γ3 ∪Γ4 ∪Γ5 ∪Γ6 ∪Γ7✳ ❋✐❣✉r❡✽✿❈♦♥t♦✉r Γ = Γ0 ∪Γ1 ∪Γ2 ∪Γ3 ∪Γ4 ∪Γ5 ∪Γ6 ∪Γ7✳ = − Z ∞ 0 τ µ τ C ρµe−jµπ + 1  ψ(ρejπ) |ψ(ρejπ)|2 e−ρtdρ, ✭✼✺✮ ✭✼✺✮ s✐♥❝❡ψ(ρe−jπ) = ¯ψ(ρejπ)✱✇❤❡r❡❜❛r ❞❡♥♦t❡s ❝♦♠♣❧❡①❝♦♥❥✉❣❛t✐♦♥✱s♦t❤❛t s✐♥❝❡ψ(ρe−jπ) = ¯ψ(ρejπ)✱✇❤❡r❡❜❛r ❞❡♥♦t❡s ❝♦♠♣❧❡①❝♦♥❥✉❣❛t✐♦♥✱s♦t❤❛t 1 2πj (IΓ3 + IΓ5) = 1 π Z ∞ 0 τ µρ1+µ sin(µπ) |ψ(ρejπ)|2 e−ρtdρ, ✭✼✻✮ ✭✼✻✮ ❛❝❝♦r❞✐♥❣t♦✭✼✹✮❛♥❞✭✼✺✮✳ ❛❝❝♦r❞✐♥❣t♦✭✼✹✮❛♥❞✭✼✺✮✳ ❙✐♥❝❡✐♥t❡❣r❛❧❛❧♦♥❣Γ0 ✐s ❣✐✈❡♥❜②✭✻✺✮❛♥❞s✐♥❝❡✐♥t❡❣r❛❧s ❛❧♦♥❣Γ3 ❛♥❞Γ5 ❛r❡❣✐✈❡♥❜②✭✼✻✮✱✇❤✐❧❡ ✐♥t❡❣r❛❧s ❛❧♦♥❣❛❧❧♦t❤❡r ❝♦♥t♦✉rs ♦♥❋✐❣✉r❡✽t❡♥❞t♦③❡r♦❛s R →∞❛♥❞r →0✱t❤❡❈❛✉❝❤②r❡s✐❞✉❡s t❤❡♦r❡♠✭✼✸✮t❛❦❡s t❤❡❢♦r♠ g(2) C (t) + 1 π Z ∞ 0 τ µρ1+µ sin(µπ) |ψ(ρejπ)|2 e−ρtdρ = Res  ˆg(2) C (s) est, s0  + Res  ˆg(2) C (s) est, ¯s0  , ✭✼✼✮ ✭✼✼✮ ✇❤❡r❡t❤❡r❡s✐❞✉❡s ❛r❡❝❛❧❝✉❧❛t❡❞❛s ✇❤❡r❡t❤❡r❡s✐❞✉❡s ❛r❡❝❛❧❝✉❧❛t❡❞❛s Res  ˆg(2) C (s) est, s0  = τ µ τ C sµ + 1  est d dsψ(s) s=s0 = τ µ τ C sµ 0 + 1  ejtIms0 d dsψ(s) s=s0 e−|Res0|t, Res  ˆg(2) C (s) est, ¯s0  = τ µ τ C sµ + 1  est d dsψ(s) s=¯s0 = τ µ τ C ¯sµ 0 + 1  e−jtIms0 d dsψ(s) s=¯s0 e−|Res0|t, s♦t❤❛t s♦t❤❛t Res  ˆg(2) C (s) est, s0  + Res  ˆg(2) C (s) est, ¯s0  = 2 Re τ µ τ C sµ 0 + 1 d dsψ(s) s=s0 ejtIms0 ! ❆✳✷ ▲❛♣❧❛❝❡tr❛♥s❢♦r♠✐♥✈❡rs✐♦♥♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(2) C e−|Res0|t Res  ˆg(2) C (s) est, s0  + Res  ˆg(2) C (s) est, ¯s0  = 2 Re τ µ τ C sµ 0 + 1 d dsψ(s) s=s0 ejtIms0 ! e−|Res0|t s✐♥❝❡ d dsψ(s) s=¯s0 = d dsψ(s) s=s0✱✐♠♣❧②✐♥❣❜②✭✼✼✮ s✐♥❝❡ d dsψ(s) s=¯s0 = d dsψ(s) s=s0✱✐♠♣❧②✐♥❣❜②✭✼✼✮ s✐♥❝❡ d dsψ(s) s=¯s0 = d dsψ(s) s=s0✱✐♠♣❧②✐♥❣❜②✭✼✼✮ g(2) C (t) = −1 π Z ∞ 0 τ µρ1+µ sin(µπ) |ψ(ρejπ)|2 e−ρtdρ + 2 Re τ µ τ C sµ 0 + 1 d dsψ(s) s=s0 ejtIms0 ! e−|Res0|t. ❇ ❉❡r✐✈❛t✐♦♥♦❢❛s②♠♣t♦t✐❝❢♦r♠✉❧❛❡ ■♥♦r❞❡r t♦❞❡s❝r✐❜❡t❤❡❛s②♠♣t♦t✐❝❜❡❤❛✈✐♦r ♦❢t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ˆg(1) R dB ❢♦r ❧♦✇❢r❡q✉❡♥❝✐❡s✱ ♦♥❡r❡✇r✐t❡s ✭✺✷✮✐♥t❤❡❢♦r♠ 10 1 10 ˆg(1) R (ω) dB = 1 −  1 + 2τ α ωα cos απ 2  ×  1 −2τ α ωα cos απ 2 −τ 2 α ω2α −2τ Cτ α ω1+α sin απ 2 −τ 2 C ω2 + 4τ 2 α ω2α cos2 απ 2 + 4τ 3 α ω3α cos απ 2 + . . . −8τ 3 α ω3α cos3 απ 2 + . . .  ∼τ 2 α ω2α −2τ 3 α ω3α cos απ 2 + 2τ Cτ α ω1+α sin απ 2 ❛s ω →0, ✭✼✽✮  α 2  ×  1 −2τ α ωα cos απ 2 −τ 2 α ω2α −2τ Cτ α ω1+α sin απ 2 −τ 2 C ω2 + 4τ 2 α ω2α cos2 απ 2 + 4τ 3 α ω3α cos απ 2 + . . . −8τ 3 α ω3α cos3 απ 2 + . . .  ∼τ 2 α ω2α −2τ 3 α ω3α cos απ 2 + 2τ Cτ α ω1+α sin απ 2 ❛s ω →0, ✭✼✽✮ ✭✼✽✮ ✉s✐♥❣t❤❡s❡r✐❡s ❡①♣❛♥s✐♦♥ 1 1 + x = 1 −x + x2 −x3 + . . . , ✭✼✾✮ ✭✼✾✮ ❛♥❞❜②♥❡❣❧❡❝t✐♥❣t❤❡❤✐❣❤❡r ♦r❞❡r t❡r♠s✱s♦t❤❛t ✭✼✽✮②✐❡❧❞s t❤❡❛s②♠♣t♦t✐❝❢♦r♠✉❧❛✭✺✻✮✳■♥t❤❡❝❛s❡♦❢ ❤✐❣❤❢r❡q✉❡♥❝✐❡s✱t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ˆg(1) R dB , ❣✐✈❡♥❜②✭✺✷✮✱t❛❦❡s t❤❡❢♦r♠ 10 1 10 ˆg(1) R (ω) dB = 1 − 1 τ 2 C ω2 1 + 2τ α ωα cos απ 2 1 + 2 τ α τ C 1 ω1−α sin απ 2 + τ 2 α τ 2 C 1 ω2(1−α) + 2 τ α τ 2 C 1 ω2−α cos απ 2 + 1 τ 2 C ω2 = 1 − 1 τ 2 C ω2  1 + 2τ α ωα cos απ 2  ×  1 −2 τ α τ C 1 ω1−α sin απ 2 −τ 2 α τ 2 C 1 ω2(1−α) −2 τ α τ 2 C 1 ω2−α cos απ 2 − 1 τ 2 C ω2 + 4 τ 2 α τ 2 C 1 ω2(1−α) sin2 απ 2 + . . . ❆✳✷ ▲❛♣❧❛❝❡tr❛♥s❢♦r♠✐♥✈❡rs✐♦♥♦❢tr❛♥s❢❡r ❢✉♥❝t✐♦♥ˆg(2) C ✷✽ ■t ✐s ❧❡❢t t♦♣r♦✈❡t❤❛t ✐♥t❡❣r❛❧s ❛❧♦♥❣❝♦♥t♦✉rs✿Γ1✱Γ2✱Γ4✱Γ6✱❛♥❞Γ7 t❡♥❞t♦③❡r♦❛s R →∞❛♥❞ r →0✳ r →0✳ ❚❤❡✐♥t❡❣r❛❧❛❧♦♥❣❝♦♥t♦✉r Γ1✱♣❛r❛♠❡t❡r✐③❡❞❜②s = p + jR✱✇✐t❤p ∈(0, p0) ❛s R →∞✱②✐❡❧ IΓ1 = lim R→∞ Z Γ1 ˆg(2) C (s)estds = lim R→∞ Z 0 p0 τ µ τ C (p + jR)µ + 1 ψ(p + jR) e(p+jR)tdp, s♦t❤❛t |IΓ1| ≤lim R→∞ Z p0 0 τ µ τ C (p + jR)µ + 1 |ψ(p + jR)| eptdp ≤lim R→∞ Z p0 0 1 τ CReptdp →0 ❛s R →∞, s✐♥❝❡❢♦r |s| = p p2 + R2 ∼R ❛♥❞arg s = arctan R p ∼π 2 ✱♦♥❡❤❛s s✐♥❝❡❢♦r |s| = p p2 + R2 ∼R ❛♥❞arg s = arctan R p ∼π 2 ✱♦♥❡❤❛s τ µ τ C Rµej µπ 2 + 1 ∼τ µ τ C Rµ ❛♥❞ ψ(Rej π 2 ) ∼τ µR1+µ ❛s R →∞, ✇✐t❤ψ ❣✐✈❡♥❜②✭✻✼✮✱❛♥❞t❤❡r❡❢♦r❡IΓ1 →0 ❛s R →∞✳❙✐♠✐❧❛r ❛r❣✉♠❡♥t❛t✐♦♥❣✐✈❡s IΓ7 →0 ❛s R →∞✳ ❚❤❡✐♥t❡❣r❛❧❛❧♦♥❣❝♦♥t♦✉r Γ2✱♣❛r❛♠❡t❡r✐③❡❞❜②s = Rejϕ✱✇✐t❤ϕ ∈( π 2 , π) ❛s R →∞✱②✐❡❧❞ ✇✐t❤ψ ❣✐✈❡♥❜②✭✻✼✮✱❛♥❞t❤❡r❡❢♦r❡IΓ1 →0 ❛s R →∞✳❙✐♠✐❧❛r ❛r❣✉♠❡♥t❛t✐♦♥❣✐✈❡s IΓ7 →0 ❛s R →∞✳ ❚❤❡✐♥t❡❣r❛❧❛❧♦♥❣❝♦♥t♦✉r Γ2✱♣❛r❛♠❡t❡r✐③❡❞❜②s = Rejϕ✱✇✐t❤ϕ ∈( π 2 , π) ❛s R →∞✱②✐❡❧❞ IΓ2 = lim R→∞ Z Γ2 ˆg(2) C (s)estds = lim R→∞ Z π π 2 τ µ τ C Rµejµϕ + 1 ψ(Rejϕ) eRtejϕjRejϕdϕ, s♦t❤❛t |IΓ2| ≤lim R→∞ Z π π 2 τ µ τ C Rµejµϕ + 1 |ψ(Rejϕ)| R eRt cos ϕdϕ ≤lim R→∞ Z π π 2 1 τ C eRt cos ϕdϕ →0 ❛s R →∞, s✐♥❝❡cos ϕ < 0 ❢♦r ϕ ∈( π 2 , π)✱✇❤✐❧❡ s✐♥❝❡cos ϕ < 0 ❢♦r ϕ ∈( π 2 , π)✱✇❤✐❧❡ τ µ τ C Rµejµϕ + 1 ∼τ µ τ C Rµ ❛♥❞ ψ(Rejϕ) ∼τ µR1+µ ❛s R →∞, ✇✐t❤ψ ❣✐✈❡♥❜②✭✻✼✮✱s♦t❤❛t IΓ2 →0 ❛s R →∞✳❙✐♠✐❧❛r ❛r❣✉♠❡♥t❛t✐♦♥❣✐✈❡s IΓ6 →0 ❛s R →∞✳ ❚❤❡✐♥t❡❣r❛❧❛❧♦♥❣❝♦♥t♦✉r Γ4✱♣❛r❛♠❡t❡r✐③❡❞❜②s = rejϕ✱✇✐t❤ϕ ∈( π 2 , π) ❛s r →0✱②✐❡❧❞ ✇✐t❤ψ ❣✐✈❡♥❜②✭✻✼✮✱s♦t❤❛t IΓ2 →0 ❛s R →∞✳❙✐♠✐❧❛r ❛r❣✉♠❡♥t❛t✐♦♥❣✐✈❡s IΓ6 →0 ❛s R →∞✳ ❚❤❡✐♥t❡❣r❛❧❛❧♦♥❣❝♦♥t♦✉r Γ4✱♣❛r❛♠❡t❡r✐③❡❞❜②s = rejϕ✱✇✐t❤ϕ ∈( π 2 , π) ❛s r →0✱②✐❡❧❞ IΓ4 = lim r→0 Z Γ4 ˆg(2) C (s)estds = lim r→0 Z −π π τ µ τ C rµejµϕ + 1 ψ(rejϕ) ertejϕjrejϕdϕ, s♦t❤❛t |IΓ4| ≤lim r→0 Z π −π τ µ τ C rµejµϕ + 1 |ψ(rejϕ)| r ert cos ϕdϕ ≤lim r→0 Z π −π rdϕ →0 ❛s r →0, s✐♥❝❡❜②✭✻✼✮♦♥❡❤❛s ψ(rejϕ) ∼1 ❛s r →0✱s♦t❤❛t IΓ4 →0 ❛s r →0✳ ✷✾ ❇ ❉❡r✐✈❛t✐♦♥♦❢❛s②♠♣t♦t✐❝❢♦r♠✉❧❛❡  ∼1 − 1 τ 2 C ω2  2τ α ωα cos απ 2 −2 τ 2 α τ C ω2α−1 sin(απ) + 2 τ 3 α τ 2 C ω3α−2 cos απ 2  4 sin2 απ 2 −1  + 1  ❛s ω →∞, ✭✽✵✮ ✭✽✵✮ ✉s✐♥❣t❤❡s❡r✐❡s ❡①♣❛♥s✐♦♥✭✼✾✮✉♣t♦t❤❡q✉❛❞r❛t✐❝t❡r♠s ❛♥❞❜②♥❡❣❧❡❝t✐♥❣t❤❡❤✐❣❤❡r ♦r❞❡r t❡r♠s✱s♦ t❤❛t t❤❡❛s②♠♣t♦t✐❝❢♦r♠✉❧❛✭✺✼✮❢♦❧❧♦✇s ❢r♦♠✭✽✵✮❜②♥❡❣❧❡❝t✐♥❣t❤❡t❡r♠s ✐♥❜r❛❝❦❡t ❤❛✈✐♥❣♥❡❣❛t✐✈❡ ♣♦✇❡rs✳ ❚❤❡❛s②♠♣t♦t✐❝❡①♣❛♥s✐♦♥✭✺✽✮❢♦r t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t arg ˆg(1) R ✐♥t❤❡❝❛s❡♦❢❧♦✇❢r❡q✉❡♥❝✐❡s ❢♦❧❧♦✇s ❢r♦♠✭✺✸✮✱r❡✇r✐tt❡♥✐♥t❤❡❢♦r♠ tan arg ˆg(1) R (ω) = tan απ 2 1 + τ C τ α ω1−α 1 sin απ 2 1 + τ α ωα 1 cos απ 2 + 2τ C ω tan απ 2 + τ 2 C τ α ω2−α 1 cos απ 2 = tan απ 2  1 + τ C τ α ω1−α 1 sin απ 2  ×  1 −τ α ωα 1 cos απ 2 −2τ C ω tan απ 2 −τ 2 C τ α ω2−α 1 cos απ 2 + τ 2 α ω2α 1 cos2 απ 2 + . . . ❇ ❉❡r✐✈❛t✐♦♥♦❢❛s②♠♣t♦t✐❝❢♦r♠✉❧❛❡  ✸✵ ∼tan απ 2  1 −τ α ωα 1 cos απ 2 + τ 2 α ω2α 1 cos2 απ 2 + τ C τ α ω1−α 1 sin απ 2  ❛s ω →0, ✉s✐♥❣t❤❡s❡r✐❡s ❡①♣❛♥s✐♦♥✭✼✾✮✉♣t♦t❤❡q✉❛❞r❛t✐❝t❡r♠s✱❜②♥❡❣❧❡❝t✐♥❣t❤❡❤✐❣❤❡r ♦r❞❡r t❡r♠s✱❛♥❞❜② s❡❧❡❝t✐♥❣t❡r♠s ❤❛✈✐♥❣t❤❡❞♦♠✐♥❛♥t ❝♦♥tr✐❜✉t✐♦♥✱✇❤✐❧❡t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t arg ˆg(1) R , ❣✐✈❡♥❜② ✭✺✸✮✱❢♦r ❤✐❣❤❢r❡q✉❡♥❝✐❡s tr❛♥s❢♦r♠s ✐♥t♦ ✉s✐♥❣t❤❡s❡r✐❡s ❡①♣❛♥s✐♦♥✭✼✾✮✉♣t♦t❤❡q✉❛❞r❛t✐❝t❡r♠s✱❜②♥❡❣❧❡❝t✐♥❣t❤❡❤✐❣❤❡r ♦r❞❡r t❡r♠s✱❛♥❞❜② s❡❧❡❝t✐♥❣t❡r♠s ❤❛✈✐♥❣t❤❡❞♦♠✐♥❛♥t ❝♦♥tr✐❜✉t✐♦♥✱✇❤✐❧❡t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t arg ˆg(1) R , ❣✐✈❡♥❜② ✭✺✸✮✱❢♦r ❤✐❣❤❢r❡q✉❡♥❝✐❡s tr❛♥s❢♦r♠s ✐♥t♦ tan arg ˆg(1) R (ω) = 1 τ C ω 1 + τ α τ C 1 ω1−α sin απ 2 1 + 2 τ α τ C 1 ω1−α sin απ 2 + τ 2 α τ 2 C 1 ω2(1−α) + τ α τ 2 C 1 ω2−α cos απ 2 = 1 τ C ω  1 + τ α τ C 1 ω1−α sin απ 2  ×  1 −2 τ α τ C 1 ω1−α sin απ 2 −τ 2 α τ 2 C 1 ω2(1−α) −τ α τ 2 C 1 ω2−α cos απ 2  ∼ 1 τ C ω  1 −τ α τ C 1 ω1−α sin απ 2  ❛s ω →∞, ✉s✐♥❣t❤❡s❡r✐❡s ❡①♣❛♥s✐♦♥✭✼✾✮✉♣t♦t❤❡❧✐♥❡❛r t❡r♠s ❛♥❞❜②♥❡❣❧❡❝t✐♥❣t❤❡❤✐❣❤❡r ♦r❞❡r t❡r♠s✱②✐❡❧❞✐♥❣ t❤❡❛s②♠♣t♦t✐❝❡①♣❛♥s✐♦♥✭✺✾✮✳ ❚❤❡❛s②♠♣t♦t✐❝s ✭✻✶✮♦❢t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥♠♦❞✉❧✉s ˆg(2) R dB ✐s ❡❛s✐❧②♦❜t❛✐♥❡❞❢r♦♠✭✺✹✮❜②r❡t❛✐♥✐♥❣ t✇♦t❡r♠s ❝♦♥t❛✐♥✐♥❣t❤❡❧❛r❣❡st ♣♦✇❡rs ❢♦r ❧♦✇❢r❡q✉❡♥❝✐❡s ❛♥❞s♠❛❧❧❡st ♣♦✇❡rs ❢♦r ❤✐❣❤❢r❡q✉❡♥❝✐❡s✱✇❤✐❧❡ t❤❡tr❛♥s❢❡r ❢✉♥❝t✐♦♥❛r❣✉♠❡♥t arg ˆg(2) R , ❣✐✈❡♥❜②✭✺✺✮✱tr❛♥s❢♦r♠s ✐♥t♦t❤❡❛s②♠♣t♦t✐❝❡①♣❛♥s✐♦♥s ✭✻✷✮ ❜②♥❡❣❧❡❝t✐♥❣t❤❡❛♣♣r♦♣r✐❛t❡t❡r♠s ✐♥t❤❡❞❡♥♦♠✐♥❛t♦r ♦❢tan arg ˆg(2) R ✿τ Cτ µω1+µ ✐♥t❤❡❝❛s❡♦❢❧♦✇❛♥❞ τ C sin µπ 2 ✐♥t❤❡❝❛s❡♦❢❤✐❣❤❢r❡q✉❡♥❝✐❡s✳ ❉✐s❝❧♦s✉r❡♦❢♣♦t❡♥t✐❛❧❝♦♥✢✐❝ts ♦❢✐♥t❡r❡st ✲❡t❤✐❝❛❧❛♥❞✜♥❛♥❝✐❛❧ ❋✉♥❞✐♥❣✿❚❤✐s ✇♦r❦✐s s✉♣♣♦rt❡❞❜②t❤❡❙❡r❜✐❛♥▼✐♥✐str②♦❢❙❝✐❡♥❝❡✱❊❞✉❝❛t✐♦♥❛♥❞❚❡❝❤♥♦❧♦❣✐❝❛❧❉❡✲ ✈❡❧♦♣♠❡♥t ✉♥❞❡r ❣r❛♥ts ✹✺✶✲✵✸✲✻✽✴✷✵✷✵✲✶✹✭❙▼❈✮✱❚❘✸✷✵✺✺✭❑❍✮✱❛♥❞✹✺✶✲✵✸✲✾✴✷✵✷✶✲✶✹✴✷✵✵✶✷✺✭❉❩✮✳ ❈♦♥✢✐❝t ♦❢✐♥t❡r❡st✿❚❤❡❛✉t❤♦rs ❞❡❝❧❛r❡t❤❛t t❤❡②❤❛✈❡♥♦❝♦♥✢✐❝t ♦❢✐♥t❡r❡st✳ ❬✸❪❆✳❆❧❧❛❣✉✐✱❚✳❏✳❋r❡❡❜♦r♥✱❆✳❙✳❊❧✇❛❦✐❧✱▼✳❊✳❋♦✉❞❛✱❇✳❏✳▼❛✉♥❞②✱❆✳●✳❘❛❞✇❛♥✱❩✳❙❛✐❞✱ ❛♥❞▼✳❆✳❆❜❞❡❧❦❛r❡❡♠❛✳❘❡✈✐❡✇♦❢❢r❛❝t✐♦♥❛❧✲♦r❞❡r ❡❧❡❝tr✐❝❛❧❝❤❛r❛❝t❡r✐③❛t✐♦♥♦❢s✉♣❡r❝❛♣❛❝✐t♦rs✳ ❏♦✉r♥❛❧♦❢P♦✇❡r ❙♦✉r❝❡s✱✹✵✵✿✹✺✼✕✹✻✼✱✷✵✶✽✳ ❘❡❢❡r❡♥❝❡s ❬✶❪❏✳❆❜❛t❡❛♥❞P✳P✳❱❛❧❦ó✳▼✉❧t✐✲♣r❡❝✐s✐♦♥▲❛♣❧❛❝❡tr❛♥s❢♦r♠✐♥✈❡rs✐♦♥✳■♥t❡r♥❛t✐♦♥❛❧❏♦✉r♥❛❧❢♦r ◆✉♠❡r✐❝❛❧▼❡t❤♦❞s ✐♥❊♥❣✐♥❡❡r✐♥❣✱✻✵✿✾✼✾✕✾✾✸✱✷✵✵✹✳ ❬✷❪❆✳❆❧❧❛❣✉✐✱❆✳❙✳❊❧✇❛❦✐❧✱▼✳❊✳❋♦✉❞❛✱❛♥❞❆✳●✳❘❛❞✇❛♥✳❈❛♣❛❝✐t✐✈❡❜❡❤❛✈✐♦r ❛♥❞st♦r❡❞❡♥❡r❣② ✐♥s✉♣❡r❝❛♣❛❝✐t♦rs ❛t ♣♦✇❡r ❧✐♥❡❢r❡q✉❡♥❝✐❡s✳❏♦✉r♥❛❧♦❢P♦✇❡r ❙♦✉r❝❡s✱✸✾✵✿✶✹✷✕✶✹✼✱✷✵✶✽✳ ❬✸❪❆✳❆❧❧❛❣✉✐✱❚✳❏✳❋r❡❡❜♦r♥✱❆✳❙✳❊❧✇❛❦✐❧✱▼✳❊✳❋♦✉❞❛✱❇✳❏✳▼❛✉♥❞②✱❆✳●✳❘❛❞✇❛♥✱❩✳❙❛✐❞✱ ❛♥❞▼✳❆✳❆❜❞❡❧❦❛r❡❡♠❛✳❘❡✈✐❡✇♦❢❢r❛❝t✐♦♥❛❧✲♦r❞❡r ❡❧❡❝tr✐❝❛❧❝❤❛r❛❝t❡r✐③❛t✐♦♥♦❢s✉♣❡r❝❛♣❛❝✐t♦rs✳ ❏♦✉r♥❛❧♦❢P♦✇❡r ❙♦✉r❝❡s✱✹✵✵✿✹✺✼✕✹✻✼✱✷✵✶✽✳ ✸✶ ❬✹❪❆✳❆❧❧❛❣✉✐✱❉✳❩❤❛♥❣✱❛♥❞❆✳❙✳❊❧✇❛❦✐❧✳❙❤♦rt✲t❡r♠♠❡♠♦r②✐♥❡❧❡❝tr✐❝❞♦✉❜❧❡✲❧❛②❡r ❝❛♣❛❝✐t♦rs✳ ❆♣♣❧✐❡❞P❤②s✐❝s ▲❡tt❡rs✱✶✶✸✿✷✺✸✾✵✶✕✶✕✺✱✷✵✶✽✳ ❬✺❪▼✳❈✳❇♦➨❦♦✈✐➣✱❚✳❇✳➆❡❦❛r❛✱❇✳▲✉t♦✈❛❝✱▼✳❉❛❦♦✈✐➣✱P✳❉✳▼❛♥❞✐➣✱❛♥❞▼✳P✳▲❛③❛r❡✈✐➣✳❆♥❛❧②s✐s ♦❢ ❡❧❡❝tr✐❝❛❧❝✐r❝✉✐ts ✐♥❝❧✉❞✐♥❣❢r❛❝t✐♦♥❛❧♦r❞❡r ❡❧❡♠❡♥ts✳■♥✻t❤▼❡❞✐t❡rr❛♥❡❛♥❈♦♥❢❡r❡♥❝❡♦♥❊♠❜❡❞❞❡❞ ❈♦♠♣✉t✐♥❣✭▼❊❈❖✮✱❇❛r✱▼♦♥t❡♥❡❣r♦✱✷✵✶✼✳ ❬✻❪❳✳❈❤❡♥✱❨✳❈❤❡♥✱❇✳❩❤❛♥❣✱❛♥❞❉✳◗✐✉✳❆♠♦❞❡❧✐♥❣❛♥❞❛♥❛❧②s✐s ♠❡t❤♦❞❢♦r ❢r❛❝t✐♦♥❛❧✲♦r❞❡r ❉❈✲❉❈❝♦♥✈❡rt❡rs✳■❊❊❊❚r❛♥s❛❝t✐♦♥s ♦♥P♦✇❡r ❊❧❡❝tr♦♥✐❝s✱✸✷✿✼✵✸✹✕✼✵✹✹✱✷✵✶✼✳ ❬✼❪❆✳❉③✐❡❧✐➠s❦✐✱●✳❙❛r✇❛s✱❛♥❞❉✳❙✐❡r♦❝✐✉❦✳❈♦♠♣❛r✐s♦♥❛♥❞✈❛❧✐❞❛t✐♦♥♦❢✐♥t❡❣❡r ❛♥❞❢r❛❝t✐♦♥❛❧ ♦r❞❡r ✉❧tr❛❝❛♣❛❝✐t♦r ♠♦❞❡❧s✳❆❞✈❛♥❝❡s ✐♥❉✐✛❡r❡♥❝❡❊q✉❛t✐♦♥s✱✷✵✶✶✿✶✶✿✶✕✶✺✱✷✵✶✶✳ ❬✽❪❖✳❊❧✇②✱▲✳❆✳❙❛✐❞✱❆✳❍✳▼❛❞✐❛♥✱❛♥❞❆✳●✳❘❛❞✇❛♥✳❆❧❧♣♦ss✐❜❧❡t♦♣♦❧♦❣✐❡s ♦❢t❤❡❢r❛❝t✐♦♥❛❧✲ ♦r❞❡r ❲✐❡♥♦s❝✐❧❧❛t♦r ❢❛♠✐❧②✉s✐♥❣❞✐✛❡r❡♥t ❛♣♣r♦①✐♠❛t✐♦♥t❡❝❤♥✐q✉❡s✳❈✐r❝✉✐ts✱❙②st❡♠s✱❛♥❞❙✐❣♥❛❧ Pr♦❝❡ss✐♥❣✱✸✽✿✸✾✸✶✕✸✾✺✶✱✷✵✶✾✳ ❬✾❪▼✳❊✳❋♦✉❞❛✱❆✳❆❧❧❛❣✉✐✱❆✳❙✳❊❧✇❛❦✐❧✱❙✳❉❛s✱❈✳Ps②❝❤❛❧✐♥♦s✱❛♥❞❆✳●✳❘❛❞✇❛♥✳◆♦♥❧✐♥❡❛r ❝❤❛r❣❡✲✈♦❧t❛❣❡r❡❧❛t✐♦♥s❤✐♣✐♥❝♦♥st❛♥t ♣❤❛s❡❡❧❡♠❡♥t✳ ■♥t❡r♥❛t✐♦♥❛❧❏♦✉r♥❛❧♦❢❊❧❡❝tr♦♥✐❝s ❛♥❞ ❈♦♠♠✉♥✐❝❛t✐♦♥s ✭❆❊Ü✮✱✶✶✼✿✶✺✸✶✵✹✕✶✕✹✱✷✵✷✵✳ ❬✶✵❪❘✳●❛rr❛♣♣❛✱❊✳❑❛s❧✐❦✱❛♥❞▼✳P♦♣♦❧✐③✐♦✳ ❊✈❛❧✉❛t✐♦♥♦❢❢r❛❝t✐♦♥❛❧✐♥t❡❣r❛❧s ❛♥❞❞❡r✐✈❛t✐✈❡s ♦❢ ❡❧❡♠❡♥t❛r②❢✉♥❝t✐♦♥s✿❖✈❡r✈✐❡✇❛♥❞t✉t♦r✐❛❧✳▼❛t❤❡♠❛t✐❝s✱✼✿✹✵✼✕✶✕✷✶✱✷✵✶✾✳ ❬✶✶❪❋✳●ó♠❡③✱❏✳❘♦s❛❧❡s✱❛♥❞▼✳●✉í❛✳RLC ❡❧❡❝tr✐❝❛❧❝✐r❝✉✐t ♦❢♥♦♥✲✐♥t❡❣❡r ♦r❞❡r✳❈❡♥tr❛❧❊✉r♦♣❡❛♥ ❏♦✉r♥❛❧♦❢P❤②s✐❝s✱✶✶✿✶✸✻✶✕✶✸✻✺✱✷✵✶✸✳ ❬✶✷❪❘✳●♦r❡♥✢♦❛♥❞❋✳▼❛✐♥❛r❞✐✳ ❋r❛❝t✐♦♥❛❧❈❛❧❝✉❧✉s✿■♥t❡❣r❛❧❛♥❞❉✐✛❡r❡♥t✐❛❧❊q✉❛t✐♦♥s ♦❢❋r❛❝✲ t✐♦♥❛❧❖r❞❡r✳■♥✿❋r❛❝t❛❧s ❛♥❞❋r❛❝t✐♦♥❛❧❈❛❧❝✉❧✉s ✐♥❈♦♥t✐♥✉✉♠▼❡❝❤❛♥✐❝s ✭❡❞s ❆✳❈❛r♣✐♥t❡r✐✱❋✳ ▼❛✐♥❛r❞✐✮✱✈♦❧✉♠❡✸✼✽♦❢❈■❙▼❈♦✉rs❡s ❛♥❞▲❡❝t✉r❡◆♦t❡s✳❙♣r✐♥❣❡r ❱❡r❧❛❣✱❲✐❡♥❛♥❞◆❡✇❨♦r❦✱ ✶✾✾✼✳ ❬✶✸❪▼✳●✉í❛✱❏✳❘♦s❛❧❡s✱❛♥❞❋✳●ó♠❡③✳❆♥❛❧②s✐s ♦♥t❤❡t✐♠❡❛♥❞❢r❡q✉❡♥❝②❞♦♠❛✐♥❢♦r t❤❡RC ❡❧❡❝tr✐❝ ❝✐r❝✉✐t ♦❢❢r❛❝t✐♦♥❛❧♦r❞❡r✳❈❡♥tr❛❧❊✉r♦♣❡❛♥❏♦✉r♥❛❧♦❢P❤②s✐❝s✱✶✶✿✶✸✻✻✕✶✸✼✶✱✷✵✶✸✳ ❬✶✹❪❑✳❍❛➨❦❛✱❉✳❩♦r✐❝❛✱❛♥❞❙✳▼✳❈✈❡t✐➣❛♥✐♥✳❋r❛❝t✐♦♥❛❧RLC ❝✐r❝✉✐t ✐♥tr❛♥s✐❡♥t ❛♥❞st❡❛❞②st❛t❡ r❡❣✐♠❡s✳❈♦♠♠✉♥✐❝❛t✐♦♥s ✐♥◆♦♥❧✐♥❡❛r ❙❝✐❡♥❝❡❛♥❞◆✉♠❡r✐❝❛❧❙✐♠✉❧❛t✐♦♥✱✾✻✿✶✵✺✻✼✵✕✶✕✶✼✱✷✵✷✶✳ ❬✶✺❪❆✳❏❛❦✉❜♦✇s❦❛❛♥❞❏✳❲❛❧❝③❛❦✳❆♥❛❧②s✐s ♦❢t❤❡tr❛♥s✐❡♥t st❛t❡✐♥❛❝✐r❝✉✐t ✇✐t❤s✉♣❡r❝❛♣❛❝✐t♦r✳ P♦③♥❛♥❯♥✐✈❡rs✐t②♦❢❚❡❝❤♥♦❧♦❣②❆❝❛❞❡♠✐❝❏♦✉r♥❛❧s✳❊❧❡❝tr✐❝❛❧❊♥❣✐♥❡❡r✐♥❣✱✽✶✿✼✶✕✼✼✱✷✵✶✺✳ ❬✶✻❪❆✳❏❛❦✉❜♦✇s❦❛❛♥❞❏✳❲❛❧❝③❛❦✳❆♥❛❧②s✐s ♦❢t❤❡tr❛♥s✐❡♥t st❛t❡✐♥❛s❡r✐❡s ❝✐r❝✉✐t ♦❢t❤❡❝❧❛ss RLβCα✳ ❈✐r❝✉✐ts✱❙②st❡♠s✱❛♥❞❙✐❣♥❛❧Pr♦❝❡ss✐♥❣✱✸✺✿✶✽✸✶✕✶✽✺✸✱✷✵✶✻✳ ✸✷ ❬✶✼❪❆✳❏❛❦✉❜♦✇s❦❛✲❈✐s③❡❦❛♥❞❏✳❲❛❧❝③❛❦✳❆♥❛❧②s✐s ♦❢t❤❡tr❛♥s✐❡♥t st❛t❡✐♥❛♣❛r❛❧❧❡❧❝✐r❝✉✐t ♦❢t❤❡ ❝❧❛ss RLβCα✳❆♣♣❧✐❡❞▼❛t❤❡♠❛t✐❝s ❛♥❞❈♦♠♣✉t❛t✐♦♥✱✸✶✾✿✷✽✼✕✸✵✵✱✷✵✶✽✳ ❬✶✽❪■✳❙✳❏❡s✉s ❛♥❞❏✳❆✳❚✳▼❛❝❤❛❞♦✳❉❡✈❡❧♦♣♠❡♥t ♦❢❢r❛❝t✐♦♥❛❧♦r❞❡r ❝❛♣❛❝✐t♦rs ❜❛s❡❞♦♥❡❧❡❝tr♦❧②t❡ ♣r♦❝❡ss❡s✳◆♦♥❧✐♥❡❛r ❉②♥❛♠✐❝s✱✺✻✿✹✺✕✺✺✱✷✵✵✾✳ ❬✶✾❪❨✳❏✐❛♥❣✱❇✳❩❤❛♥❣✱❳✳❙❤✉✱❛♥❞❩✳❲❡✐✳❋r❛❝t✐♦♥❛❧✲♦r❞❡r ❛✉t♦♥♦♠♦✉s ❝✐r❝✉✐ts ✇✐t❤♦r❞❡r ❧❛r❣❡r t❤❛♥ ♦♥❡✳❚♦❛♣♣❡❛r ✐♥✿❏♦✉r♥❛❧♦❢❆❞✈❛♥❝❡❞❘❡s❡❛r❝❤✱✷✵✷✵✳ ❬✷✵❪❆✳❑❛rt❝✐✱❆✳❆❣❛♠❜❛②❡✈✱◆✳❍❡r❡♥❝s❛r✱❛♥❞❑✳◆✳❙❛❧❛♠❛✳❙❡r✐❡s✲✱♣❛r❛❧❧❡❧✲✱❛♥❞✐♥t❡r✲❝♦♥♥❡❝t✐♦♥ ♦❢s♦❧✐❞✲st❛t❡❛r❜✐tr❛r②❢r❛❝t✐♦♥❛❧✲♦r❞❡r ❝❛♣❛❝✐t♦rs✿❚❤❡♦r❡t✐❝❛❧st✉❞②❛♥❞❡①♣❡r✐♠❡♥t❛❧✈❡r✐✜❝❛t✐♦♥✳ ■❊❊❊❆❝❝❡ss✱✻✿✶✵✾✸✸✕✶✵✾✹✸✱✷✵✶✽✳ ❬✷✶❪▼✳❙✳❑r✐s❤♥❛✱❙✳❉❛s✱❑✳❇✐s✇❛s✱❛♥❞❇✳●♦s✇❛♠✐✳❋❛❜r✐❝❛t✐♦♥♦❢❛❢r❛❝t✐♦♥❛❧♦r❞❡r ❝❛♣❛❝✐t♦r ✇✐t❤ ❞❡s✐r❡❞s♣❡❝✐✜❝❛t✐♦♥s✿❆st✉❞②♦♥♣r♦❝❡ss ✐❞❡♥t✐✜❝❛t✐♦♥❛♥❞❝❤❛r❛❝t❡r✐③❛t✐♦♥✳■❊❊❊❚r❛♥s❛❝t✐♦♥s ♦♥❊❧❡❝tr♦♥❉❡✈✐❝❡s✱✺✽✿✹✵✻✼✕✹✵✼✸✱✷✵✶✶✳ ❬✷✷❪❏✳❆✳❚✳▼❛❝❤❛❞♦❛♥❞❆✳▼✳❙✳❋✳●❛❧❤❛♥♦✳❋r❛❝t✐♦♥❛❧♦r❞❡r ✐♥❞✉❝t✐✈❡♣❤❡♥♦♠❡♥❛❜❛s❡❞♦♥t❤❡ s❦✐♥❡✛❡❝t✳◆♦♥❧✐♥❡❛r ❉②♥❛♠✐❝s✱✻✽✿✶✵✼✕✶✶✺✱✷✵✶✷✳ ❬✷✸❪❱✳▼❛rt②♥②✉❦❛♥❞▼✳❖rt✐❣✉❡✐r❛✳❋r❛❝t✐♦♥❛❧♠♦❞❡❧♦❢❛♥❡❧❡❝tr♦❝❤❡♠✐❝❛❧❝❛♣❛❝✐t♦r✳❙✐❣♥❛❧Pr♦❝❡ss✲ ✐♥❣✱✶✵✼✿✸✺✺✕✸✻✵✱✷✵✶✺✳ ❬✷✹❪❉✳▼♦♥❞❛❧❛♥❞❑✳❇✐s✇❛s✳P❛❝❦❛❣✐♥❣♦❢s✐♥❣❧❡✲❝♦♠♣♦♥❡♥t ❢r❛❝t✐♦♥❛❧♦r❞❡r ❡❧❡♠❡♥t✳■❊❊❊❚r❛♥s❛❝✲ t✐♦♥s ♦♥❉❡✈✐❝❡❛♥❞▼❛t❡r✐❛❧s ❘❡❧✐❛❜✐❧✐t②✱✶✸✿✼✸✕✽✵✱✷✵✶✸✳ ❬✷✺❪▼✳❆✳▼♦r❡❧❡s ❛♥❞❘✳▲❛✐♥❡③✳ ▼❛t❤❡♠❛t✐❝❛❧♠♦❞❡❧❧✐♥❣♦❢❢r❛❝t✐♦♥❛❧♦r❞❡r ❝✐r❝✉✐t ❡❧❡♠❡♥ts ❛♥❞ ❜✐♦✐♠♣❡❞❛♥❝❡❛♣♣❧✐❝❛t✐♦♥s✳❈♦♠♠✉♥✐❝❛t✐♦♥s ✐♥◆♦♥❧✐♥❡❛r ❙❝✐❡♥❝❡❛♥❞◆✉♠❡r✐❝❛❧❙✐♠✉❧❛t✐♦♥✱✹✻✿✽✶✕ ✽✽✱✷✵✶✼✳ ❬✷✻❪❘✳Pr❛s❛❞✱❑✳❑♦t❤❛r✐✱❛♥❞❯✳▼❡❤t❛✳❋❧❡①✐❜❧❡❢r❛❝t✐♦♥❛❧s✉♣❡r❝❛♣❛❝✐t♦r ♠♦❞❡❧❛♥❛❧②③❡❞✐♥t✐♠❡ ❞♦♠❛✐♥✳■❊❊❊❆❝❝❡ss✱✼✿✶✷✷✻✷✻✕✶✷✷✻✸✸✱✷✵✶✾✳ ❬✷✼❪❘✳Pr❛s❛❞✱❯✳▼❡❤t❛✱❛♥❞❑✳❑♦t❤❛r✐✳❱❛r✐♦✉s ❛♥❛❧②t✐❝❛❧♠♦❞❡❧s ❢♦r s✉♣❡r❝❛♣❛❝✐t♦rs✿❛♠❛t❤❡♠❛t✐❝❛❧ st✉❞②✳❘❡s♦✉r❝❡✲❊✣❝✐❡♥t ❚❡❝❤♥♦❧♦❣✐❡s✱✶✿✶✕✶✺✱✷✵✷✵✳ ❬✷✽❪❏✳❏✳◗✉✐♥t❛♥❛✱❆✳❘❛♠♦s✱❛♥❞■✳◆✉❡③✳▼♦❞❡❧✐♥❣♦❢❛♥❊❉▲❈✇✐t❤❢r❛❝t✐♦♥❛❧tr❛♥s❢❡r ❢✉♥❝t✐♦♥s ✉s✐♥❣▼✐tt❛❣✲▲❡✤❡r ❡q✉❛t✐♦♥s✳▼❛t❤❡♠❛t✐❝❛❧Pr♦❜❧❡♠s ✐♥❊♥❣✐♥❡❡r✐♥❣✱✷✵✶✸✿✽✵✼✵✸✹✕✶✕✼✱✷✵✶✸✳ ❬✷✾❪❆✳●✳❘❛❞✇❛♥✳❘❡s♦♥❛♥❝❡❛♥❞q✉❛❧✐t②❢❛❝t♦r ♦❢t❤❡RLαCα ❢r❛❝t✐♦♥❛❧❝✐r❝✉✐t✳■❊❊❊❏♦✉r♥❛❧♦♥ ❊♠❡r❣✐♥❣❛♥❞❙❡❧❡❝t❡❞❚♦♣✐❝s ✐♥❈✐r❝✉✐ts ❛♥❞❙②st❡♠s✱✸✿✸✼✼✕✸✽✺✱✷✵✶✸✳ ❬✸✵❪❆✳●✳❘❛❞✇❛♥❛♥❞▼✳❊✳❋♦✉❞❛✳❖♣t✐♠✐③❛t✐♦♥♦❢❢r❛❝t✐♦♥❛❧✲♦r❞❡r RLC ✜❧t❡rs✳❈✐r❝✉✐ts✱❙②st❡♠s ❛♥❞❙✐❣♥❛❧Pr♦❝❡ss✐♥❣✱✸✷✿✷✵✾✼✕✷✶✶✽✱✷✵✶✸✳ ✸✸ ❬✸✶❪❆✳●✳❘❛❞✇❛♥❛♥❞❑✳◆✳❙❛❧❛♠❛✳P❛ss✐✈❡❛♥❞❛❝t✐✈❡❡❧❡♠❡♥ts ✉s✐♥❣❢r❛❝t✐♦♥❛❧LβCα ❝✐r❝✉✐t✳■❊❊❊ ❚r❛♥s❛❝t✐♦♥s ♦♥❈✐r❝✉✐ts ❛♥❞❙②st❡♠s ■✿❘❡❣✉❧❛r P❛♣❡rs✱✺✽✿✷✸✽✽✕✷✸✾✼✱✷✵✶✶✳ ❬✸✷❪❆✳●✳❘❛❞✇❛♥❛♥❞❑✳◆✳❙❛❧❛♠❛✳❋r❛❝t✐♦♥❛❧✲♦r❞❡r RC ❛♥❞RL ❝✐r❝✉✐ts✳❈✐r❝✉✐ts✱❙②st❡♠s ❛♥❞ ❙✐❣♥❛❧Pr♦❝❡ss✐♥❣✱✸✶✿✶✾✵✶✕✶✾✶✺✱✷✵✶✷✳ ❬✸✸❪❆✳●✳❘❛❞✇❛♥✱❆✳▼✳❙♦❧✐♠❛♥✱❛♥❞❆✳❙✳❊❧✇❛❦✐❧✳❉❡s✐❣♥❡q✉❛t✐♦♥s ❢♦r ❢r❛❝t✐♦♥❛❧✲♦r❞❡r s✐♥✉s♦✐❞❛❧♦s✲ ❝✐❧❧❛t♦rs✿❋♦✉r ♣r❛❝t✐❝❛❧❝✐r❝✉✐t ❡①❛♠♣❧❡s✳■♥t❡r♥❛t✐♦♥❛❧❏♦✉r♥❛❧♦❢❈✐r❝✉✐t ❚❤❡♦r②❛♥❞❆♣♣❧✐❝❛t✐♦♥s✱ ✸✻✿✹✼✸✕✹✾✷✱✷✵✵✽✳ ❬✸✹❪▼✳❙✳❙❛r❛❢r❛③❛♥❞▼✳❙✳❚❛✈❛③♦❡✐✳❘❡❛❧✐③❛❜✐❧✐t②♦❢❢r❛❝t✐♦♥❛❧✲♦r❞❡r ✐♠♣❡❞❛♥❝❡s ❜②♣❛ss✐✈❡❡❧❡❝tr✐❝❛❧ ♥❡t✇♦r❦s ❝♦♠♣♦s❡❞♦❢❛❢r❛❝t✐♦♥❛❧❝❛♣❛❝✐t♦r ❛♥❞RLC ❝♦♠♣♦♥❡♥ts✳■❊❊❊❚r❛♥s❛❝t✐♦♥s ♦♥❈✐r❝✉✐ts ❛♥❞❙②st❡♠s ■✿❘❡❣✉❧❛r P❛♣❡rs✱✻✷✿✷✽✷✾✕✷✽✸✺✱✷✵✶✺✳ ❬✸✺❪■✳❙❝❤ä❢❡r ❛♥❞❑✳❑rü❣❡r✳▼♦❞❡❧❧✐♥❣♦❢❝♦✐❧s ✉s✐♥❣❢r❛❝t✐♦♥❛❧❞❡r✐✈❛t✐✈❡s✳❏♦✉r♥❛❧♦❢▼❛❣♥❡t✐s♠❛♥❞ ▼❛❣♥❡t✐❝▼❛t❡r✐❛❧s✱✸✵✼✿✾✶✕✾✽✱✷✵✵✻✳ ❬✸✻❪❩✳▼✳❙❤❛❤✱▼✳❨✳❑❛t❤❥♦♦✱❋✳❆✳❑❤❛♥❞❛②✱❑✳❇✐s✇❛s✱❛♥❞❈✳Ps②❝❤❛❧✐♥♦s✳❆s✉r✈❡②♦❢s✐♥❣❧❡❛♥❞ ♠✉❧t✐✲❝♦♠♣♦♥❡♥t ❢r❛❝t✐♦♥❛❧✲♦r❞❡r ❡❧❡♠❡♥ts ✭❋❖❊s✮❛♥❞t❤❡✐r ❛♣♣❧✐❝❛t✐♦♥s✳▼✐❝r♦❡❧❡❝tr♦♥✐❝s ❏♦✉r♥❛❧✱ ✽✹✿✾✕✷✺✱✷✵✶✾✳ ❬✸✼❪▼✳❙♦✇❛✳❆s✉❜✐♥t❡r✈❛❧✲❜❛s❡❞♠❡t❤♦❞❢♦r ❝✐r❝✉✐ts ✇✐t❤❢r❛❝t✐♦♥❛❧♦r❞❡r ❡❧❡♠❡♥ts✳❇✉❧❧❡t✐♥♦❢t❤❡ P♦❧✐s❤❆❝❛❞❡♠②♦❢❙❝✐❡♥❝❡s ❚❡❝❤♥✐❝❛❧❙❝✐❡♥❝❡s✱✻✷✿✹✹✾✕✹✺✹✱✷✵✶✹✳ ❬✸✽❪▼✳❙♦✇❛✳✑❣❝❞❆❧♣❤❛✑✕❛s❡♠✐✲❛♥❛❧②t✐❝❛❧♠❡t❤♦❞❢♦r s♦❧✈✐♥❣❢r❛❝t✐♦♥❛❧st❛t❡❡q✉❛t✐♦♥s✳P♦③♥❛♥ ❯♥✐✈❡rs✐t②♦❢❚❡❝❤♥♦❧♦❣②❆❝❛❞❡♠✐❝❏♦✉r♥❛❧s✳❊❧❡❝tr✐❝❛❧❊♥❣✐♥❡❡r✐♥❣✱✾✻✿✷✸✶✕✷✹✷✱✷✵✶✽✳ ❬✸✾❪❚✳P✳❙t❡❢❛➠s❦✐❛♥❞❏✳●✉❧❣♦✇s❦✐✳ ❙✐❣♥❛❧♣r♦♣❛❣❛t✐♦♥✐♥❡❧❡❝tr♦♠❛❣♥❡t✐❝♠❡❞✐❛❞❡s❝r✐❜❡❞ ❜②❢r❛❝t✐♦♥❛❧✲♦r❞❡r ♠♦❞❡❧s✳ ❈♦♠♠✉♥✐❝❛t✐♦♥s ✐♥◆♦♥❧✐♥❡❛r ❙❝✐❡♥❝❡❛♥❞◆✉♠❡r✐❝❛❧❙✐♠✉❧❛t✐♦♥✱ ✽✷✿✶✵✺✵✷✾✕✶✕✶✻✱✷✵✷✵✳ ❬✹✵❪❘✳❙üÿ❡✱❆✳❉♦♠❤❛r❞t✱❛♥❞▼✳❘❡✐♥❤❛r❞✳❈❛❧❝✉❧❛t✐♦♥♦❢❡❧❡❝tr✐❝❛❧❝✐r❝✉✐ts ✇✐t❤❢r❛❝t✐♦♥❛❧❝❤❛r❛❝✲ t❡r✐st✐❝s ♦❢❝♦♥str✉❝t✐♦♥❡❧❡♠❡♥ts✳❋♦rs❝❤■♥❣❡♥✐❡✉r✇❡s✱✻✾✿✷✸✵✕✷✸✺✱✷✵✵✺✳ ❬✹✶❪❱✳❱✳❯❝❤❛✐❦✐♥✱❆✳❙✳❆♠❜r♦③❡✈✐❝❤✱❘✳❚✳❙✐❜❛t♦✈✱❙✳❆✳❆♠❜r♦③❡✈✐❝❤✱❛♥❞❊✳❱✳▼♦r♦③♦✈❛✳▼❡♠✲ ♦r②❛♥❞♥♦♥❧✐♥❡❛r tr❛♥s♣♦rt ❡✛❡❝ts ✐♥❝❤❛r❣✐♥❣✲❞✐s❝❤❛r❣✐♥❣♦❢❛s✉♣❡r❝❛♣❛❝✐t♦r✳❚❡❝❤♥✐❝❛❧P❤②s✐❝s✱ ✻✶✿✷✺✵✕✷✺✾✱✷✵✶✻✳ ❬✹✷❪❏✳❲❛❧❝③❛❦❛♥❞❆✳❏❛❦✉❜♦✇s❦❛✳ ❘❡s♦♥❛♥❝❡✐♥s❡r✐❡s ❢r❛❝t✐♦♥❛❧♦r❞❡r RLβCα ❝✐r❝✉✐t✳ Pr③❡❣❧→❞ ❊❧❡❦tr♦t❡❝❤♥✐❝③♥②✱✾✵✿✷✶✵✕✷✶✸✱✷✵✶✹✳ ❬✹✸❪▲✳❩❤♦✉✱❩✳❚❛♥✱❛♥❞◗✳❩❤❛♥❣✳❆❢r❛❝t✐♦♥❛❧✲♦r❞❡r ♠✉❧t✐❢✉♥❝t✐♦♥❛❧n✲st❡♣❤♦♥❡②❝♦♠❜RLC ❝✐r❝✉✐t ♥❡t✇♦r❦✳❋r♦♥t✐❡rs ♦❢■♥❢♦r♠❛t✐♦♥❚❡❝❤♥♦❧♦❣②❛♥❞❊❧❡❝tr♦♥✐❝❊♥❣✐♥❡❡r✐♥❣✱✶✽✿✶✶✽✻✕✶✶✾✻✱✷✵✶✼✳ ✸✹ Figures g Figure 1 "Please see the Manuscript PDF ¦le for the complete ¦gure caption". "Please see the Manuscript PDF ¦le for the complete ¦gure caption". Figure 2 "Please see the Manuscript PDF ¦le for the complete ¦gure caption". "Please see the Manuscript PDF ¦le for the complete ¦gure caption". Figure 3 "Please see the Manuscript PDF ¦le for the complete ¦gure caption". Figure 3 "Please see the Manuscript PDF ¦le for the complete ¦gure caption". "Please see the Manuscript PDF ¦le for the complete ¦gure caption". "Please see the Manuscript PDF ¦le for the complete ¦gure caption". ure 4 ease see the Manuscript PDF ¦le for the complete ¦gure caption". Figure 4 "Please see the Manuscript PDF ¦le for the complete ¦gure caption". Figure 4 "Please see the Manuscript PDF ¦le for the complete ¦gure caption". "Please see the Manuscript PDF ¦le for the complete ¦gure caption". Figure 5 "Please see the Manuscript PDF ¦le for the complete ¦gure caption". Figure 6 "Please see the Manuscript PDF ¦le for the complete ¦gure caption". Figure 7 "Please see the Manuscript PDF ¦le for the complete ¦gure caption". Figure 7 "Please see the Manuscript PDF ¦le for the complete ¦gure caption". "Please see the Manuscript PDF ¦le for the complete ¦gure caption". "Please see the Manuscript PDF ¦le for the complete ¦gure caption". Figure 8 "Please see the Manuscript PDF ¦le for the complete ¦gure caption". Figure 8 "Please see the Manuscript PDF ¦le for the complete ¦gure caption". "Please see the Manuscript PDF ¦le for the complete ¦gure caption".
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https://www.nature.com/articles/s41598-018-19835-8.pdf
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Hypomagnesemia and clinical benefits of anti-EGFR monoclonal antibodies in wild-type KRAS metastatic colorectal cancer: a systematic review and meta-analysis
Scientific reports
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Hypomagnesemia and clinical benefits of anti-EGFR monoclonal antibodies in wild-type KRAS metastatic colorectal cancer: a systematic review and meta- analysis Meng-Chiao Hsieh   1,2,3, Chun-Feng Wu4, Chun-Wei Chen5, Chung-Sheng Shi6, W Shih H 1 7 & F Ch K 2 4 Received: 31 May 2017 Accepted: 9 January 2018 Published: xx xx xxxx Received: 31 May 2017 Accepted: 9 January 2018 Published: xx xx xxxx Meng-Chiao Hsieh   1,2,3, Chun-Feng Wu4, Chun-Wei Chen5, Chung-Sheng Shi6, Wen-Shih Huang1,7 & Feng-Che Kuan2,4 Hypomagnesemia is a recognized side-effect of cetuximab- or panitumumab-based chemotherapy for metastatic colorectal cancer (mCRC). The clinical relevance of hypomagnesemia is under debate. Thus, a systematic review and meta-analysis of retrospective studies and randomized clinical trials (RCTs) comparing hypomagnesemia with normal magnesium levels in wild-type KRAS mCRC was performed. One RCT, two retrospective studies, and two American Society of Clinical Oncology (ASCO) and European Society for Medical Oncology (ESMO) conference presentations from phase III RCTs involving 1723 patients were included in this study. Patients with hypomagnesemia demonstrated better progression-free survival (PFS) (Hazard ratio [HR]: 0.64; 95% confidence interval [CI]: 0.47–0.88), overall survival (OS) (HR: 0.72; 95% CI: 0.53–0.92), and objective response rate (ORR) (Risk ratio [RR]: 1.81; 95% confidence interval [CI]: 1.30–2.52). By subgroup analysis, frontline, later lines or combination therapy with hypomagnesemia were associated with PFS benefits (HR: 0.78; 95% CI: 0.62–0.98; HR: 0.60; 95% CI: 0.40–0.90; HR: 0.62; 95% CI: 0.41–0.94, respectively). In patients with wild-type KRAS mCRC, hypomagnesemia is associated with better clinical benefits of PFS, OS and ORR when treated with cetuximab- or panitumumab-based chemotherapy. Future clinical trials should corroborate its predictive role. Personalized medicine has an important role in the treatment of colorectal cancer, which accounts for 8.5% of all cancer-related deaths worldwide1. The activation of the epidermal growth factor receptor (EGFR) and down- stream signaling of the Ras-Raf-MAP, PI3K, and Akt pathways are associated with the promotion of tumor growth, invasion, metastases, and the inhibition of apoptosis2–4. Cetuximab and panitumumab are monoclo- nal antibodies (MoA) that target the extracellular domain of EGFR and provide survival benefits in metastatic colorectal cancer (mCRC)5–8. The mutated Ras gene, mostly seen in the Kirsten rat sarcoma viral oncogene (KRAS) with point mutations in codon 12 and 13 of exon 2, leads to constitutive activation and confer resistance against these biologic agents9,10. Although KRAS mutation is a negative predictive marker, the wild-type KRAS 1Division of Colon and Rectal Surgery, Department of Surgery, Chang Gung Memorial Hospital Chiayi Branch, Chiayi, Taiwan. 2Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan. 3Centre for Evidence-Based Medicine, Chang Gung Memorial Hospital Chiayi Branch, Chiayi, Taiwan. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 31 May 2017 Accepted: 9 January 2018 Published: xx xx xxxx Methods D Data source and search strategy. This study involved a comprehensive search of all published eligible stud- ies and conference presentations that reported the PFS, OS, and ORR for wide-type KRAS mCRC patients with hypomagnesemia after treatment with cetuximab- or panitumumab-based chemotherapy. The searched included MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials (CENTRAL) for relevant trials from inception to 01 April 2017. The search strategy were “(hypomagnesemia OR magnesium reduction) AND (cetux- imab OR panitumumab OR EGFR) AND (survival OR outcome OR efficacy) AND (colon cancer OR rectal can- cer OR colorectal cancer)” without restrictions for language and gender. To identify unpublished studies, the US National Institutes of Health trials register (http://clinicaltrial.gov) and conference abstracts from proceeding of the American Society of Clinical Oncology (ASCO) and the European Society for Medical Oncology (ESMO) were also searched. The ethical approval was waived because all analyses were based on previous published studies. Inclusion and exclusion criteria. Studies or conference presentations that investigated wild-type KRAS mCRC treated with cetuximab- or panitumumab-based chemotherapy were included. Those with available data for the events and incidences of hypomagnesemia and those with available Hazard ratio (HR), 95% confidence interval (CI), or Kaplan-Meier survival analysis that compared outcomes in the hypomagnesemia and normal magnesium level groups were also included. The references from included trials were also checked to identify relevant trials. Studies that did not meet these criteria were excluded. Data extraction and quality assessment. Two authors independently extracted the available data from the included trials using a standardized data collection form: first author, year of publication or conference pres- entation, study design, intervention type, study population per group, clinical setting (type of chemotherapy, definition of hypomagnesemia), and outcome data (PFS, OS, and ORR). i yp g ), ( , , ) Hypomagnesemia was defined according to the CTCAE (Common Terminology Criteria for Adverse Events) or magnesium reduction level27. Grade 1 hypomagnesemia was from the lower limit of normal to 1.2 mg/dL, Grade 2 was <1.2 to 0.9 mg/dL, Grade 3 was <0.9 to 0.7 mg/dL, and Grade 4 was <0.7 mg/dL. Those with grades 1–4 hypomagnesemia or > 50% magnesium reduction was arbitrarily classified as the hypomagnesemia group. Methods D Observed differences in ORR that could also predict clinically relevant therapeutic benefits and evaluations were done in most trails, according to the RECIST (Response Evaluation Criteria in Solid Tumours) guidelines28.h g ( p ) g A third author resolved discrepancies in opinion. The quality of methodology was assessed using the Cochrane Collaboration tool for randomized controlled trials (RCTs) and methodological index for non-randomized stud- ies (MINORS)29,30. Hypomagnesemia and clinical benefits of anti-EGFR monoclonal antibodies in wild-type KRAS metastatic colorectal cancer: a systematic review and meta- analysis Meng-Chiao Hsieh   1,2,3, Chun-Feng Wu4, Chun-Wei Chen5, Chung-Sheng Shi6, W Shih H 1 7 & F Ch K 2 4 4Department of Hematology and Oncology, Chang Gung Memorial Hospital Chiayi Branch, Chiayi, Taiwan. 5Department of Gastroenterology and Hepatology, Chang-Gung Memorial Hospital, Taoyuan, Taiwan. 6Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang-Gung University, Taoyuan, Taiwan. 7College of Medicine, Chang Gung University, Taoyuan, Taiwan. Wen-Shih Huang and Feng-Che Kuan contributed equally to this work. Correspondence and requests for materials should be addressed to W.-S.H. (email: wen1204@adm.cgmh.org. tw) or F.-C.K. (email: mnpq8893@gmail.com) SCIEnTIFIC Reports | (2018) 8:2047 | DOI:10.1038/s41598-018-19835-8 1 www.nature.com/scientificreports/ does not always guarantee clinical benefits with anti-EGFR MoAs4,11–13. Besides, there are proposed positive predictive markers in patients with wild-type KRAS, such as EGFR amplification and overexpression of EGFR ligands14–16. Through both negative and positive selection that makes it possible to define the population that benefits more from these anti-EGFR MoAs. i Hypomagnesemia is an appreciated side effect of cetuximab and panitumumab treatment, as reported in ran- domized trials and meta-analysis17–20. Though magnesium is essential to organism, how hypomagnesemia relates to efficacy of these anti-EGFR MoAs and what effect magnesium levels have on cancer progression is an uncertain area of research with contradicting results21–26. Whether hypomagnesemia is associated with superior or inferior outcomes remains equivocal in these retrospective or prospective clinical trials. To elucidate the role and clinical benefits of hypomagnesemia in wild-type KRAS mCRC, a systematic review and meta-analysis of current clinical trials was performed. SCIEnTIFIC Reports | (2018) 8:2047 | DOI:10.1038/s41598-018-19835-8 Statistical analysis y The Review Manager 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration, 2014) was used for meta-analysis. The HRs and 95% CIs were extracted for all included trials and subgroups. Data from independ- ent assessment of PFS and OS were pooled together for time-to-event outcomes analysis when both types of reviews were available. A random-effects (RE) model was used to calculate pooled HRs, 95% CIs, and p values31. A generic inverse variance (IV) meta-analysis was analyzed using log HRs and standard errors (SEs), estimated as described in the Cochrane handbook for systemic reviews of intervention, if the HRs is quoted in a trial together with a 95% CIs or p values30. If the HRs, 95% CIs, and p values were not reported, the HRs and SEs were estimated from Kaplan-Meier survival curve, patient number, censored data, or number at risk table reported32. For ORR analysis, risk ratio (RR) and 95% CI for binomial data and dichotomous meta-analysis were calculated with Mantel-Haenszel test under the random-effect model. A two-sided p < 0.05 was set as statistically significant. f p y gi For heterogeneity tests, the chi-square (chi2) and I2 inconsistency statistics were used33,34. A p < 0.10 was con- sidered significant heterogeneity. Moreover, I2 values of 0–24.9%, 25–49.9%, 50–74%, and 75–100% were consid- ered as none, low, moderate, and high heterogeneity, respectively33,34. Data availability. All data generated or analyzed during this study are included in these published article or unpublished meeting abstracts (ref 22–26 with DOI or web link below). 22. https://doi.org/10.1016/S0959-8049(11)71740–3 22. https://doi.org/10.1016/S0959-8049(11)71740–3 23 htt //d i /10 1093/ / d 550 g 23. https://doi.org/10.1093/annonc/mdq550 g 24. https://doi.org/10.1093/annonc/mds577 p g 25. http://meetinglibrary.asco.org/content/140232-158 p g y g 26. https://doi.org/10.1007/s00280-016-3039-1 SCIEnTIFIC Reports | (2018) 8:2047 | DOI:10.1038/s41598-018-19835-8 2 2 www.nature.com/scientificreports/ Figure 1. The PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) flow diagra35. Figure 1. The PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) flow diagra35. Results li ibl grade 1–4 hypomagnesemia Cetuximab group: 95/408 Panitumumab group: 143/353 Burkes et al.22 PRIME Conference presentation from phase III RCT Panitumumab (6 mg/kg every 2 weeks) with FOLFOX4 vs. FOLFOX4 alone in the first- line treatment of mCRC Grade 0 vs. grade 1–4 hypomagnesemia 168/154 Table 1. Characteristics of included trials for meta-analysis. Hypo-mg: hypo-magnesemia, Normo-mg: normo-magnesemia, FOLFOX: folinic acid, fluorouracil& oxaliplatin, FOLFIRI: folinic acid, fluorouracil& irinotecan, mCRC: metastatic colorectal cancer, NCIC CTG/AGITG CO. 17: National Cancer Institute of Canada Clinical Trials Group and Australasian Gastrointestinal Trials Group CO. 17, RCT: randomized control trial, BSC: best supportive care, ASPECCT: A Study of Panitumumab Efficacy and Safety Compared to Cetuximab, PRIME: Panitumumab Randomized Trial in Combination with Chemotherapy for Metastatic Colorectal Cancer to Determine Efficacy. *The panitumumab and cetuximab arm in ASPECCT was separated for analysis. Table 1. Characteristics of included trials for meta-analysis. Hypo-mg: hypo-magnesemia, Normo-mg: normo-magnesemia, FOLFOX: folinic acid, fluorouracil& oxaliplatin, FOLFIRI: folinic acid, fluorouracil& irinotecan, mCRC: metastatic colorectal cancer, NCIC CTG/AGITG CO. 17: National Cancer Institute of Canada Clinical Trials Group and Australasian Gastrointestinal Trials Group CO. 17, RCT: randomized control trial, BSC: best supportive care, ASPECCT: A Study of Panitumumab Efficacy and Safety Compared to Cetuximab, PRIME: Panitumumab Randomized Trial in Combination with Chemotherapy for Metastatic Colorectal Cancer to Determine Efficacy. *The panitumumab and cetuximab arm in ASPECCT was separated for analysis. Table 1. Characteristics of included trials for meta-analysis. Hypo-mg: hypo-magnesemia, Normo-mg: normo-magnesemia, FOLFOX: folinic acid, fluorouracil& oxaliplatin, FOLFIRI: folinic acid, fluorouracil& irinotecan, mCRC: metastatic colorectal cancer, NCIC CTG/AGITG CO. 17: National Cancer Institute of Canada Clinical Trials Group and Australasian Gastrointestinal Trials Group CO. 17, RCT: randomized control trial, BSC: best supportive care, ASPECCT: A Study of Panitumumab Efficacy and Safety Compared to Cetuximab, PRIME: Panitumumab Randomized Trial in Combination with Chemotherapy for Metastatic Colorectal Cancer to Determine Efficacy. *The panitumumab and cetuximab arm in ASPECCT was separated for analysis. Analysis of hypomagnesemia and PFS benefit. There were five trials of 1723 wild-type KRAS mCRC patients who received either cetuximab- or panitumumab-based chemotherapy, including 568 patients (33.0%) with clinical hypomagnesemia and 1155 patients (67.0%) with normal serum magnesium levels. In the hypo- magnesemia group, anti-EGFR MoA-based chemotherapy was statistically significantly associated with 36% reduction in the risk of diseases progression or death (HR: 0.64; 95% CI: 0.47-0.88; p = 0.006) (Fig. 2a). Results li ibl Eligible studies. The PRISMA study flow diagram were shown in Fig. 135. A total of 157 records were identi- fied after searching the MEDLINE, EMBASE, and CENTRAL databases. Another 16 records were identified from the United States National Institutes of Health trials register databases and from conference abstracts of ASCO and ESMO. After removing 29 duplicates and 139 exclusions due to irrelevant results of hypomagnesemia, lack of placebo, or mixed mutant type KRAS analysis, only five trials were incorporated in this meta-analysis. These were two conference presentations of phase III RCTs22,25, two retrospective studies23,26 and one phase III RCT24.h p p p p The original setting of CO. 175,6, PRIME7 and ASPECCT8 were multiple-center and open label phase III RCTs that had a low risk of bias when appraised using the Cochrane Collaboration’s tool36. As two non-randomized control trial, Vincenzi et al.23 and Fujii et al.26, also had a low risk of bias when appraised using the MINORS appraisal tool29.h pp The subgroup PFS and OS analyses of cetuximab-based chemotherapy were reported by Vincenzi et al., CO.17, and ASPECCT, while panitumumab-based chemotherapy was reported by PRIME and ASPECCT. The patients in the Fujii’s trial contained either cetuximab-based or panitumumab-based chemotherapy which analyzed PFS outcome. All trials, except CO. 17, reported ORR. The characteristics of the included trials were summarized in Table 1. SCIEnTIFIC Reports | (2018) 8:2047 | DOI:10.1038/s41598-018-19835-8 3 www.nature.com/scientificreports/ Author Trial name Type of study Study design Definition of hypomagnesemia No. of Patients (Hypo- mg/ Normo-mg) Fujii et al.26 N/A Retrospective Cetuximab or Panitumumab with mFOLFOX or FOLFIRI vs. mFOLFOX or FOLFIRI in the first-line treatment of mCRC Grade 0 vs. grade 1–4 hypomagnesemia 14/29 Vincenzi et al.23 N/A Retrospective Cetuximab (400 mg/m2 loading dose, then 250 mg/m2 weekly) with irinotecan in the third-line treatment of mCRC  > 50% vs. < 50% reduction compared with basal value within 1 months from treatment 95/48 Vickers et al.24 NCIC CTG/ AGITG CO. 17 RCT Cetuximab (400 mg/m2 loading dose, then 250 mg/ m2 weekly) with BSC vs. BSC alone in the third-line treatment of mCRC Grade 0 vs. grade 1–4 hypomagnesemia 53/163 Price et al.25 ASPECCT Conference presentation from phase III RCT Panitumumab (6 mg/kg every 2 weeks) vs. Cetuximab (400 mg/ m2 loading dose, then 250 mg/ m2 weekly) in the third-line treatment of mCRC* Grade 0 vs. Results li ibl After frontline or later-lines of anti-EGFR MoA-based chemotherapy, patient with hypomagnesemia gained significant PFS benefit (frontline, HR: 0.78; 95% CI: 0.62-0.98; p = 0.03 and later-lines, HR: 0.60; 95% CI: 0.40–0.90; p = 0.01) (Fig. 2b). Subgroup analysis of hypomagnesemia after anti-EGFR MoA monotherapy or combination therapy also showed significant PFS benefit in combination therapy (HR: 0.62; 95% CI: 0.41–0.94; p = 0.03) and a trend for PFS benefit in monotherapy (HR: 0.68; 95% CI: 0.41–1.14; p = 0.14) (Fig. 2c). Analysis of hypomagnesemia and OS benefit. Fujii et al. did not report OS and only four trials were included in this meta-analysis. There was also significant OS benefit in patients with hypomagnesemia after anti-EGFR MoA-based chemotherapy (HR: 0.72; 95% CI: 0.56–0.92; p = 0.008) (Fig. 3a). Subgroup analysis of patients with hypomagnesemia after panitumumab-based chemotherapy revealed significant OS benefit (HR: 0.64; 95% CI: 0.53–0.76; p < 0.001). However, patients with hypomagnesemia after cetuximab-based chemother- apy had no OS benefit (HR: 0.86; 95% CI: 0.51–1.45; p = 0.58). Subgroup analysis of patients with hypomagne- semia after frontline anti-EGFR MoA-based chemotherapy revealed significant OS benefit (frontline, HR: 0.68; 95% CI: 0.52–0.90; p = 0.007 and later-lines, HR: 0.75; 95% CI: 0.54–1.04; p = 0.09) (Fig. 3b). Subgroup analysis of patients with hypomagnesemia after combination therapy showed significant OS benefit (combination therapy, HR: 0.64; 95% CI: 0.52–0.78; p < 0.001 and monotherapy, HR: 0.86; 95% CI: 0.54–1.36; p = 0.51) (Fig. 3c). Analysis of hypomagnesemia and ORR benefit. The CO. 17 trial did not report ORR and only four tri- als were included in this meta-analysis. In patients with hypomagnesemia, anti-EGFR MoA-based chemotherapy had significantly better ORR (RR: 1.81; 95% CI: 1.30 to 2.52; p < 0.001) (Fig. 4a). Moreover, patients with hypo- magnesemia irrespective of later-lines of anti-EGFR MoA-based chemotherapy had significant ORR (frontline, RR: 1.48; 95% CI: 0.92–2.37; p = 0.11 and later-lines, HR: 2.05; 95% CI: 1.44–2.91; p < 0.001) (Fig. 4b). Subgroup analysis revealed significant ORR benefit in anti-EGFR MoA monotherapy and combination therapy (mono- therapy, HR: 1.83; 95% CI: 1.41–2.37; p < 0.001 and combination therapy, HR: 1.94; 95% CI: 1.02–3.68; p = 0.04) (Fig. 4c). The HRs or RRs for all of the different comparisons were summarized in Fig. 5. Discussionh Discussion The KRAS mutation is a deeply-rooted negative predictive marker in mCRC treated with cetuximab or pan- itumumab9,10. Recently, extended RAS mutation (in addition to exons 3 and 4 of KRAS and exons 2, 3, 4 of SCIEnTIFIC Reports | (2018) 8:2047 | DOI:10.1038/s41598-018-19835-8 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Analysis of progression-free survival (PFS) benefit in patients with hypomagnesemia or normo- magnesemia. (a) Analysis of PFS in terms of cetuximab- or panitumumab-based chemotherapy. Pooled analysis showed significant PFS benefit in patients with hypomagnesemia. (b) Analysis of PFS in terms of frontline or later-lines of anti-EGFR MoA-based chemotherapy revealed significant PFS benefit in both frontline and later- lines therapy. (c) Analysis of PFS in terms of anti-EGFR MoA monotherapy or combination therapy revealed significant PFS benefit in combination therapy and a trend for PFS benefit in monotherapy. Squares, study- specific hazard ratios (size of the square reflected the study-specific statistical weight); Horizontal lines, 95% CIs; Diamond, summary hazard ratios estimate with its 95% CI. Figure 2. Analysis of progression-free survival (PFS) benefit in patients with hypomagnesemia or normo- magnesemia. (a) Analysis of PFS in terms of cetuximab- or panitumumab-based chemotherapy. Pooled analysis showed significant PFS benefit in patients with hypomagnesemia. (b) Analysis of PFS in terms of frontline or later-lines of anti-EGFR MoA-based chemotherapy revealed significant PFS benefit in both frontline and later- lines therapy. (c) Analysis of PFS in terms of anti-EGFR MoA monotherapy or combination therapy revealed significant PFS benefit in combination therapy and a trend for PFS benefit in monotherapy. Squares, study- specific hazard ratios (size of the square reflected the study-specific statistical weight); Horizontal lines, 95% CIs; Diamond, summary hazard ratios estimate with its 95% CI. Figure 2. Analysis of progression-free survival (PFS) benefit in patients with hypomagnesemia or normo- magnesemia. (a) Analysis of PFS in terms of cetuximab- or panitumumab-based chemotherapy. Pooled analysis showed significant PFS benefit in patients with hypomagnesemia. (b) Analysis of PFS in terms of frontline or later-lines of anti-EGFR MoA-based chemotherapy revealed significant PFS benefit in both frontline and later- lines therapy. (c) Analysis of PFS in terms of anti-EGFR MoA monotherapy or combination therapy revealed significant PFS benefit in combination therapy and a trend for PFS benefit in monotherapy. Squares, study- specific hazard ratios (size of the square reflected the study-specific statistical weight); Horizontal lines, 95% CIs; Diamond, summary hazard ratios estimate with its 95% CI. Figure 2. Analysis of progression-free survival (PFS) benefit in patients with hypomagnesemia or normo- magnesemia. (a) Analysis of PFS in terms of cetuximab- or panitumumab-based chemotherapy. Pooled analysis showed significant PFS benefit in patients with hypomagnesemia. www.nature.com/scientificreports/ (b) Analysis of PFS in terms of frontline or later-lines of anti-EGFR MoA-based chemotherapy revealed significant PFS benefit in both frontline and later- lines therapy. (c) Analysis of PFS in terms of anti-EGFR MoA monotherapy or combination therapy revealed significant PFS benefit in combination therapy and a trend for PFS benefit in monotherapy. Squares, study- specific hazard ratios (size of the square reflected the study-specific statistical weight); Horizontal lines, 95% CIs; Diamond, summary hazard ratios estimate with its 95% CI. Figure 2. Analysis of progression-free survival (PFS) benefit in patients with hypomagnesemia or normo- magnesemia. (a) Analysis of PFS in terms of cetuximab- or panitumumab-based chemotherapy. Pooled analysis showed significant PFS benefit in patients with hypomagnesemia. (b) Analysis of PFS in terms of frontline or later-lines of anti-EGFR MoA-based chemotherapy revealed significant PFS benefit in both frontline and later- lines therapy. (c) Analysis of PFS in terms of anti-EGFR MoA monotherapy or combination therapy revealed significant PFS benefit in combination therapy and a trend for PFS benefit in monotherapy. Squares, study- specific hazard ratios (size of the square reflected the study-specific statistical weight); Horizontal lines, 95% CIs; Diamond, summary hazard ratios estimate with its 95% CI. neuroblastoma RAS viral oncogene [NRAS]) is proposed as an even more potent negative predictive marker13. Stepwise strategy with negative4,11–13 and positive selection14–16 seems to help define the most benefit for anti-EGFR MoAs, but not simply “opt-in” strategy in patients with wild-type KRAS tumors. This study suggests that hypomagnesemia in patients with wild-type KRAS is associated with better PFS (HR: 0.64; 95% CI: 0.47– 0.88), OS (HR: 0.72; 95% CI: 0.56–0.92), and ORR (RR: 1.81; 95% CI: 1.30–2.52) when treated with cetuximab- or panitumumab-based regimen. Previous literatures only indicate the risk and incidence of hypomagnesemia in anti-EGFR MoAs, but not its relationship with clinical efficacy, such as PFS, OS and ORR in this study17–20. In SCIEnTIFIC Reports | (2018) 8:2047 | DOI:10.1038/s41598-018-19835-8 5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Analysis of overall survival (OS) benefit in patients with hypomagnesemia or normo-magnesemia. (a) Analysis of OS in terms of cetuximab- or panitumumab-based chemotherapy. Pooled analysis showed significant OS benefit in patients with hypomagnesemia. (b) Analysis of OS in terms of frontline or later-lines of anti-EGFR MoA-based chemotherapy revealed significant OS benefit in frontline therapy and a trend for OS benefit in later-lines therapy. (c) Analysis of OS in terms of anti-EGFR MoA monotherapy or combination therapy revealed significant OS benefit in combination therapy and a trend for OS benefit in monotherapy. Figure 3. Analysis of overall survival (OS) benefit in patients with hypomagnesemia or normo-magnesemia. (a) Analysis of OS in terms of cetuximab- or panitumumab-based chemotherapy. Pooled analysis showed significant OS benefit in patients with hypomagnesemia. (b) Analysis of OS in terms of frontline or later-lines of anti-EGFR MoA-based chemotherapy revealed significant OS benefit in frontline therapy and a trend for OS benefit in later-lines therapy. (c) Analysis of OS in terms of anti-EGFR MoA monotherapy or combination therapy revealed significant OS benefit in combination therapy and a trend for OS benefit in monotherapy. igure 3. Analysis of overall survival (OS) benefit in patients with hypomagnesemia or normo-magnesemia. Figure 3. Analysis of overall survival (OS) benefit in patients with hypomagnesemia or normo-magnesemia. (a) Analysis of OS in terms of cetuximab- or panitumumab-based chemotherapy. Pooled analysis showed significant OS benefit in patients with hypomagnesemia. (b) Analysis of OS in terms of frontline or later-lines of anti-EGFR MoA-based chemotherapy revealed significant OS benefit in frontline therapy and a trend for OS benefit in later-lines therapy. (c) Analysis of OS in terms of anti-EGFR MoA monotherapy or combination therapy revealed significant OS benefit in combination therapy and a trend for OS benefit in monotherapy. Figure 3. Analysis of overall survival (OS) benefit in patients with hypomagnesemia or normo-magnesemia. (a) Analysis of OS in terms of cetuximab- or panitumumab-based chemotherapy. Pooled analysis showed significant OS benefit in patients with hypomagnesemia. (b) Analysis of OS in terms of frontline or later-lines of anti-EGFR MoA-based chemotherapy revealed significant OS benefit in frontline therapy and a trend for OS benefit in later-lines therapy. (c) Analysis of OS in terms of anti-EGFR MoA monotherapy or combination therapy revealed significant OS benefit in combination therapy and a trend for OS benefit in monotherapy. Figure 3. www.nature.com/scientificreports/ Analysis of overall survival (OS) benefit in patients with hypomagnesemia or normo-magnesemia. (a) Analysis of OS in terms of cetuximab- or panitumumab-based chemotherapy. Pooled analysis showed significant OS benefit in patients with hypomagnesemia. (b) Analysis of OS in terms of frontline or later-lines of anti-EGFR MoA-based chemotherapy revealed significant OS benefit in frontline therapy and a trend for OS benefit in later-lines therapy. (c) Analysis of OS in terms of anti-EGFR MoA monotherapy or combination therapy revealed significant OS benefit in combination therapy and a trend for OS benefit in monotherapy. addition, serum magnesium levels are easy to check and follow-up during active treatment. Thus, it is useful in decision-making for tailor-made treatment. Skin rash, another well-documented adverse effect of anti-EGFR MoAs, is reportedly predictive of better clinical benefits in mCRC patients37. However, anti-EGFR MoAs are not beneficial in patients with mutated KRAS and in clinical practice, the severity of skin rash may be difficult to document among different observers. gf Subgroup analysis reveals that panitumumab has a trend for PFS (panitumumab, HR: 0.60; 95% CI: 0.35–1.03 and cetuximab, HR: 0.71; 95% CI: 0.36–1.41) and significant OS (panitumumab, HR: 0.64; 95% CI: 0.53–0.76 and cetuximab, HR: 0.86; 95% CI: 0.51–1.45) benefits in patients with hypomagnesemia. On the other hand, cetuximab had significant ORR (cetuximab, RR: 2.19; 95% CI: 1.02–4.69 and panitumumab, RR: 1.57; 95% CI: 0.95–2.58). The differences of these two anti-EGFR MoAs mainly resulted from the negative impact of CO.17 on PFS and OS24. Although they performed the analyses in a phase III trial, RCT CO.17, substantial cases (10.6%) were excluded due to missing serum magnesium levels and KRAS status compared to the ASPECCT or PRIME trials (<2.8%)22,25. Thus, it is immature here to interpret the differences of these two anti-EGFR MoAs in view of hypomagnesemia and clinical efficacy. SCIEnTIFIC Reports | (2018) 8:2047 | DOI:10.1038/s41598-018-19835-8 6 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. Summary of Findings (*The panitumumab and cetuximab arm in ASPECCT was separated for analysis) (a) Analysis of PFS and OS in terms of cetuximab- or panitumumab-based chemotherapy. (b) A of ORR in terms of cetuximab- or panitumumab-based chemotherapy. Figure 5. Summary of Findings (*The panitumumab and cetuximab arm in ASPECCT was separated for analysis) (a) Analysis of PFS and OS in terms of cetuximab- or panitumumab-based chemotherapy. (b) Analysis of ORR in terms of cetuximab- or panitumumab-based chemotherapy. Figure 5. Summary of Findings (*The panitumumab and cetuximab arm in ASPECCT was separated for analysis) (a) Analysis of PFS and OS in terms of cetuximab- or panitumumab-based chemotherapy. (b) Analysis of ORR in terms of cetuximab- or panitumumab-based chemotherapy. magnesiotropic hormone, which in turn, makes tumor more susceptible to anti-EGFR MoAs14,16,39. With diverg- ing roles of magnesium in tumor biology, careful preclinical models are awaited to demonstrate this underlying mechanism. magnesiotropic hormone, which in turn, makes tumor more susceptible to anti-EGFR MoAs14,16,39. With diverg- ing roles of magnesium in tumor biology, careful preclinical models are awaited to demonstrate this underlying mechanism.h This study has some limitations. First, not all included trials are RCTs and some are non-full-published meet- ing posters or abstracts22,25. However, CO.17, PRIME, and ASPECCT are randomized, controlled, phase III trials in original design5–8. Future full publications with unpublished data are likely to be relatively modest and are unlikely to substantively change the results reported here. In addition, the trials included here are not based on an individual patient data (IPD) but abstracted data from published literatures40. Despite attempts to contact the authors for original data, data from the studies by Vickers et al. and Vincenzi et al. are missing. Regulatory author- ities should play an important role in support such an IPD meta-analysis and re-evaluate the extent of hypo- magnesemia and its predictive role in anti-EGFR MoAs. Funnel plots, Egger’s test and sensitivity analysis were not performed for each pairwise comparison due to less than 10 studies included in this meta-analysis. Instead, all available studies were enrolled for analysis. Lastly, hypomagnesemia is associated with better outcomes in patients with the wild-type KRAS mCRC in this study, but this warrants further validation in extended RAS mutation population. Prospectively designed study is needed to elucidate the magnitude of hypomagnesemia and its predictive role on cetuximab and panitumumab, and how magnesium supplementation can influence these outcomes. Conclusions In patients with wild-type KRAS mCRC, hypomagnesemia is associated with better clinical benefits of PFS, OS and ORR when treated with cetuximab- or panitumumab-based chemotherapy. Future clinical trials should cor- roborate its predictive role. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Analysis of objective response rate (ORR) benefit in patients with hypomagnesemia or normo- magnesemia. (a) Analysis of ORR in terms of cetuximab- or panitumumab-based chemotherapy. Pooled analysis showed significant ORR benefit in patients with hypomagnesemia. (b) Analysis of ORR in terms of frontline or later-lines of anti-EGFR MoA-based chemotherapy revealed significant ORR benefit in later- lines therapy and a trend for OS benefit in frontline therapy. (c) Analysis of ORR in terms of anti-EGFR MoA monotherapy or combination therapy revealed significant ORR benefit in both monotherapy and combination therapy. Figure 4. Analysis of objective response rate (ORR) benefit in patients with hypomagnesemia or normo- magnesemia. (a) Analysis of ORR in terms of cetuximab- or panitumumab-based chemotherapy. Pooled analysis showed significant ORR benefit in patients with hypomagnesemia. (b) Analysis of ORR in terms of frontline or later-lines of anti-EGFR MoA-based chemotherapy revealed significant ORR benefit in later- lines therapy and a trend for OS benefit in frontline therapy. (c) Analysis of ORR in terms of anti-EGFR MoA monotherapy or combination therapy revealed significant ORR benefit in both monotherapy and combination h Figure 4. Analysis of objective response rate (ORR) benefit in patients with hypomagnesemia or normo- magnesemia. (a) Analysis of ORR in terms of cetuximab- or panitumumab-based chemotherapy. Pooled analysis showed significant ORR benefit in patients with hypomagnesemia. (b) Analysis of ORR in terms of frontline or later-lines of anti-EGFR MoA-based chemotherapy revealed significant ORR benefit in later- lines therapy and a trend for OS benefit in frontline therapy. (c) Analysis of ORR in terms of anti-EGFR MoA monotherapy or combination therapy revealed significant ORR benefit in both monotherapy and combination therapy. Nonetheless, cetuximab and panitumumab can lead to hypomagnesemia in varying extent18,20. Although the mechanism of hypomagnesemia may lie in the interaction of anti-EGFR MoA, with transient receptor poten- tial cation channel, subfamily M, and member 6 (TRPM6) in the intestine and distal collecting tubules, there is little known about the difference between cetuximab and panitumumab, such as immunogenic properties, half-life, and dosing intervals38. Analyses of frontline, later-lines, monotherapy, and combination therapy sug- gest that cetuximab and panitumumab may possess different properties related to hypomagnesemia and efficacy. The underlying mechanism of hypomagnesium and this favorable response toward cancer treatment is lack- ing and not well elucidated21, and it is inferred that hypomagnesemia induces upregulation of EGFR ligands as SCIEnTIFIC Reports | (2018) 8:2047 | DOI:10.1038/s41598-018-19835-8 7 3. Heinemann, V., Stintzing, S., Kirchner, T., Boeck, S. & Jung, A. Clinical relevance of EGFR- and KRAS-status in colorectal cancer patients treated with monoclonal antibodies directed against the EGFR. Cancer Treat Rev. 35(3), 262–271 (2009). 4. Bertotti, A. et al. The genomic landscape of response to EGFR blockade in colorectal carcinoma. Nature. 526(7572), 263–267 (2015). 5. Jonker, D. J. et al. Cetuximab for the treatment of colorectal cancer. N Engl J Med. 357(20), 2040–2048 (2007). References 1. 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Randomized, open-label, phase 3 study of panitumumab (Pmab) with FOLFOX4 vs FOLFOX4 alone as 1st-line treatment for metastatic colorectal cancer (mCRC) – the role of hypomagnesemia (Hypomag) on efficacy. Eur J Cancer. 47(sup1), S420: 6095 poster (2011).ifi p 3. Vincenzi, B. et al. Early magnesium modifications as a surrogate marker of efficacy of cetuximab-based anticancer treatment in KRAS wild-type advanced colorectal cancer patients. Ann Oncol. 22(5), 1141–1146 (2011). yp p ( ) ( ) 24. Vickers, M. M. et al. Association of hypomagnesemia with inferior survival in a phase III, randomized study of cetuximab plus best supportive care versus best supportive care: NCIC CTG/AGITG CO.17. Ann Oncol. 24(4), 953–960 (2013). 24. Vickers, M. M. et al. Association of hypomagnesemia with inferior survival in a phase III, randomized study of cetuximab p supportive care versus best supportive care: NCIC CTG/AGITG CO.17. Ann Oncol. 24(4), 953–960 (2013). yp g p y supportive care versus best supportive care: NCIC CTG/AGITG CO.17. Ann Oncol. 24(4), 953–960 (2013). pp pp 25. Price, T. J. et al. Randomized phase 3 study of panitumumab vs. cetuximab in chemo-refractory wild-type KRA colorectal cancer: outcomes by hypomagnesemia in ASPECCT. J Clin Oncol 33, abstract 3587 (2015).fi 5. Author Contributions F.C.K. was responsible for concept and study design. M.C.H., C.F.W., C.W.C., C.S.S., W.S.H. and F.C.K. were responsible for publication retrieve and data extraction. Statistical analyses: M.C.H. and F.C.K. were responsible for statistical analyses. All authors drafted and approved the submission of the manuscript. SCIEnTIFIC Reports | (2018) 8:2047 | DOI:10.1038/s41598-018-19835-8 9 www.nature.com/scientificreports/ SCIEnTIFIC Reports | (2018) 8:2047 | DOI:10.1038/s41598-018-19835-8 Additional Informationh Competing Interests: The authors declare that they have no competing interests. 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The interaction of migration range, regional economic development level on social capital and public health services of internal migrants: evidence from the China Migrant Dynamic Survey in 2017
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The interaction of migration range, regional economic development level on social capital and public health services of internal migrants: evidence from the China Migrant Dynamic Survey in 2017 Zhen Yang  Tongji University Cheng-hua Jiang  (  jchtongji@163.com ) Tongji University Jiansheng Hu  Jinggangshan University Zhen Yang  Tongji University Cheng-hua Jiang  (  jchtongji@163.com ) Tongji University Jiansheng Hu  Jinggangshan University Research Article Research Article Keywords: Internal Migrants, Migration Range, Regional Economic Development Level, Social Capital, Public Health Services Posted Date: November 12th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-309989/v5 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Title page The interaction of migration range, regional economic development level on social capital and public health services of internal migrants: evidence from the China Migrant Dynamic Survey in 2017 Zhen Yang 1, 2 , Cheng-hua Jiang 1,* , Jiansheng Hu2 1School of medicine, Tongji university, Shanghai 200092, China; 2School of medicine, Jinggangshan university, Jian,Jiangxi 343009, China *correspondence:Cheng-hua Jiang School of medicine, Tongji university, No 1239 Siping Road, Yangpu District, Shanghai 200092, China Phone:+8618917266778 E-mail:jchtongji@163.com Title page 1 The interaction of migration range, regional economic development level on social capital and public health services of internal migrants: evidence from the China Migrant Dynamic Survey in 2017 Zhen Yang 1, 2 , Cheng-hua Jiang 1,* , Jiansheng Hu2 1School of medicine, Tongji university, Shanghai 200092, China; 2School of medicine, Jinggangshan university, Jian,Jiangxi 343009, China *correspondence:Cheng-hua Jiang School of medicine, Tongji university, No 1239 Siping Road, Yangpu District, Shanghai 200092, China Phone:+8618917266778 E-mail:jchtongji@163.com The interaction of migration range, regional economic development level on social capital and public health services of internal migrants: evidence from the China Migrant Dynamic Survey in 2017 1 1 Abstract Background Studies have confirmed that migration range (MR), regional economic development level (REDL) and social capital have impacts on the internal migrants' (IMs') accessibility of National Essential Public Health Services (NEPHS), but no research has explored the interaction of the four variables. Method A cross-sectional sample of 115412 IMs from the China Migrant Dynamic Survey in 2017 was selected. The sampling method was layered, multi-stage, and probability proportional to size. Logistic regression was conducted by SPSS22.0, sex, residence duration, community type, and education as the control variables, REDL and MR as the moderating variables, social capital as independent variable and awareness of NEPHS and registration of health records (RHR) as the dependent variables. Results In high income provinces (HIPs), the intra-provincial IMs' CSC, civic participation, social participation, NEPHS awareness and RHR were 4.73±1.736, 49.3%, 55.3%, 61.1%, and 31.9%, and the inter-provincial IMs' levels were 4.01±1.713, 40.5%, 47.2%, 54.6%, and 26.8%; In low-and middle- income provinces (LMIPs), the intra-provincial and inter-provincial IMs' levels were 5.13±1.767, 46.1%, 48.1%, 65.6%, 35.5%, and 5.02±1.775, 41.5%, 41.5%, 62.06%, 34.1%, respectively. In the complete regression model, the interaction of MR, REDL and CSC was not significant, while the interaction of MR, REDL and SSC was significant. In the group regression model, the ORs of CSC, civic participation, and social participation were significantly greater than 1 both in HIPs and LMIPs. However, the interaction of MR and SSC was significant in HIPs but not significant in LMIPs. Conclusions The social capital and NEPHS utilization of inter-provincial IMs was significantly lower than that of intra-provincial IMs, and these gaps were more prominent in HIPs. Social capital had a positive effect on IMs' NEPHS utilization, and this effect was significantly moderated by MR and REDL. The next focus of the equalization of public services should be the primary health resources supply and the IMs' social capital construction of HIPs. Keywords: Internal Migrants, Migration Range, Regional Economic Development Level, Social Capital, Public Health Services Background Social development drives global mobility. Migration can be internal or international, and the internal migrants (IMs) are about four times the size of international migrants [1]. Due to institutional restrictions, IMs in some low - and middle- income countries (LMICs) are not treated equally as natives, and they need to face many obstacles in health resources utilization. Studies from China, India, Indonesia and other LMICs show that the IMs have insufficient access to local public health services [2-6], and this has become a risk affecting the health of IMs [2]. How to 2 effectively improve the IMs' accessibility to health services has become an important challenge for LMICs. One way to address this challenge is to explore the influencing factors and how they work. Social capital is a social determinant of health [7], it can influence individual health outcomes by influencing access to health services [8]. Different types of social capital affect health services utilization by influencing the availability of health services in communities, the availability and effectiveness of outreach resources between health-care providers and communities they serve, and care-seeking behavior of individuals in those communities [9]. Migration means a loss of the original social network and a reduction of social participation in the new environment [10]. Surveys show that migrants generally lack social capital, which limits their access to local health services[11, 12]. Perkins et al [13] pointed out that social capital may be particularly vital to health outcomes as extended webs of social ties often are the principal source of various resources in LMICs. Therefore, social capital may play a prominent role in IMs' access to health services in LMICs, and some relevant studies support this point [6, 14-18 ]. Not everyone has access to the same sources of social capital and not everyone will benefit in the same way [19]. Discussion of the relationship between social capital and health must be set in context to be more effective. Carpiano and Moore [20] suggested that social capital and health knowledge base can be better served by asking three foundational questions of (a) how, (b) for whom, and (c) in which contexts does social capital work, rather than focusing primarily on whether social capital provides some universal health benefit. In fact, existing studies on the relationship between social capital and IMs' accessibility to health services have not considered these three questions comprehensively. Background The relationship between social capital and health is not consistent across countries [21], which could be attributed to the differences in economy, culture and system among these countries [22]. Similarly, there are differences in regional economic development levels (REDL) and cultural practices in different regions of the same country. These gaps are likely to influence the relationship between social capital and access to health services, but few studies have demonstrated this. During the past three decades, China has experienced the largest migration in human history, with hundreds of millions of rural inhabitants moving temporarily or permanently to cities. Internal migration is inevitable and essential for the economic and social prosperity of China. IMs exceeded 240 million in 2017 [23], and the number was stable with a slight decline in recent years. In 2009, the central government initiated the National Essential Public Health Services (NEPHS), the project is provided free of charge to all residents, including IMs who have lived there for more than six months [24]. NEPHS includes health records, health education, immunization for children, and chronic disease management etc. Since 2009, the government has successively introduced measures to strengthen the equalization of NEPHS [25-27]. Efforts have paid off, and the IMs' NEPHS utilization level is rapidly improving [28]. The new situation promotes the government's working mode to change from flooding to precise [27], and the focus has gradually shifted to how to achieve the NEPHS equalization within the IMs. Researchers are beginning to conduct more studies of population differentiation[3, 4, 14, 15, 28-33]. Existing studies have confirmed that both migration range (MR) and REDL have a significant impact on NEPHS utilization. Studies have consistently concluded that the NEPHS utilization of inter-provincial IMs is lower than that of intra-provincial IMs [3, 28-33]. However, opinions on the relationship between REDL and IMs' NEPHS utilization are inconsistent: some believe that the 3 IMs' NEPHS utilization in high income provinces (HIPs) is lower than that in low- and middle-income provinces (LMIPs) [28-33], but Yang Xin [4] does not think so. Surprisingly, these studies almost analyzed MR and REDL as independent factors, without considering possible interactions between the two variables. On the other hand, studies provide important clues for further exploring the relationship among MR, REDL and social capital. International migrants research suggests acculturation affects the social network construction of migrants [34]. Background Among factors that affect the social integration of migrants, cultural exclusion is an important source of prejudice, discrimination and social isolation suffered by individuals in the process of migration [35]. In fact, dialects, diets and customs are different among provinces in China, and the problem of acculturation of IMs also exists. However, there are few studies on the relationship between acculturation and social capital of IMs. Yang and Zhang [36] found that being familiar with dialect can help IMs better integrate into society, while Xiao et al. [37] found that the IMs in HIPs has the lowest level of psychological integration. However, studies do not provide direct evidence that MR and REDL affect social capital. To sum up, given MR and REDL may have a significant impact on the IMs' social capital and health services utilization, and social capital is closely related to health services utilization, we speculate that different MR and REDL create different contexts for IMs, and these different contexts may significantly affect the mechanism of social capital on service utilization. To test this hypothesis, we adopted a retrospective cross-sectional survey ，a sample of the China Migrant Dynamic Survey in 2017 was analyzed, and we want to verify four hypotheses: (1) IMs' social capital may be significantly affected by MR and REDL; (2) MR and REDL have a significant interaction on IMs' NEPHS utilization; (3) Social capital has a significant impact on the IMs' NEPHS utilization; (4) The relationship between social capital and NEPHS utilization of IMs is significantly moderated by MR and REDL. This study was the first to explore the impact of MR and REDL on IMs' access to public health services from the perspective of social capital based on a national sample. Our study can provide evidence for the Chinese government to deepen the equalization of NEPHS for IMs, and can also provide references for other LMICs to deal with the health problems of migrants. Utilization of NEPHS Awareness of NEPHS is a prerequisite for NEPHS utilization [3]. Awareness of NEPHS was set as an outcome variable, and the question was "Have you heard of the NEPHS?" and the answer was "yes or no". Another outcome variable was registration of health records (RHR). RHR is one of the service priorities and reflects the actual utilization of NEPHS by the IMs. IMs can voluntarily register for a health record account at the local community health service center, and their health information will be recorded. The question was "Have you registered health records at the destination?" and the answer was "yes or no". Social capital refers to the resources and benefits received through connections with others, either as individuals or groups, it can be distinguished into two dimensions: cognitive social capital (CSC) and structural social capital (SSC) [7]. The CSC generally refers to IMs' perceptions, beliefs, and attitudes toward their destination, with corresponding measures focused mainly on the concepts of generalized and particularized trust [38]. In this study, social capital was limited to the destination, and it was a localized social capital that reflects the social resources available to the IMs there. There were six questions in the survey: "I like the city/place I live now", "I am concerned about the changes in the city/place I live now", "I am very willing to blend with the local people and become a part of them", "I think the local people are willing to accept me as a part of them", "I feel locals look down on outsiders" (reverse scoring) and "I feel like I'm already a local". The answer to each question was "1=totally disagree, 2=disagree, 3=basically agree, and 4=totally agree". The internal consistency coefficient α =0.786. According to the distribution of scores, CSC was divided into eight levels: 1 (6-14 points), 2 (15-16 points), 3 (17 points), 4 (18 points), 5 (19-20 points), 6 (21-22 points), 7 (23 points), and 8 (24 points). SSC refers to the presence of formal opportunity structures or activities in which individuals build or strengthen their social connections [38]. The SSC of this survey included civic participation and social participation in the destination. Methods Data A retrospective cross-sectional study was used in this study. The data was obtained from the China Migrant Dynamic Survey in 2017 provided by the Migrant Population Service Center. China Migrant Dynamic Survey is an annual national sample survey of the IMs organized by the National Health Commission from 2009, with an annual sample size of approximately 200000 households. China Migrant Dynamic Survey adopts the layered, multi-stage, and probability proportional to size (PPS) sampling method. This study adopted the individual questionnaire A of China Migrant Dynamic Survey, which was uniformly printed and distributed by the National Health Commission. The questionnaire A includes basic information about respondent's demography, perception of the destination, the state of social interaction, and utilization status of NEPHS, etc. Full-time investigators collected the questionnaire data through household interviews, and each respondent gave informed consent before commencing the interview. Dates were entered through the migrant population health and household planning dynamic monitoring system, input data was subjected to multiple checks to ensure quality. 4 The target population of China Migrant Dynamic Survey is the inflow population aged 15 and above who came to live in the local area (county or city) one month before the survey. In this study, the inclusion conditions of sample were set as "18-59 years of age, residence duration more than one year". Beijing, Tianjin and Shanghai, the three cities have only inter-provincial IMs, which do not meet the objective of this study, so the samples of these three cities were excluded. After the quality audit, 115412 people were finally included. In addition, we introduced GDP per capita to reflect the REDL of each provincial region, and GDP per capita is based on 2017 data from the National Bureau of Statistics. Table 1 Characteristics of the sample, in 2017, China (N=115412). Table 1 Characteristics of the sample, in 2017, China (N=115412). Characteristics of the sample Table 1 shows that the NEPHS utilization level of IMs is low. The awareness of NEPHS was 59.0%, while the RHR rate was even lower to 30.1%. In terms of sample composition, the proportion of migrant population was higher among males, urban communities, residents duration > 3 years, years of education ≤9, inter-provincial migrants and those from HIPs. The IMs' CSC was high, 79.9% of IMs had a positive evaluation of the destination (≥18points), but IMs' SSC was low. In the past year, 56.4% of them did not participate in the listed civic activities, and 51.5% did not participate in the listed organizational activities. Statistical analysis First, we described the distribution characteristics of all the included variables (table 1). Secondly, cross-table and chi-square tests were used to verify the influence of sex, education, residence duration, community type, MR and REDL on IMs' awareness of NEPHS and RHR (table 2). Thirdly, we examined the interaction of MR and REDL on the IMs' social capital and NEPHS utilization, statistical methods including two-way ANOVA and logistic regression analysis (table 3, 4). Fourthly, we used sex, residence duration, education, community type as the control variables, MR and REDL as the moderating variables, and awareness of NEPHS and RHR as the dependent variables for a hierarchical logistic regression analysis (table 5) to discuss the degree and direction of the interaction of MR, REDL, social capital on IMs' NEPHS utilization. Finally, we conducted grouping logistic regression according to REDL (table 6), sex, residence duration, community type, and education as the control variables, MR as the moderating variables, and awareness of NEPHS and RHR as the dependent variables. Sampling weights were included in all analyses to adjust for the complex survey design. In logistic regression analysis model, Odds ratios (OR) were presented. All the analyses were performed using SPSS 22.0. Utilization of NEPHS Questions of the former were: Since 2016, "have you made suggestions to your unit/community/village or supervised the unit/community/ village affairs management?", " have you participated in property donation, blood donation, volunteer activities, etc.?", "have you reported the situation/put forward policy suggestions to relevant government departments in various ways?", "have you posted online comments on national affairs and social events or participated in related discussions?", "have you participated in party/youth league organization activities and party branch meetings?". Respondents were assigned a "yes" if they participated in any of these tasks, and a "no" if they did not. The question of social participation was "Have you participated in any of the following activities in the past year: trade unions, volunteer associations, homecoming associations, fellow-students association, 5 home town chamber of commerce, others?". Respondents were assigned a "yes" if they participated in any of these organizations, and a "no" if they did not. Demographic variables As mentioned above, MR and REDL variables have significant impacts on social capital and service utilization. Meanwhile, studies have also confirmed that sex, education, community type, residence duration, have significant impacts on IMs' NEPHS [3, 4, 28-33]. So all of these variables are included in the analysis. Education was divided into two categories: ≤9 and >9 years groups. The residence duration was divided into three groups: ≤3 years, 3-10 years and ≥ 10 years. Community types were divided into urban and rural communities. MR was divided into inter-provincial and intra-provincial migration. REDL was divided into two groups according to per capita GDP in 2017, five provinces with a per capita GDP of more than RMB 70,000 were classified as HIPs, and the other 23 provinces were classified as LMIPs. Statistical analysis Results of univariate analysis Table 2 shows that sex, community type, residence duration, education, MR, REDL and social capital all have significant impacts on IMs' NEPHS utilization. The awareness of NEPHS and RHR of inter-provincial IMs were significantly lower than those of intra-provincial IMs. 6 Compared with LMIPs, the IMs' rates of NEPHS awareness and RHR in HIPs were significantly lower. The impact of social capital, civic participation and social participation on NEPHS utilization was more prominent than other variables. With the increase of CSC, the NEPHS utilization level of IMs also increased, but the relationship is nonlinear. Table 2 Impact of sex, community type, community type, education, MR, REDL and social capital on utilization of NEPHS (N=115412). Interaction of MR and REDL on social capital and utilization of NEPHS According to table 3, we found that there was a significant difference in the IMs' social capital between different REDL groups. The IMs of HIPs had lower CSC (t=77.268, p<0.001) and civic participation (Χ2=22.258, p<0.001) than those of the IMs of LMIPs, but the former had higher social participation (Χ2=128.583, p<0.001). The IMs' NEPHS utilization level was also significantly different between HIPs and LMIPs, the NEPHS awareness (Χ2=627.718, p<0.001) and RHR (Χ2=388.239, p<0.001) of the former were lower than those of the latter. At the same time, we can also find that the level of intra-provincial IMs in the three dimensions of social capital was significantly better than that of inter-provincial IMs, and this gap was greater in HIPs. The interaction among MR, REDL and NEPHS Utilization was similar. Table 3 Distribution of social capital and NEPHS utilization levels in different MR and REDL groups (N=115412). Table 3 visually describes the influence of MR and REDL on social capital and NEPHS Utilization, and then we further analyze the direction and intensity of this effect. Two-way ANOVA results showed that the main effect of MR (F=645.074, p<0.001) and REDL (F=3187.650, p<0.001) on CSC was significant, and the interaction effect of MR and REDL on CSC was also significant (F=658.215, p<0.001). Since civic participation, social participation, awareness of NEPHS and RHR were dichotomous variables, we took these four variables as dependent variables, MR, REDL and MR*REDL as independent variables for logistic regression analysis. Table 4 shows that the main effects of MR on four dependent variables were all significantly negative, the main effects of REDL on four dependent variables were also significant but the direction was not consistent. MR*REDL had significant negative effects on CSC, civic participation, social participation, and awareness of NEPHS, but had no significant effect on RHR. Table 4 Logistic regression results of MR, REDL and MR*REDL on social capital and utilization of NEPHS (N=115412). Table 5 shows that when awareness of NEPHS was taken as the dependent variable, block 2 compared with block 1, Omnibus test Χ2 increased from 2729.821 (p<0.001) to 8680.254 (p<0.001), Cox & Snell R2 increased from 0.023 to 0.072, Hosmer & Lemeshow test Χ2 decreased from 63.315 (p<0.001) to 5.954 (p>0.05). With RHR as the dependent variable, block 2 compared with block 1, Omnibus test Χ2 increased from 2195.950 (p<0.001) to 6484.332 (p<0.001), Cox & Snell R2 increased from 0.019 to 0.055, Hosmer & Lemeshow test Χ2 decreased from 209.463 (p<0.001) to 23.646 (p<0.01). Obviously, with the introduction of social capital, the explanatory power and fitting degree of the model both have been significantly improved. When controlling 7 for other variables, CSC, civic participation and social participation all had a positive impact on NEPHS utilization. In the regression model with awareness of NEPHS as the dependent variable, THE OR values of MR*CSC, REDL*civic participation, MR*REDL*civic participation and MR*REDL*social participation were significant. In the regression model with RHR as the dependent variable, the OR values of REDL*social participation, MR*REDL*civic participation and MR*REDL*social participation were significant. However, the OR value of MR*REDL*CSC was not significant in both regression models. Table 5 Logistic regression results of sex, community type, residence duration, MR, REDL and social capital on NEPHS utilization (N=115412). Table 3 Distribution of social capital and NEPHS utilization levels in different MR and REDL groups (N=115412). To further clarify the relationship between MR, REDL, social capital and NEPHS utilization presented in table 5. We conducted grouping logistic regression analysis according to REDL (table 6). In model 1, the omnibus test Χ2 was 1963.520 (p<0.001), Cox & Snell R2 was 0.054, Hosmer & Lemeshow Χ2 was 13.431(p>0.05). In Model 2, the corresponding three indexes were 6217.905 (p<0.001), 0.075, 20.740 (p<0.01). In model 3, the corresponding three indexes were 1289.293 (p<0.001), 0.036, 10.105 (p>0.05). In model 4, the corresponding three indexes were 5074.320 (p<0.001), 0.061, 43.297 (p<0.001). The value of Cox & Snell R2 was always larger in the HIPs. Table 6 showed that the situation of Model 1 was similar to model 3, that was, the three main effects of social capital were significantly positive. In Model 2, the three main effects and three interaction effects of social capital were significantly positive, while in Model 4, the situation was very similar, the three main effects and two interaction effects were significantly positive. Combined with the information in table 5 and 6, MR and REDL had a significant moderating effect on the relationship between SSC and NEPHS utilization. Table 6 Logistic regression results of sex, community type, residence duration, education, MR and social capital on awareness of NEPHS and RHR in two groups of REDL (N=115412). Table 6 Logistic regression results of sex, community type, residence duration, education, MR and social capital on awareness of NEPHS and RHR in two groups of REDL (N=115412). Discussion However, it is noteworthy that MR difference of social capital was larger in HIPs, and there is no direct or indirect explanation for this phenomenon in existing studies. According to a survey [23], the HIPs attract more than 54.8% of the country's migrant population, of which 78.2% are inter-provincial migrants. In order to control the population size, HIPs have introduced strict household registration access standards. The inter-provincial IMs in HIPs face the dual difficulties of acculturation and institutional exclusion when constructing social capital, which may be one of the reasons why MR gap of IMs' social capital is larger in HIPs. This study supports the conclusion that the NEPHS utilization level of inter-provincial IMs is significantly lower than that of intra-provincial IMs [3, 28-33]. With regard to REDL, we support that the NEPHS utilization of IMs in HIPs is low [28-33]. Some studies believe that the low level of NEPHS utilization in HIPs is due to the insufficient supply [28, 31]. NEPHS is mainly provided by primary health institutions. Thanks to the support of the central government, primary medical service resources in LMIPs are also abundant, and even have advantages in per capita resources [42]. Our findings provide new evidence for this assertion: the NEPHS utilization of both inter-provincial and intra-provincial IMs was lower in HIPs than in LMIPs. Therefore, we believe that although the higher proportion of inter-provincial IMs (78.2%) would affect the overall NEPHS utilization level of the HIPs group, the shortage of per capita resources in these regions may be one of the main reasons for limiting the NEPHS level of the IMs. So it is urgent for the HIPs to rapidly increase their primary health resources to address the shortage of NEPHS supply. The conclusion of this study on the relationship between social capital and NEPHS utilization was consistent with previous research [6, 14-18]. Social capital has an important informational function, and it can influence health information through three mechanisms: increased information exposure, enhanced seeking abilities, and reinforced health culture or norms embedded in social networks [43]. Therefore, social capital had a greater impact on awareness of NEPHS than RHR. Discussion There were three main findings:(1) The social capital level of the inter-provincial IMs was significantly lower than that of the intra-provincial IMs, and the gap was larger in the HIPs. (2) The NEPHS utilization level of inter-provincial IMs was significantly lower than that of intra-provincial IMs, and the gap was larger in HIPs too. (3) The social capital of IMs had a positive impact on their NEPHS utilization, and the relationship between SSC and NEPHS utilization was significantly moderated by MR and REDL. This study found that the social capital of IMs was characterized by high CSC and low SSC, which was consistent with previous studies [33, 39, 40]. Yip et al. have pointed out that high CSC and low SSC may be a universal characteristic of Chinese people [41]. Migration means a loss of the original social capital for migrants [10], and this loss may be even more pronounced on the inter-provincial IMs. We found that the average level of social capital of inter-provincial IMs was lower than that of intra-provincial IMs. Bourdieu pointed out that culture is one of the key factors affecting social capital [22]. Malacculturation can lead to loss of social networks [34]. China has a vast territory, and there are often visible differences in dialects, diets and customs among provinces. This cultural difference makes it easier for people in the same province to identify with 8 each other. A survey [36] finds that, compared with those who have migrated to areas with the same dialect as those of their origin, those who have migrated to areas with different dialects have greater difficulty in social integration. This can also be verified by the lower CSC of the inter-provincial IMs in this study. REDL also had a significant impact on the social capital of IMs. IMs in HIPs and those in LMIPs face different macro institutional environments. Xiao et al. [37] argued that, compared with other regions, IMs from HIPs have the lowest level of psychological integration, but their level of economic integration and political integration is not low. We have some similar results to this study that the CSC of the IMs in HIPs was significantly lower than that of the IMs in LMIPs, but the difference in SSC between the two groups was not the case. Strength and Limitations From the perspective of ecology, this study comprehensively examined the interaction mechanism among environmental factors (REDL, MR), individual factors (individual social capital) and NEPHS utilization of IMs through a national sample. This study also revealed that the inter-provincial IMs in HIPs are in a disadvantageous position in social capital and NEPHS utilization, which provides a breakthrough for the subsequent equalization of public services for the IMs. Our study provides valuable clues for further exploring the relationship between acculturation, institutional exclusion, resources supply and IMs' health service utilization. Our findings also provide useful implications for other LMICs in dealing with the equalization of health services for IMs. However, this study also had two limitations: First, we used cross-sectional survey data in 2017, which could not reflect the latest status of China's IMs and made it difficult for us to make convincing causal inference about the path of variables. Subsequent studies need to pay attention to the change trend of these variables in the time dimension. Secondly, we only found the possible relationship between MR and acculturation, REDL and institutional exclusion, resource supply, but could not provide direct evidence. Subsequent studies need to improve the measurement of variables. Discussion We can also see that CSC had a lower impact on NEPHS utilization than SSC, both of which were lower than we expected, and the reasons for this maybe: (a) High CSC of migrant population leads to ceiling effect; (b) The IMs' social network is homogeneous and closed [44], so their SSC quality is poor [45]. The explanatory power of social capital to IMs' NEPHS utilization was smaller in LMIPs and larger in HIPs. Viswanath et al. [46] found a stronger relationship between social capital and access to health information in socioeconomic disadvantaged communities. Perkins et al. [13] noted that individuals in LMICs rely more on social networks to access health services. This study found that social capital played a more important role for inter provincial IMs in HIPs than LMIPs. A more general conclusion might be that the impact of social capital on access to health services is more important in under-resourced areas. 9 This study also found that social capital has both a main effect and buffer effect on IMs' NEPHS utilization in HIPs, but only main effect in LMIPs. The latter is in line with Cohen and Wills' points [47], while the former supports Fried and Tiegs' conclusion [48]. The original reason may be that the gap in the social capital of inter-provincial and intra-provincial migrants is small in LMIPs and large in HIPs. MR is related to acculturation, while REDL reflects the intensity of institutional exclusion, so MR and REDL essentially reflect the IMs' socioeconomic status. Uphoff et al [19] proposed three paths by which socioeconomic status could affect the relationship between social capital and health outcomes: (a) A more significant social capital benefit on the health of disadvantaged persons in society, and no effects or limited health benefits for those in positions higher up in the social ladder. (b) People with a low socioeconomic status will generally have less social capital, and the capital available to them cannot be used effectively for health benefits. (c) Social capital might benefit the better-off in society while excluding people with a lower socioeconomic status. Obviously, the relationship between social capital and health services of IMs in HIPs conformed to path a. Conclusions The level of social capital and NEPHS utilization of inter-provincial IMs was significantly lower than that of intra-provincial IMs, and these gaps were more prominent in HIPs. Social capital had a significant positive effect on NEPHS utilization of IMs. In LMIPs, SSC had only a main effect, while in HIPs, SSC had both a main effect and buffer effect. We believe that SSC is more important for people of lower socioeconomic status to access health services in resource-poor settings. Therefore, the next step of equalization of public services for IMs should focus on HIPs. One is to increase the supply of grass-roots health services; the other is to pay attention to the construction of social networks for inter-provincial IMs. Authors' Information Zhen Yang. (1) School of medicine, Tongji university, Shanghai 200092, China; Zhen Yang. (1) School of medicine, Tongji university, Shanghai 200092, China; (2) School of medicine, Jinggangshan university, Jian, Jiangxi 343009, China. Chenghua Jiang. (1) School of medicine, Tongji university, Shanghai 200092, China. Jiansheng Hu. (2)School of medicine, Jinggangshan university, Jian, Jiangxi 343009, Ch (2) School of medicine, Jinggangshan university, Jian, Jiangxi 343009, China. Chenghua Jiang. (1) School of medicine, Tongji university, Shanghai 200092, China. h ( ) h l f d h Jiansheng Hu. (2)School of medicine, Jinggangshan university, Jian, Jiangxi 343009, China. Abbreviations CSC: Cognitive Social Capital 10 RHR: Registration of Health Records HIPs: high income provinces IMs: Internal Migrants LMICs: low- and middle- income Countries LMIPs: low- and middle- income provinces MR: Migration Range NEPHS: National Essential Public Health Services REDL: Regional Economic Development Level SSC: Structural Social Capital Authors’ contributions All authors participated in the design of the study. Z Y carried out the statistical analysis and composed the first draft. CH J gave opinions for modification. JS H was responsible for data sorting and manuscript editing. All authors read and approved the final manuscript. Competing interests No competing interests in this study. Availability of data and materials Since the data used in this paper were provided by the Migrant Population Service Center, which is the top agency governing migrant population issues in China, we had to sign a legally binding agreement with the agency that we will not share any original data with any third parties. However, interested researchers can apply for access to the data at http://www.ldrk.org.cn/. Ethics approval and consent to participate The “National Internal Migrant Dynamic Monitoring Survey, 2017” data is publicly available to authorized researchers who have been given permission by the Migrant Population Service Center, and written informed consents were obtained from all participants. The analysis of public access data was exempted by the local IRB; as this involved analyzing de-identified existing data, ethical approval was not required. 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Ein Beitrag zur Serumbehandlnng des Tetanus
Deutsche medizinische Wochenschrift/Deutsche Medizinische Wochenschrift
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public-domain
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I. Januar. Frostschauer will Patientin nicht gehabt haben, Kopfschmerzen fehlten; sie fühlte sich nur matt und unbehaglich, besonders als sich am Abend vereinzelt Zuckungen ein- stellten. Am 10. September nahmen alle Symptome an Intensität zu. St t 11 S t b 1903 P ti ti li t p y p Status praesens vom 11. September 1903: Patientin liegt regungs- lo im Bett. Da Sensorium ist frei. Die Stirn ist gerunzelt, die Ge- ichtczüge zeigen das Starre und Unbewegliche einer Maske. Die ver- (9lgerten I'iipilleii reagieren auf Lichteinfall fast gar nicht. Es besteht starker Trismus, die Zahareilien liegen fest aufeinander, der Unter- kiefer Ist gänzlich unbeweglich. Der etwas rückwärts gebeugte Kopf kann weder aktiv noch passiv auch nur um 1 cm verschoben werden. Der Rücken ist .tark iiacli vorn gekrümmt, sodall man bequem die Faust zwischen Rücken un(l Bett hindurchschieben kann. Also aus- gesprochener Opisthotoniis. Die Bauchdecken sind hart und gespannt. Die Aritie sind im Ellenbogengelenk rechtwinklig gebeugt, die Hände teheri i mäßiger Flexion, und die Fingei befinden sich gleichfalls in halber Beugestellung. Die Arme können in engen Grenzen mühsam bewegt werden. Die iiiiteren Extremitäten sind aktiv und passiv uji- beweglich. Die Atinutig ist frei, der Pulsschlag regelmäßig, kräftig, oll ich zähle 7() Schläge in der Minute. Die tonische Starre des Körpers wiid in Paiiseii von etwa 2-3 Minuten von heftigen Zuckungen unterbrochen, dic .stoliartig den ganzen Körper erschüttern und von einem Stithneuu bigleitet siuid. Die kioniseluen Zuckungen sind vor- wiegeuid in der linken Körpereite lokalisiert, sodaß der Leib bei jederiu Stoll nach ueclits iibeuzuikippen scheint. Jede Berührung und jedes laute Geräusch löst sofort einen klonischen Krampf aus. Es winde deshalb die tra1.lc, die hart an dein Häuschen, das die Kranke beherbergte, orüberfülurte, für F'uuhrwerk gesperrt, eine Malregel, die sich ausführen lieI, weil die Dorfstraf3e cirka lOfl in vorher eine Neben- stral3e abzweigte. die, einen IIäuseikomplex iiuselartig umschliel3end, später wieder in die Hauptstral3e einmündete. Jede Zuckung veu- tirsachte der Patientin Schmerzen. Appetit ist vorhanden, besonders lebhaft ist (las Durstgefiihl. p , September. Es ist im Laufe des 20. September nur noch eine solche gewaltige, das Leben im höchsten Grade gefährdende Zuckung aufgetreten. Temperatur 37,6-38,5° C. K f k l idli h d h d g p September. Wohlbefinden. Kopf kann leidlich gedreht und auch etwas voruuüber gebeugt werden, Opisthotoniis geringer, Zuckungen selten und schwach. Temperatur abends 37,6° C. S b A d H d id k d b September. I. Januar. I. Januar. der als alle früheren, der die erschreckten Eltern veranlaßte, mich rufen zu lassen. Sogleich nach meinem Eintreffen erfolgte ein neuer grß1icher Krampfanfall, sodaß ich selbst beobachten konnte: dei' Körper ist wie eine Gerte gebogen und schwebt in der Luft, nur der Kopf und die Schultern und die Füße bilden die Stützpunkte. Das Gesicht ist blau verfärbt, die Atmung sistiert, der Puis ist nicht zu fühlen. Der Anfall dauert wohl 20 Sekunden. Dann fängt Patientin wieder an zu atmen unter lautem Stöhnen. Sie fühlt sich matt und müde und klagt über Schmerzen in allen Gliedern. Sofort Injektion der noch vorhandenen 100 A.-E. und eine Morphiumin.jektion von 0,02. Die Chlou'al-Morphin-Lösung, die nur noch drei- bis vierstitndlich ver- abfolgt war, wird wieder stündlich gegeben. Der tonische Spasmuis scheint mir nach dem Anfall in allen Muskelgebieten geringer zu sein. Höchste Temperatur 38,7°. der als alle früheren, der die erschreckten Eltern veranlaßte, mich rufen zu lassen. Sogleich nach meinem Eintreffen erfolgte ein neuer grß1icher Krampfanfall, sodaß ich selbst beobachten konnte: dei' Körper ist wie eine Gerte gebogen und schwebt in der Luft, nur der Kopf und die Schultern und die Füße bilden die Stützpunkte. Das Gesicht ist blau verfärbt, die Atmung sistiert, der Puis ist nicht zu fühlen. Der Anfall dauert wohl 20 Sekunden. Dann fängt Patientin wieder an zu atmen unter lautem Stöhnen. Sie fühlt sich matt und müde und klagt über Schmerzen in allen Gliedern. Sofort Injektion der noch vorhandenen 100 A.-E. und eine Morphiumin.jektion von 0,02. Die Chlou'al-Morphin-Lösung, die nur noch drei- bis vierstitndlich ver- abfolgt war, wird wieder stündlich gegeben. Der tonische Spasmuis scheint mir nach dem Anfall in allen Muskelgebieten geringer zu sein. Höchste Temperatur 38,7°. des 8. September will die Kranke beim Pufferessen (Kartoffelkuchen) bemerkt haben, daß ihr das Kauen schwer fiel. Sie habe mit gutem Appetit gegessen und sich, abgesehen von einer geringen Müdigkeit, ganz wohl gefühlt., auch die Nacht leidlich geschlafen. Am anderen Morgen verließ sie das Bett, fand aber, daß sie den Mund nur noch wenig öffnen und den Kopf mühsam drehen und bewegen konnte. Im Laufe des Tages soll auch der Rücken steif geworden sein und die Gesichtszüge sollen nach Angabe der Eltern einen starren Ausdruck angenommen haben. Die Kranke mußte das Bett aufsuchen, weil auch die Beine schwer beweglich wurden. DEUTSCHE MEDIZINISCHE WOCHENSCHRWT. DEUTSCHE MEDIZINISCHE WOCHENSCHRWT. No. I 22 22 Dieses Dokument wurde zum persönlichen Gebrauch heruntergeladen. Vervielfältigung nur mit Zustimmung des Verlages. Aus der Praxis. Ein Beitrag zur SerumbehandJung des Tetanus. Von Dr. K. Walistabe in Güsten (Anhalt). Solange ich Arzt bin (seit 1889), habe ich drei T etanusfäfle ge- sehen und von diesen einen selbständig behandelt. Die beiden ersten Erkrankungen fallen in die serumlose Zeit, sie endeten t;dlic1i. T)er letzte Fall, dessen Krankengeschichte ich hier geben will, wurde mit Antitoxin behandelt und verdankt wohl dem Serum seinen Ausgang in Heilung. Am 11 September 1903 wurde ich zu der 18 Jahre alten Luise li Aus der Praxis. Ein Beitrag zur SerumbehandJung des Tetanus. Von Dr. K. Walistabe in Güsten (Anhalt). Solange ich Arzt bin (seit 1889), habe ich drei T etanusfäfle ge- sehen und von diesen einen selbständig behandelt. Die beiden ersten Erkrankungen fallen in die serumlose Zeit, sie endeten t;dlic1i. T)er letzte Fall, dessen Krankengeschichte ich hier geben will, wurde mit Antitoxin behandelt und verdankt wohl dem Serum seinen Ausgang in Heilung. A 11 S t b 1903 d i h d 18 J h lt L i li Solange ich Arzt bin (seit 1889), habe ich drei T etanusfäfle ge- sehen und von diesen einen selbständig behandelt. Die beiden ersten Erkrankungen fallen in die serumlose Zeit, sie endeten t;dlic1i. T)er letzte Fall, dessen Krankengeschichte ich hier geben will, wurde mit Antitoxin behandelt und verdankt wohl dem Serum seinen Ausgang in Heilung. A 11 S b 1903 d i h d 18 J h l L i li g Am 11. September 1903 wurde ich zu der 18 Jahre alten Luise li.. Tochter des Grundbesitzers Friedrich R. zu R., gerufen. Am Abend DEUTSCHE MEDIZINISCHE WOCHENSCHRIFT. 23 I. Januar. darübem' kaun nuan uuuur eine Vermutung haben. Eine couupieremude Wirkung hat das Serum uuicht gehabt, wie uviu' eine solche von dein Diphtheritisserumnu gewöhnt sind. Vielleicht deshalb nicht, uveil es zu 51)ät zur Anwendung gekommen ist, Aber den Eindruck habe ich gewonnen, daß durch das Antitoxiuu dem Organismus Zeit gewonnen wurde, seine Truppen xii sammeln mind dem Feind entgegen zu werfen, Jugend welche Folge- eischeinuungen der Seuuuminjektionen habe ich nicht gesehen. Oh dei Fall zu deuu schwersten zu rechncuu ist, mud ich dahin- stellen, weil ich zu wenig Tetanuisfälle gesehen habe. Welcheur Ver- lauf die Erkrankung chine Antitoxin genommen hätte. darübem' kaun nuan uuuur eine Vermutung haben. Eine couupieremude Wirkung hat das Serum uuicht gehabt, wie uviu' eine solche von dein Diphtheritisserumnu gewöhnt sind. Vielleicht deshalb nicht, uveil es zu 51)ät zur Anwendung gekommen ist, Aber den Eindruck habe ich gewonnen, daß durch das Antitoxiuu dem Organismus Zeit gewonnen wurde, seine Truppen xii sammeln mind dem Feind entgegen zu werfen, Jugend welche Folge- eischeinuungen der Seuuuminjektionen habe ich nicht gesehen. 'rlieuapie: Injektion von (1,02 Morph. mur. Ordination: Chlorai liydrati 1(1,00, Morph. muir. ),l : 20(.) Aqu. dest. .. zweistündlich ein Eßlöffel. Urn 5 Uhr nachmittags bestellte ich telegraphisch bei der Firma Dr. Siebert und Dr. Ziegeubein in Marburg 200 A-E. des 1-lehringschen Tetanus.sernms, das am anderen Morgen hier eintraf. Chl i 'rlieuapie: Injektion von (1,02 Morph. mur. Ordination: Chlorai liydrati 1(1,00, Morph. muir. ),l : 20(.) Aqu. dest. .. zweistündlich ein Eßlöffel. Urn 5 Uhr nachmittags bestellte ich telegraphisch bei der Firma Dr. Siebert und Dr. Ziegeubein in Marburg 200 A-E. des 1-lehringschen Tetanus.sernms, das am anderen Morgen hier eintraf. i i Chl i September. Status idem. Patientin steht unter Chlorai Morphin-Wirkung. Die tonische Starre ist dieselbe vie gestern, die Zuckungen treten regelmäßig ein. Es wurden 100 A-E, in die Sub- clavikulargegend eingespritzt. Die Medikation bleiht dieselbe. S t b K i A d Wi d 100 A E i di lb September. Status idem. Patientin steht unter Chlorai Morphin-Wirkung. Die tonische Starre ist dieselbe vie gestern, die Zuckungen treten regelmäßig ein. Es wurden 100 A-E, in die Sub- clavikulargegend eingespritzt. Die Medikation bleiht dieselbe. S b K i A d Wi d 100 A E i di lb September. Keine Anderung. Wieder 100 A.E. in dieselbe Korpergegend. Die Temperatur, die am Il. September 3$0 betrug, schwankte auuu 12. und 13. September zwischen 37,7 und 38,21) C. 4. Oktober. Die Kranke kann mit TJuuterstiitzuung einige schliir- fende Schritte machen. Oh d i F ll d h h i d i h d hi 30. September. Patientin hat nui' noch dreimal erbrochen. Tern- peratuur normal. I. Januar. Arme und Hände weiden gestreckt und gebeugt, (lie Beine lassen sich passiv bewegen. Allgemeinbefinden glut, ruhiger Schlaf, reichliche Nahrungsaufnahme. Temperatur 37 bis 37,9° C. Nuit hin und wieder ein Eßlöffel des Narkoticums. In der Nacht zum 24. September heftiges Erbrechen. Die Kranke speit alle 10-15 Minuten cirka 50 g eines grünlichen dünnflüssigen Schleimes aus, ohne daß ein Wiirgen vorhergeht. Legt unan die Hauud in der Magengegeuud leicht auf die fast ganz erschlaffte Bauiehdecke, so fühlt man deutlich, lyle ein kugeliges Gebilde dagegen schnellt. Ohne Zweifel ist es der Magen. der durch ruuckartige Kouutraktioneuu seinen Inhalt herausschleuudert, Es wird eneu'gisch Chloral-Morphiuu gegeben. Temperatur 37,2 bis 38.2° C. 25. und 26. September. Das Erbrechen dauert ungeschwächt fort. Aligemeinbefinden nicht gerade schlecht. Temperatur schwankt zwischeuu 37,4 und 38,4° C. bi b i i h d i l h 27. bis 29. September. Die Pausen zwischen den einzelnen Brech- akten werden länger, cirka Stunde. Patientin klagt über Hunger und trinkt aus einer Tasse oder einem Glase gierig Milch, Kakao und Brühe mit Ei; aber sie behält noch fast nichts bei sich. Temperatur 37,2 bis 37,90 C. 30. September. Patientin hat nui' noch dreimal erbrochen. Tern- peratuur normal. 1 Ok b Di S i ll M i k l bi i h l 1. Oktober. Die Starre in allen Muiskelgebieten weicht langsauru. Ok b P i i i j B tt d tillt i Diagnose: Tetanus. Eine Verletzung ist am ganzen Körper trotz cörigen Siicliens nicht aufzufinden. Tndes gibt die Kranke an. dali einige Tage vor dem Erscheinen dei ersten Krankheitssvmptome ihre Nase und die Oberlippe etwas gerötet und geschwollen waren, als venn mau den Schnupfen bekommt". Es ist wohl nicht ganz von der 1-fand zu wei.en, ilaß die Patientin, die mit Feldarbeit beschäftigt war, eine Excoriation an der Nase gehabt hat, die die Eingangspforte für die Tetanuserreger ge\veseuu ist.. g g 2 Oktober. Patientin sitzt auifu'ec'ht jun Bett und stillt eigen- häuudig ihren wiitenden Hunger mit flüssiger nuud breiigeu' Nahrung 4 Ok b Di K k k i TJ ii i i hlii 4. Oktober. Die Kranke kann mit TJuuterstiitzuung einige schliir- fende Schritte machen. Oh d i F ll d h h i d i h d hi Oh dei Fall zu deuu schwersten zu rechncuu ist, mud ich dahin- stellen, weil ich zu wenig Tetanuisfälle gesehen habe. Welcheur Ver- lauf die Erkrankung chine Antitoxin genommen hätte. 1. Oktober. Die Starre in allen Muiskelgebieten weicht langsauru. Ok b P ti ti it t j B tt d tillt i 2 Oktober. Patientin sitzt auifu'ec'ht jun Bett und stillt eigen- häuudig ihren wiitenden Hunger mit flüssiger nuud breiigeu' Nahrung i k k i ii i i hlii I. Januar. K i B ll di h k i September. Keine Besserung, allerdings auch keine Ver- schlimmerung. Temperatur zwischen 38 und 38,20. ich ließ noch ein- mal 200 A-E. schicken. S b id A f i t t b i A f September. Status idem. Atmung frei, setzt nur beim Auf- treten der klonischen Zuckungen einen Augenblick aus. Nahrungs- aufnahme genügend, Patientin schlürft durch einen Strohhalm, dei durch eine Zahnlücke in den Mund eingeführt wird, gierig Milch, Kakao, Brühe nut Ei, Wein und Wasser. Es verden 100 A.E. eingespritzt. bi 19 S b R l äßi k äfti H täti k it P l bi 19. September. Regelmäßige, kuäftige Herztätigkeit, Puls M5. Temperatur schwankt zwischen 37,5 unid 38,50 C. Die Zuckungen nehmen an Häufigkeit und intensität langsam, aber merklich ab. Patientin schläft viel und erwacht nur etwa alle Stunden, wenn eine stärkere Zuckung den Körper erschüttert. Obgleich die klonischen Stöße viel schwächer auftreten, sieht man deutlich, daß die Nacken- muskulatur und beide Arme, aber fast ausschließlich die linke Körper- seite und das linke Bein betroffen werden, während die tonische Starre rechts und links gleich stark ausgeprägt ist. Der Kopf kann schon ein wenig bewegt werden, die Zahnreihen werden '/. cm auseinander- gebracht, die Arme können gestreckt werden, die Beine lassen sich passiv etwas beugen und der Opisthotonus ist geringer. flber Schmerzen klagt die Kranke wenig mehr. Da traf in der Nacht vorn 19. zum 20. September den Körper ein Stoß, furchtbarer und anhalten-
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Performance Evaluation of RapiSure (EDGC) COVID-19 S1 RBD IgG/Neutralizing Ab Test for the Rapid Detection of SARS-CoV-2 Antibodies
Diagnostics
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Keywords: COVID-19 antibodies; neutralizing antibodies; SARS-CoV-2; rapid chromatographic immunoassay Citation: Kim, H.N.; Yoon, J.; Jang, W.S.; Lim, C.S. Performance Evaluation of RapiSure (EDGC) COVID-19 S1 RBD IgG/Neutralizing Ab Test for the Rapid Detection of SARS-CoV-2 Antibodies. Diagnostics 2023, 13, 643. https://doi.org/ 10.3390/diagnostics13040643 Academic Editor: Lusheng Song Received: 12 December 2022 Revised: 25 January 2023 Accepted: 7 February 2023 Published: 9 February 2023 Citation: Kim, H.N.; Yoon, J.; Jang, W.S.; Lim, C.S. Performance Evaluation of RapiSure (EDGC) COVID-19 S1 RBD IgG/Neutralizing Ab Test for the Rapid Detection of SARS-CoV-2 Antibodies. Diagnostics 2023, 13, 643. https://doi.org/ 10.3390/diagnostics13040643 diagnostics diagnostics diagnostics diagnostics Article Performance Evaluation of RapiSure (EDGC) COVID-19 S1 RBD IgG/Neutralizing Ab Test for the Rapid Detection of SARS-CoV-2 Antibodies Ha Nui Kim , Jung Yoon, Woong Sik Jang and Chae Seung Lim * Ha Nui Kim , Jung Yoon, Woong Sik Jang and Chae Seung Lim * Department of Laboratory Medicine, Korea University College of Medicine, Seoul 08308, Republic of Korea * Correspondence: malarim@korea.ac.kr; Tel.: +82-2-2626-3245; Fax: +82-2-2626-1465 Abstract: The accurate detection of anti-neutralizing SARS-CoV-2 antibodies can aid in the under- standing of the development of protective immunity against COVID-19. This study evaluated the diagnostic performance of the RapiSure (EDGC) COVID-19 S1 RBD IgG/Neutralizing Ab Test. Using the 90% plaque reduction neutralization test (PRNT90) as a reference, 200 serum samples collected from 78 COVID-19-positive and 122 COVID-19-negative patients were divided into 76 PRNT90- positive and 124 PRNT90-negative groups. The ability of the RapiSure test to detect antibodies was compared to that of the STANDARD Q COVID-19 IgM/IgG Plus test and that of PRNT90. The positive, negative, and overall percent agreement between the RapiSure and STANDARD Q test was 95.7%, 89.3%, and 91.5%, respectively, with a Cohen’s kappa of 0.82. The RapiSure neutralizing antibody test results revealed a sensitivity of 93.4% and a specificity of 100% compared to the PRNT results, with an overall percent agreement of 97.5% and Cohen’s kappa of 0.95. The diagnostic perfor- mance of the RapiSure test was in good agreement with the STANDARD Q COVID-19 IgM/IgG Plus test and comparable to that of the PRNT. The RapiSure S1 RBD IgG/Neutralizing Ab Test was found to be convenient and reliable and, thus, can provide valuable information for rapid clinical decisions during the COVID-19 pandemic. 1. Introduction The coronavirus disease (COVID-19) pandemic still remains a global concern even after 2 years after the first reported case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [1]. Molecular diagnostic tests for SARS-CoV-2 infection and serological tests for the corresponding antibody responses are essential for patient man- agement. Serological tests are especially useful in public health management in terms of pandemic surveillance, assessment of vaccine effectiveness, and evaluation of immune responses [2,3]. Academic Editor: Lusheng Song Received: 12 December 2022 Revised: 25 January 2023 Accepted: 7 February 2023 Published: 9 February 2023 p Humoral and cell-mediated responses are the two arms of the adaptive immune system, which protect against infection and reduce disease severity [4]. Protection is mainly mediated by neutralizing antibodies (nAbs) against SARS-CoV-2 [5]. Almost all patients with COVID-19 develop detectable nAbs after 3–4 weeks of illness [6,7]. Among the virus neutralization assays, which measure serum nAb titers, the plaque reduction neutralization test (PRNT) is considered the gold standard due to its high sensitivity [8]. However, PRNT is technically demanding, time-consuming, and requires biosafety level 3 facilities and trained personnel [9]. Recently, an ELISA-based neutralization assay was developed, but it requires additional equipment, such as a microplate reader [10]. Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). The humoral response to SARS-CoV-2 infection is directed against the viral spike (S) and nucleocapsid proteins [11]. The S protein is responsible for viral entry by interacting with the angiotensin-converting enzyme 2 (ACE2) receptor via a receptor-binding domain https://www.mdpi.com/journal/diagnostics Diagnostics 2023, 13, 643. https://doi.org/10.3390/diagnostics13040643 Diagnostics 2023, 13, 643 2 of 11 2 of 11 (RBD), located in its S1 domain [12,13]. The RBD is the most immunodominant epitope of nAbs [14], and the detection of anti-RBD antibodies is employed in tests, such as point- of-care testing (POCT) [15,16]. Unlike ELISA, POCT does not require sample preparation and is more cost-effective, rapid, and easy to use. Fast and convenient evaluation of SARS- CoV-2 antibodies can aid in determining the level of immune status during the ongoing response to the pandemic. In this study, the diagnostic performance of the RapiSure (EDGC) COVID-19 S1 RBD IgG/Neutralizing Ab Test (EDGC, Incheon, Republic of Korea) was evaluated and compared to that of the STANDARD Q COVID-19 IgM/IgG Plus test (SD Biosensor, Suwon, Republic of Korea), the first diagnostic device to detect SARS-CoV-2 antibodies approved by Ministry of Foods and Drug Safety in Korea. 2.1. Sample Collection Since the COVID-19 pandemic, COVID-19 RT-PCR tests, including the STANDARD M nCoV Real-Time Detection kit (SD Biosensor, Suwon, Republic of Korea) and Allplex 2019- nCoV Real-time PCR (Seegene, Seoul, Republic of Korea), have been routinely implemented at Korea University Guro Hospital. This study used 200 serum samples from patients who visited the Korea University Guro Hospital with respiratory symptoms from December 2020 to September 2021. Using RT-PCR analysis, the samples were divided into COVID- 19-positive (n = 78) and -negative groups (n = 122); for each positive sample, the number of days after illness onset was recorded. The samples were analyzed for the presence of nAbs against SARS-CoV-2, with the PRNT used as a reference method. Before testing, the collected serum samples were stored at −70 ◦C. This study was approved by the Institutional Review Board of the Korea University Guro Hospital (2021GR0481). 2.2. RapiSure COVID-19 S1 RBD IgG/Neutralizing Ab Test 2 3 P f C i f th R iS T t ith th PRNT d th STANDARD Q (c) (c) re 1. Schematic diagram and images of the RapiSure COVID-19 S1 RBD IgG/Neutra (a) S1 RBD IgG; (b) neutralizing antibody; and (c) images of the RapiSure COVID-1 Neutralizing Ab Test using representative serum samples with positive, weakly pos tive results from left to right. Figure 1. Schematic diagram and images of the RapiSure COVID-19 S1 RBD IgG/Neutralizing Ab Test. (a) S1 RBD IgG; (b) neutralizing antibody; and (c) images of the RapiSure COVID-19 S1 RBD IgG/Neutralizing Ab Test using representative serum samples with positive, weakly positive, and negative results from left to right. Performance Comparison of the RapiSure Test with the PRNT and the STAND 2.3. Performance Comparison of the RapiSure Test with the PRNT and the STANDARD Q COVID-19 IgM/IgG Plus Test VID-19 IgM/IgG Plus Test The PRNT was used as a reference method to confirm the presence of ser RS-CoV-2 nAbs. The PRNT was performed as previously described at the Dep Microbiology of Korea University where a biosafety level three facility is avail highest serum dilution that resulted in 90% (PRNT90) and 50% (PRNT50) redu l plaque numbers when compared to controls was determined. PRNT90 and s that were diluted by at least 1:20 were considered positive for anti-SAR bs [18]. For five positive samples that had a PRNT90 titer dilution of 1:160, se The PRNT was used as a reference method to confirm the presence of serum anti- SARS-CoV-2 nAbs. The PRNT was performed as previously described at the Department of Microbiology of Korea University where a biosafety level three facility is available [17]. The highest serum dilution that resulted in 90% (PRNT90) and 50% (PRNT50) reductions in viral plaque numbers when compared to controls was determined. PRNT90 and PRNT50 titers that were diluted by at least 1:20 were considered positive for anti-SARS-CoV-2 nAbs [18]. For five positive samples that had a PRNT90 titer dilution of 1:160, serial dilutions were used to measure the limit of detection (LoD) in the RapiSure test. Positive PRNT samples were divided into low- and high-titer groups and used to evaluate the performance of the RapiSure test. p p s were used to measure the limit of detection (LoD) in the RapiSure test. NT samples were divided into low- and high-titer groups and used to eva ormance of the RapiSure test. 2.2. RapiSure COVID-19 S1 RBD IgG/Neutralizing Ab Test The RapiSure COVID-19 S1 RBD IgG/Neutralizing Ab Test (RapiSure test) is a rapid, portable qualitative chromatographic colloidal gold-based lateral flow immunoassay that uses two test lanes: one for the detection of S1 RBD IgG antibody and the other for the detection of anti-SARS-CoV-2 nAbs. The test is initiated by adding 25 µL of serum sample to the sample collection sites of the test cassette. In each test lane, the sample then moves chromatographically through a filter pad, a conjugate pad, a nitrocellulose membrane that contains a control (C) band and a test (T) band, and a moisture absorption pad. In the S1 RBD IgG test lane, the C and T bands are coated with goat anti-chicken-IgY and mouse monoclonal anti-human IgG antibodies, respectively. If anti-S1 RBD antibod- ies are present, they form immune complexes with gold-labeled S1 RBD antigen. The complexes then react with the anti-human IgG antibody in the T band and develop a red line. In the nAb test lane, the presence of anti-SARS-CoV-2 nAbs is indicated by the absence of a colored line in the T band. The SARS-CoV-2 nAbs form immune complexes with gold-labeled S1 protein. These immune complexes cannot interact with ACE2 antigen in the T band, resulting in a weak or absent red line. The C band in both the test lanes turns from blue to red when the sample passes through the membrane, indicating a valid test. To avoid false results, the results were read 10–15 min after test initiation. A schematic diagram and pictures of the RapiSure test are shown in Figure 1. 3 of 11 Diagnostics 2023, 13, 643 s 2023, 13, 643 (a) (b) Figure 1. Cont. (a) (a) (b) 4 of 11 Diagnostics 2023, 13, 643 , , (c) gure 1. Schematic diagram and images of the RapiSure COVID-19 S1 RBD IgG/Neutra est. (a) S1 RBD IgG; (b) neutralizing antibody; and (c) images of the RapiSure COVID-1 G/Neutralizing Ab Test using representative serum samples with positive, weakly pos egative results from left to right. Figure 1. Schematic diagram and images of the RapiSure COVID-19 S1 RBD IgG/Neutralizing Ab Test. (a) S1 RBD IgG; (b) neutralizing antibody; and (c) images of the RapiSure COVID-19 S1 RBD IgG/Neutralizing Ab Test using representative serum samples with positive, weakly positive, and negative results from left to right. 3. Results A total of 78 serum samples confirmed for SARS-CoV-2 infection by COVID-19 RT-PCR comprised 44 males and 34 females, with an average age of 68 years (range, 26–87 years). Two of the 78 samples were negative in the PRNT and reclassified as negative for nAbs, resulting in the 76 PRNT-positive samples. When the RapiSure S1 RBD IgG results were compared with those of RT-PCR, the sensitivity was 97.4% (76/78) (95% confidence interval (CI): 91.1–99.3%) and the specificity was 96.7% (118/122) (95% CI: 91.9–98.7%). The results of the RapiSure Neutralizing Ab Test conducted on the PRNT-positive and -negative samples showed a sensitivity of 93.4% (71/76) (95% CI: 85.5–97.2%) and a specificity of 100% (124/124) (95% CI: 97.0–100%). An overall percent agreement between RapiSure and PRNT was 97.5% (95% CI: 94.3% to 99.2%), with a κ value of 0.95. The sensitivity of the RapiSure Neutralizing Ab Test was lower than that of the S1 IgG test; both tests showed high specificity (Table 1). Table 1. Performance of RapiSure COVID-19 S1 RBD IgG/Neutralizing Ab Test compared to the results of COVID-19 RT-PCR and PRNT50/PRNT90. The RapiSure S1 RBD IgG test results were compared with those of RT-PCR, and the Neutralizing Ab test results were compared with those of PRNT, respectively. Table 1. Performance of RapiSure COVID-19 S1 RBD IgG/Neutralizing Ab Test compared to the results of COVID-19 RT-PCR and PRNT50/PRNT90. The RapiSure S1 RBD IgG test results were compared with those of RT-PCR, and the Neutralizing Ab test results were compared with those of PRNT, respectively. RT-PCR a Sensitivity (95% CI b) Specificity (95% CI) PRNT c50 and PRNT90 Sensitivity (95% CI) Specificity (95% CI) Percent Agreement (95% CI) Cohen’s Kappa (95% CI) Positive Negative Positive Negative RapiSure Positive 76 4 97.4% (91.1–99.3%) 96.7% (91.9–98.7%) 71 0 93.4% (85.5–97.2%) 100% (97.0–100%) 97.5% (94.3–99.2%) 0.95 (0.90–0.99) Negative 2 118 5 124 a COVID-19 RT-PCR: STANDARD™M nCoV Real-Time Detection kit or AllplexTM 2019-nCoV assay; b CI: Confidence interval; c Plaque reduction neutralization test. a COVID-19 RT-PCR: STANDARD™M nCoV Real-Time Detection kit or AllplexTM 2019-nCoV assay; b CI: Confidence interval; c Plaque reduction neutralization test. The sensitivity of the STANDARD Q test compared to the COVID-19 RT-PCR was 95.7% and comparable to that of the RapiSure (97.4%). However, the specificity of STAN- DARD Q was 90.8%, which was lower than RapiSure (96.7%). 2.2. RapiSure COVID-19 S1 RBD IgG/Neutralizing Ab Test Additionally, results with the previously marketed STANDARD Q C /IgG Plus test (STANDARD Q test) were compared with those of the RapiSur test. For paired comparisons, only IgG results from the STANDARD Q test w STANDARD Q test is another rapid chromatographic immunoassay that de anti-SARS-CoV-2 antibodies. The nitrocellulose membrane has three test b nd M, which are coated with anti-chicken IgY, monoclonal anti-human IgG, a nal anti-human IgM antibodies, respectively. If anti-SARS-CoV-2 antibodies Additionally, results with the previously marketed STANDARD Q COVID-19 IgM/IgG Plus test (STANDARD Q test) were compared with those of the RapiSure S1 RBD IgG test. For paired comparisons, only IgG results from the STANDARD Q test were used. The STANDARD Q test is another rapid chromatographic immunoassay that detects serum anti-SARS-CoV-2 antibodies. The nitrocellulose membrane has three test bands, C, G, and M, which are coated with anti-chicken IgY, monoclonal anti-human IgG, and monoclonal anti-human IgM antibodies, respectively. If anti-SARS-CoV-2 antibodies are present, they form immune complexes with gold-labeled recombinant SARS-CoV-2 proteins. The com- plexes are then captured by the coated isotype-specific antibodies, resulting in violet lines at the corresponding positions. According to the manufacturer, when compared to RT-PCR, the clinical sensitivity and specificity of the STANDARD Q test are 99.03% and 98.65%, respectively. Diagnostics 2023, 13, 643 5 of 11 2.4. Statistical Analysis 2.4. Statistical Analysis The sensitivity, specificity, and percent agreement were calculated based on the results of each test. The results of the RapiSure S1 RBD IgG and nAb tests were compared with those of COVID-19 RT-PCR and PRNT, respectively, to determine false positives or negatives. To determine the strength of agreement between the RapiSure and STANDARD Q tests, Cohen’s kappa (κ) was calculated using MedCalc Software version 20.110 (MedCalc Soft-ware Ltd., Ostend, Belgium). The κ value was interpreted in terms of strength of agreement as follows: <0.20 was poor, 0.20–0.40 was fair, 0.41–0.60 was moderate, 0.61–0.80 was good, and 0.81–1.00 was very good [19]. The p-value was calculated using the Chi- squared test in MedCalc, with a p-value of less than 0.05 considered statistically significant. 3. Results Table 3. Evaluation of the limit of detection of the RapiSure COVID-19 S1 IgG/Neutralizing Ab Test using positive samples with a PRNT90 titer of 1:160. The gray-shaded boxes indicated their lowest dilution factor. Table 3. Evaluation of the limit of detection of the RapiSure COVID-19 S1 IgG/Neutralizing Ab Test using positive samples with a PRNT90 titer of 1:160. The gray-shaded boxes indicated their lowest dilution factor. Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Dilution Factor S1-IgG nAb S1-IgG nAb S1-IgG nAb S1-IgG nAb S1-IgG nAb 1:2 + + + + + + + + + + 1:4 + + + + + + + + + + 1:8 + + + + + + + + + + 1:16 + + + + + + + + + + 1:32 + + + + + + + + + − 1:64 + − + + + + + + − − 1:128 − N/T a + − + + + + N/T N/T 1:256 − N/T + − − − − − N/T N/T a N/T: not tested. The 76 PRNT-positive samples were divided into three groups according to the number The 76 PRNT-positive samples were divided into three groups according to the number of days after the onset of illness: <7 days (n = 25), 8–14 days (n = 28), and >15 days after symptom onset (n = 23). The sensitivity of the RapiSure test in the <7 days group was 92.6% for S1 RBD IgG and 88.0% for nAbs. In the 8–14 days group, 100% and 92.9% were positive in the S1 RBD IgG and nAb tests, respectively. No false negatives were observed in the >15 days group (Figure 2). y g p g When the low- and high-titer PRNT-positive groups were applied to the RapiSure test, the low-titer groups of PRNT50 and PRNT90 showed significantly lower sensitivity (76.9% and 70.0%, respectively) than those of high-titer groups of PRNT50 and PRNT90 (Table 4). Table 4. Results of the RapiSure COVID-19 Neutralizing Ab Test according to low- and high-titer groups in the PRNT assay. 3. Results Using their corresponding positive and negative results, a comparison between the RapiSure and STANDARD Q tests revealed an overall concordance of 91.5% (95% CI: 86.8% to 94.6%) with a κ value of 0.82, indicating a very good strength of agreement (Table 2). The results of the LoD tests using the five positive samples with a PRNT90 titer of 1:160 are summarized in Table 3. In the S1 RBD IgG test, one sample remained positive at a 1:32 titer while the other four samples remained positive at a 1:64 titer or higher. Meanwhile, the RapiSure Neutralizing Ab Test showed that one sample remained positive at a 1:16 titer while the others remained positive at a 1:32 titer or higher. Diagnostics 2023, 13, 643 6 of 11 Diagnostics 2023, 13, 643 6 of 11 Table 2. Comparison between the RapiSure COVID-19 S1 RBD IgG test results and the IgG test results obtained using STANDARD Q COVID-19 IgM/IgG Plus. The results of the STANDARD Q IgG test were also compared to those of COVID-19 RT-PCR. STANDARD Q IgG Sensitivity (95% CI) Specificity (95% CI) Percent Agreement a (95% CI) Cohen’s Kappa (95% CI) Positive Negative Positive Negative Overall RapiSure S1 RBD IgG Positive 66 14 95.7% (88.0–98.5%) 89.3% (82.9–93.5%) 91.5% (86.8–94.6%) 0.82 (0.73–0.90) Negative 3 117 RT-PCR Positive 66 12 95.7% (87.8–99.1%) 90.8% (84.5–95.2%) 92.5% (87.9–95.7%) 0.84 (0.76–0.92) Negative 3 119 a Calculated as positive = (RapiSure and STANDARD Q both positive/STANDARD Q positive) × 100, negative = (RapiSure and STANDARD Q both negative/STANDARD Q negative) × 100, overall = (RapiSure and STANDARD Q both positive + RapiSure and STANDARD Q both negative)/total × 100. Table 3. Evaluation of the limit of detection of the RapiSure COVID-19 S1 IgG/Neutralizing Ab Test using positive samples with a PRNT90 titer of 1:160. The gray-shaded boxes indicated their lowest dilution factor. 3. Results Categorization Titer Numbers of Positive/Total Samples RapiSure nAb Results Sensitivity (95% CI) p-Value a Positive Negative PRNT50 Low titer 1:20, 1:40, 1:80 13/76 10 3 76.9% (49.7–91.8) 0.0090 High titer >1:80 63/76 61 2 96.8% (89.1–99.1) PRNT90 Low titer 1:10, 1:20, 1:40 10/76 7 3 70.0% (39.7–89.2) 0.0014 High titer >1:40 66/76 64 2 97.0% (89.3–99.2) a Calculated using Chi-squared test, a p-value less than 0.05 indicates a statistically significant difference. Table 2. Comparison between the RapiSure COVID-19 S1 RBD IgG test results and the IgG test results obtained using STANDARD Q COVID-19 IgM/IgG Plus. The results of the STANDARD Q IgG test were also compared to those of COVID-19 RT-PCR. STANDARD Q IgG Sensitivity (95% CI) Specificity (95% CI) Percent Agreement a (95% CI) Cohen’s Kappa (95% CI) Positive Negative Positive Negative Overall RapiSure S1 RBD IgG Positive 66 14 95.7% (88.0–98.5%) 89.3% (82.9–93.5%) 91.5% (86.8–94.6%) 0.82 (0.73–0.90) Negative 3 117 RT-PCR Positive 66 12 95.7% (87.8–99.1%) 90.8% (84.5–95.2%) 92.5% (87.9–95.7%) 0.84 (0.76–0.92) Negative 3 119 a Calculated as positive = (RapiSure and STANDARD Q both positive/STANDARD Q positive) × 100, negative = (RapiSure and STANDARD Q both negative/STANDARD Q negative) × 100, overall = (RapiSure and STANDARD Q both positive + RapiSure and STANDARD Q both negative)/total × 100. Table 3. Evaluation of the limit of detection of the RapiSure COVID-19 S1 IgG/Neutralizing Ab Test using positive samples with a PRNT90 titer of 1:160. The gray-shaded boxes indicated their lowest dilution factor. Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Dilution Factor S1-IgG nAb S1-IgG nAb S1-IgG nAb S1-IgG nAb S1-IgG nAb 1:2 + + + + + + + + + + 1:4 + + + + + + + + + + 1:8 + + + + + + + + + + 1:16 + + + + + + + + + + 1:32 + + + + + + + + + − 1:64 + − + + + + + + − − 1:128 − N/T a + − + + + + N/T N/T 1:256 − N/T + − − − − − N/T N/T a N/T: not tested. Table 2. Comparison between the RapiSure COVID-19 S1 RBD IgG test results and the IgG test results obtained using STANDARD Q COVID-19 IgM/IgG Plus. The results of the STANDARD Q IgG test were also compared to those of COVID-19 RT-PCR. 3. Results Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Dilution Factor S1-IgG nAb S1-IgG nAb S1-IgG nAb S1-IgG nAb S1-IgG nAb 1:2 + + + + + + + + + + 1:4 + + + + + + + + + + 1:8 + + + + + + + + + + 1:16 + + + + + + + + + + 1:32 + + + + + + + + + − 1:64 + − + + + + + + − − 1:128 − N/T a + − + + + + N/T N/T 1:256 − N/T + − − − − − N/T N/T a N/T: not tested. The 76 PRNT-positive samples were divided into three groups according to the number of days after the onset of illness: <7 days (n = 25), 8–14 days (n = 28), and >15 days after symptom onset (n = 23). The sensitivity of the RapiSure test in the <7 days group was 92.6% for S1 RBD IgG and 88.0% for nAbs. In the 8–14 days group, 100% and 92.9% were positive in the S1 RBD IgG and nAb tests, respectively. No false negatives were observed in the >15 days group (Figure 2). When the low- and high-titer PRNT-positive groups were applied to the RapiSure test, the low-titer groups of PRNT50 and PRNT90 showed significantly lower sensitivity (76.9% and 70.0%, respectively) than those of high-titer groups of PRNT50 and PRNT90 (Table 4). Table 4. Results of the RapiSure COVID-19 Neutralizing Ab Test according to low- and high-titer groups in the PRNT assay. Categorization Titer Numbers of Positive/Total Samples RapiSure nAb Results Sensitivity (95% CI) p-Value a Positive Negative PRNT50 Low titer 1:20, 1:40, 1:80 13/76 10 3 76.9% (49.7–91.8) 0.0090 High titer >1:80 63/76 61 2 96.8% (89.1–99.1) PRNT90 Low titer 1:10, 1:20, 1:40 10/76 7 3 70.0% (39.7–89.2) 0.0014 High titer >1:40 66/76 64 2 97.0% (89.3–99.2) a Calculated using Chi-squared test, a p-value less than 0.05 indicates a statistically significant difference. Table 2. Comparison between the RapiSure COVID-19 S1 RBD IgG test results and the IgG test results obtained using STANDARD Q COVID-19 IgM/IgG Plus. The results of the STANDARD Q IgG test were also compared to those of COVID-19 RT-PCR. 3. Results STANDARD Q IgG Sensitivity (95% CI) Specificity (95% CI) Percent Agreement a (95% CI) Cohen’s Kappa (95% CI) Positive Negative Positive Negative Overall RapiSure S1 RBD IgG Positive 66 14 95.7% (88.0–98.5%) 89.3% (82.9–93.5%) 91.5% (86.8–94.6%) 0.82 (0.73–0.90) Negative 3 117 RT-PCR Positive 66 12 95.7% (87.8–99.1%) 90.8% (84.5–95.2%) 92.5% (87.9–95.7%) 0.84 (0.76–0.92) Negative 3 119 a Calculated as positive = (RapiSure and STANDARD Q both positive/STANDARD Q positive) × 100, negative = (RapiSure and STANDARD Q both negative/STANDARD Q negative) × 100, overall = (RapiSure and STANDARD Q both positive + RapiSure and STANDARD Q both negative)/total × 100. Table 3. Evaluation of the limit of detection of the RapiSure COVID-19 S1 IgG/Neutralizing Ab Test using positive samples with a PRNT90 titer of 1:160. The gray-shaded boxes indicated their lowest dilution factor. Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Dilution Factor S1-IgG nAb S1-IgG nAb S1-IgG nAb S1-IgG nAb S1-IgG nAb 1:2 + + + + + + + + + + 1:4 + + + + + + + + + + 1:8 + + + + + + + + + + 1:16 + + + + + + + + + + 1:32 + + + + + + + + + − 1:64 + − + + + + + + − − 1:128 − N/T a + − + + + + N/T N/T 1:256 − N/T + − − − − − N/T N/T a N/T: not tested. The 76 PRNT-positive samples were divided into three groups according to the number of days after the onset of illness: <7 days (n = 25), 8–14 days (n = 28), and >15 days after symptom onset (n = 23). The sensitivity of the RapiSure test in the <7 days group was 92.6% for S1 RBD IgG and 88.0% for nAbs. In the 8–14 days group, 100% and 92.9% were positive in the S1 RBD IgG and nAb tests, respectively. No false negatives were observed in the >15 days group (Figure 2). When the low- and high-titer PRNT-positive groups were applied to the RapiSure test, the low-titer groups of PRNT50 and PRNT90 showed significantly lower sensitivity (76.9% and 70.0%, respectively) than those of high-titer groups of PRNT50 and PRNT90 (Table 4). Table 4. Results of the RapiSure COVID-19 Neutralizing Ab Test according to low- and high-titer groups in the PRNT assay. When th t t th l t 4. Discussion test, the low-titer groups of PRNT50 and PRNT90 showed significantly lower sensitivity (76.9% and 70.0%, respectively) than those of high-titer groups of PRNT50 and PRNT90 (Table 4). Table 4. Results of the RapiSure COVID-19 Neutralizing Ab Test according to low- and high-titer groups in the PRNT assay. Titer Numbers of Positive/Total Samples RapiSure nAb Results Sensitivity (95% CI) p-Value a Positive Negative 20, 1:40, 1:80 13/76 10 3 76.9% (49.7–91.8) 0.0090 >1:80 63/76 61 2 96.8% (89.1–99.1) 10, 1:20, 1:40 10/76 7 3 70.0% (39.7–89.2) 0.0014 >1:40 66/76 64 2 97.0% (89.3–99.2) a Calculated using Chi-squared test, a p-value less than 0.05 indicates a statistically significant dif- ference. 4. Discussion Timely detection and clinical decision-making processes for COVID-19 are crucial for infection control and public health management during the ongoing pandemic. The pres- Timely detection and clinical decision-making processes for COVID-19 are crucial for infection control and public health management during the ongoing pandemic. The presence of anti-SARS-CoV-2 nAbs helps protect against reinfection by the same strain [5]. Various serological assays to detect anti-SARS-CoV-2 antibodies have been marketed to date [20,21]. Currently, antibody testing is not recommended for confirming immunity after vaccination, according to the interim guidelines by the Centers for Disease Control and Prevention [15]. Moreover, the real-world application of antibody tests is hampered by the ongoing pandemic and the limited capacity of laboratory-based testing. The development of improved tests, including POCT, could substantially accelerate clinical decision-making and help monitor the effectiveness of governmental infection control strategies. Despite these potentials, POCT using lateral flow immunochromatographic assay have concerns about lower sensitivities than laboratory-based serological methods, such as ELISA and chemiluminescence immunoassay [22]. Therefore, test validity and reliability are crucial in POCT: unreliable diagnostic tests can hamper healthcare provision by failing to detect patients with SARS-CoV-2 infection or by incorrectly identifying negative patients as positive [21]. The PRNT is the gold STANDARD for assessing the presence and titer of antibodies in serum samples. However, its demanding requirements and high cost have highlighted the need for the development and validation of anti-SARS-CoV-2 antibody testing in POCT. ence of anti-SARS-CoV-2 nAbs helps protect against reinfection by the same strain [5]. Various serological assays to detect anti-SARS-CoV-2 antibodies have been marketed to date [20,21]. Currently, antibody testing is not recommended for confirming immunity after vaccination, according to the interim guidelines by the Centers for Disease Control and Prevention [15]. 3. Results Categorization Titer Numbers of Positive/Total Samples RapiSure nAb Results Sensitivity (95% CI) p-Value a Positive Negative PRNT50 Low titer 1:20, 1:40, 1:80 13/76 10 3 76.9% (49.7–91.8) 0.0090 High titer >1:80 63/76 61 2 96.8% (89.1–99.1) PRNT90 Low titer 1:10, 1:20, 1:40 10/76 7 3 70.0% (39.7–89.2) 0.0014 High titer >1:40 66/76 64 2 97.0% (89.3–99.2) a Calculated using Chi-squared test, a p-value less than 0.05 indicates a statistically significant difference. Table 4. Results of the RapiSure COVID-19 Neutralizing Ab Test according to low- and high-titer groups in the PRNT assay. a Calculated using Chi-squared test, a p-value less than 0.05 indicates a statistically significant difference. Diagnostics 2023, 13, 643 Diagnostics 2023, 13, x FO 7 of 11 7 of 11 7 of 11 7 of 11 Figure 2. Sensitivity (%) of the RapiSure COVID-19 S1 RBD IgG/Neutralizing Ab Test in three groups: <7 days, 8–14 days, and >15 days after onset of illness. Blue bars represent the results for S1 RBD IgG; gray bars represent the results for nAbs, of which each upper light-colored bar represents a false negative result. Figure 2. Sensitivity (%) of the RapiSure COVID-19 S1 RBD IgG/Neutralizing Ab Test in three groups: <7 days, 8–14 days, and >15 days after onset of illness. Blue bars represent the results for S1 RBD IgG; gray bars represent the results for nAbs, of which each upper light-colored bar represents a false negative result. Figure 2. Sensitivity (%) of the RapiSure COVID-19 S1 RBD IgG/Neutralizing Ab Test in three groups: <7 days, 8–14 days, and >15 days after onset of illness. Blue bars represent the results for S1 RBD IgG; gray bars represent the results for nAbs, of which each upper light-colored bar represents a false negative result. Figure 2. Sensitivity (%) of the RapiSure COVID-19 S1 RBD IgG/Neutralizing Ab Test in three groups: <7 days, 8–14 days, and >15 days after onset of illness. Blue bars represent the results for S1 RBD IgG; gray bars represent the results for nAbs, of which each upper light-colored bar represents a false negative result. When th t t th l t 4. Discussion Antibody titers in the recovery period (>7 days after diagnosis) are significantly higher than those in the acute phase (≤7 days) [6]. Additionally, the induction of nAbs in the acute phase has been reported to vary depending on disease severity [34]. The results of the RapiSure nAb test in the two samples were consistent with those of the PRNT; the antibody test results were consistent. Serological detection of anti-SARS-CoV-2 antibodies usually begins at the end of the first week of infection, and peak nAb detection is reached after 3–4 weeks of illness [8,35,36]. Serum collected >15 days after the onset of illness showed 100% positivity in both the RapiSure IgG and nAb tests. Meanwhile, of the samples collected 8–14 days after the onset of illness, 100% positivity was observed in the RapiSure S1 RBD IgG compared to the results of PRNT, with two false negative samples in the RapiSure nAb test. These discordant results between S1 RBD IgG and nAb test did not indicate the possibility of Omicron infection, since South Korea’s first Omicron cases were identified on 25 November 2021 [37]. However, the simultaneous measurement of anti-S1 RBD antibodies and nAb may be very useful in the detailed descriptions of immune status, especially in cases of VOC infection. There are several limitations to this study. First, sample collection occurred before Omicron became the predominant variant, hindering the relevance of the results to this variant. Second, the RapiSure test results were interpreted with the naked eye rather than any specific reading devices; therefore, the interpretation of weak test reactions, often shown as faint lines, may not be objective. However, the performance of RapiSure showed better sensitivity (97.4% vs. 95.7%) and specificity (96.7% vs. 90.8%) than the previously marketed STANDARD Q test when compared to COVID-19 RT-PCR. The ability to detect nAb was comparable to the PRNT, with an overall percent agreement of 97.5%. Nonetheless, the qualitative nature of the RapiSure test, including reduced sensitivity at an early stage (<7 days), may hamper the proper diagnosis for the level of protection. Finally, a few false positives (RapiSure: four cases, STANDARD Q test: three cases) were observed, but the causative agent that can cause cross-reactivity remained unclear. When th t t th l t 4. Discussion Moreover, the real-world application of antibody tests is hampered by the ongoing pandemic and the limited capacity of laboratory-based testing. The devel- g Since antibodies against the SARS-CoV-2 RBD are highly correlated with nAbs, anti- RBD antibody assays have been developed to assess post-infection immunity and validate vaccine effectiveness [23,24]. Anti-RBD IgG titers measured by commercial serological assays show a correlation with nAb titers, thus qualifying as a proxy marker of neutral- ization [25,26]. However, these results were obtained using the RBD of wild-type (WT) SARS-CoV-2, before the emergence of variants of concern (VOCs) [27,28]. opment of improved tests, including POCT, could substantially accelerate clinical deci- sion-making and help monitor the effectiveness of governmental infection control strate- gies. Despite these potentials, POCT using lateral flow immunochromatographic assay VOCs, especially Delta and Omicron, raised concerns about their potential for immune escape from vaccine-induced antibodies due to mutations in their S proteins [29]. In addition, there are doubts regarding the performance of previously released anti-RBD Diagnostics 2023, 13, 643 8 of 11 antibody assays that use RBD antigens derived from WT SARS-CoV-2. To address these issues, a comparative study was conducted on whether the anti-RBD IgG titers measured using commercially available kits could represent the presence of nAbs against VOCs [30]. According to the study, there was a strong correlation between anti-RBD IgG and nAbs levels against WT and pre-Omicron variants, including Alpha, Beta, and Delta. However, despite a high anti-RBD IgG titer, anti-Omicron nAbs were not observed, indicating no correlation between anti-RBD IgG and nAbs. Omicron subvariants have been reported to escape most of the anti-SARS-CoV-2 nAbs induced by vaccination and infection and elicit substantially lower nAb titers [31]. Reduced sensitivity of serological assays has been observed in assays using recombinant WT S protein for the detection of anti-RBD antibodies, suggesting an underestimation of true antibody titers [32]. The RapiSure COVID-19 S1 RBD IgG/Neutralizing Ab Test evaluated in this study detected anti-RBD antibodies and nAbs simultaneously in a single cassette. Two samples were found to be positive by RT-PCR but negative by the PRNT, with a final classification of negative; only one sample was positive for anti-S1 RBD antibodies. Both samples had less than one symptomatic day after the onset of illness. The sensitivity of immunoassays can be affected by the viral load and sample collection date, which can both affect circulating antibody levels [33]. When th t t th l t 4. Discussion According to the package insert of RapiSure, there was no cross-reactivity with anti-influenza A virus, anti-influenza B virus, anti-respiratory syncytial virus, anti-adenovirus, hepatitis B surface antigen, anti- syphilis, anti-helicobacter pylori, anti-human immunodeficiency virus, anti-hepatitis C virus, and human anti-mouse antibody positive specimens. However, the possibility of false reactivity due to coinfection with another pathogen cannot be ruled out. y p g Previously reported sensitivity and specificity of RapiSure were 96.8% and 97.7% for the S1 RBD IgG and 92.2% and 100.0% for the nAb test, respectively, which were similar to this study [38]. The limitation of POCT includes lowered and large ranges of sensitivity reported by the different POCT tests. According to the final WHO SARS-CoV-2 serology Diagnostics 2023, 13, 643 9 of 11 9 of 11 test kit evaluation that evaluated 26 rapid diagnostic tests (RDTs) using lateral flow assay, the sensitivity of IgG ranged from 77.4% to 100.0%, and specificity ranged from 81.0 to 99.0%, respectively [39]. Considering the similarity of sensitivity and specificity calculated on different populations and the performance of other RDTs, the RapiSure showed an acceptable performance. Compared to anti-RBD antibodies, the status of nAbs was a better indication of immunity against variants. However, commercially available antibody assays, which are widely used, typically measure anti-RBD IgG levels. Since the Omicron variant is currently the globally dominant strain [27], the anti-RBD IgG test alone might provide unreliable information about the immunity status following infection or vaccination. 5. Conclusions In conclusion, the RapiSure (EDGC) COVID-19 S1 RBD IgG/Neutralizing Ab Test showed reliable sensitivity and specificity as a lateral flow test, and the simultaneous detection of anti-RBD antibodies and nAbs is of advantage. This assay has the potential to be conveniently used in the Omicron era when the anti-RBD IgG test alone might be unreliable for detecting the level of protection. Author Contributions: Conceptualization, W.S.J. and C.S.L.; Formal analysis, H.N.K., J.Y., and W.S.J.; Methodology, W.S.J. and C.S.L.; Visualization, H.N.K.; Writing—original draft, H.N.K.; Writing— review and editing, H.N.K. and C.S.L. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by a grant from the Korea Health Technology R&D Project, through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (grant number, HR20C0021). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Korea University Guro Hospital (2021GR0481, 6 October 2021). Informed Consent Statement: Patient consent was waived by the Institutional Review Board of Korea University Guro Hospital as the identities of subjects are completely anonymous and there is minimal risk involved in the study. Informed Consent Statement: Patient consent was waived by the Institutional Review Board of Korea University Guro Hospital as the identities of subjects are completely anonymous and there is minimal risk involved in the study. Data Availability Statement: The data supporting the findings of this study are available from the corresponding author upon reasonable request. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 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Prévost, J.; Gasser, R.; Beaudoin-Bussières, G.; Richard, J.; Duerr, R.; Laumaea, A.; Anand, S.P.; Goyette, G.; Benlarbi, M.; Ding, S. Cross-sectional evaluation of humoral responses against SARS-CoV-2 spike. Cell Rep. Med. 2020, 1, 100126. [CrossRef] 10 of 11 Diagnostics 2023, 13, 643 10 of 11 8. Perera, R.A.; Mok, C.K.; Tsang, O.T.; Lv, H.; Ko, R.L.; Wu, N.C.; Yuan, M.; Leung, W.S.; Chan, J.M.; Chik, T.S. Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), March 2020. Eurosurveillance 2020, 25, 2000421. [CrossRef] p y y 9. Liu, J.; Liu, Y.; Xia, H.; Zou, J.; Weaver, S.C.; Swanson, K.A.; Cai, H.; Cutler, M.; Cooper, D.; Muik, A. BNT162b2-elicited neutralization of B. 1.617 and other SARS-CoV-2 variants. Nature 2021, 596, 273–275. [CrossRef] 10. Abe, K.T.; Li, Z.; Samson, R.; Samavarchi-Tehrani, P.; Valcourt, E.J.; Wood, H.; Budylowski, P.; Dupuis, A.P.; Girardin, R.C.; Rathod, B. A simple protein-based surrogate neutralization assay for SARS-CoV-2. 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Hachmann, N.; Miller, J.; Collier, A.-r.; Ventura, J.; Yu, J.; Rowe, M.; Bondzie, E.; Powers, O.; Surve, N.; Hall, K. Neutralization Escape by the SARS-CoV-2 Omicron Variants BA. 2.12. 1, BA. 4, and BA. 5. N. Engl. J. Med. 2022, 387, 86–88. [CrossRef] 11 of 11 11 of 11 Diagnostics 2023, 13, 643 32. Rössler, A.; Knabl, L.; Raschbichler, L.-M.; Peer, E.; von Laer, D.; Borena, W.; Kimpel, J. Reduced sensitivity of antibody tests after omicron infection. Lancet Microbe 2022, 4, e10–e11. [CrossRef] 33. To, K.K.-W.; Tsang, O.T.-Y.; Leung, W.-S.; Tam, A.R.; Wu, T.-C.; Lung, D.C.; Yip, C.C.-Y.; Cai, J.-P.; Chan, J.M.-C.; Chik, T.S.-H. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: An observational cohort study. Lancet Infect. Dis. 2020, 20, 565–574. [CrossRef] y f 34. Kawasuji, H.; Morinaga, Y.; Tani, H.; Kimura, M.; Yamada, H.; Yoshida, Y.; Takegoshi, Y.; Kaneda, M.; Murai, Y.; Kimoto, K. Delayed neutralizing antibody response in the acute phase correlates with severe progression of COVID-19. Sci. Rep. 2021, 11, 16535. [CrossRef] 35. Fang, F.C.; Naccache, S.N.; Greninger, A.L. The laboratory diagnosis of coronavirus disease 2019—Frequently asked questions. Clin. Infect. Dis. 2020, 71, 2996–3001. [CrossRef] 36. Cheng, M.P.; Yansouni, C.P.; Basta, N.E.; Desjardins, M.; Kanjilal, S.; Paquette, K.; Caya, C.; Semret, M.; Quach, C.; Libman, M. Serodiagnostics for Severe Acute Respiratory Syndrome–Related Coronavirus 2: A Narrative Review. Ann. Intern. Med. 2020, 173, 450–460. [CrossRef] 37. Kim, D.; Ali, S.T.; Kim, S.; Jo, J.; Lim, J.-S.; Lee, S.; Ryu, S. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References Estimation of serial interval and reproduction number to quantify the transmissibility of SARS-CoV-2 omicron variant in South Korea. Viruses 2022, 14, 533. [CrossRef] 38. Shim, H.W.; Shin, J.h.; Shin, S.C.; Lee, H.J.; So, K.S.; Lee, S.Y.; Jun, J.W.; Seo, J.K.; Lee, H.S.; Lee, S.Y. Analysis of Factors Affecting Neutralizing Antibody Production after COVID-19 Vaccination Using Newly Developed Rapid Point-of-Care Test. Diagnostics 2022, 12, 1924. [CrossRef] [ ] 39. WHO. Final WHO SARS-CoV-2 Serology Test Kit Evaluation Results. Available online: https://www.who.int/publications/m/ item/final-who-sars-cov-2-serology-test-kit-evaluation-results (accessed on 6 February 2023). Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
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Production Cost Analysis and Production Planning for Plant Factories Considering Markets
IFIP advances in information and communication technology
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Production Cost Analysis and Production Planning for Plant Factories Considering Markets Nobuhiro Sugimura, Koji Iwamura, Nguyen Quang Thinh, Kousuke Nakai, Seisuke Fukumoto, Yoshitaka Tanimizu To cite this version: Nobuhiro Sugimura, Koji Iwamura, Nguyen Quang Thinh, Kousuke Nakai, Seisuke Fukumoto, et al.. Production Cost Analysis and Production Planning for Plant Factories Considering Markets. IFIP International Conference on Advances in Production Management Systems (APMS), Sep 2015, Tokyo, Japan. pp.532-540, ￿10.1007/978-3-319-22756-6_65￿. ￿hal-01417607￿ To cite this version: Nobuhiro Sugimura, Koji Iwamura, Nguyen Quang Thinh, Kousuke Nakai, Seisuke Fukumoto, et al.. Production Cost Analysis and Production Planning for Plant Factories Considering Markets. IFIP International Conference on Advances in Production Management Systems (APMS), Sep 2015, Tokyo, Japan. pp.532-540, ￿10.1007/978-3-319-22756-6_65￿. ￿hal-01417607￿ Production Cost Analysis and Production Planning for Plant Factories Considering Markets Nobuhiro Sugimura, Koji Iwamura, Nguyen Quang Thinh, Kousuke Nakai, Seisuke Fukumoto, Yoshitaka Tanimizu Distributed under a Creative Commons Attribution 4.0 International License Nobuhiro Sugimura1, Koji Iwamura1, Nguyen Quang Thinh1, Kousuke Nakai1, Seisuke Fukumoto1 and Yoshitaka Tanimizu1 Nobuhiro Sugimura1, Koji Iwamura1, Nguyen Quang Thinh1, Kousuke Nakai1, Seisuke Fukumoto1 and Yoshitaka Tanimizu1 1 Graduate School, Osala Prefectuture University, Sakai, Japan sugimura@me.osakafu-u.ac.jp 1 Graduate School, Osala Prefectuture University, Sakai, Japan sugimura@me.osakafu-u.ac.jp Abstract. Much emphasis is now given to development of fully closed and controlled plant factories, aimed at supplying various vegetables safely and constantly. However, one of the most important issues of the plant factories is the high production costs due to the investment and equipment in the factories and the daily operations. Systematic methods are considered here increase the delivery prices of the vegetables and to reduce the running costs in the plant factories. Keywords. Plant Factories, Prodction Planning, Cost Analysis Keywords. Plant Factories, Prodction Planning, Cost Analysis HAL Id: hal-01417607 https://hal.science/hal-01417607v1 Submitted on 15 Dec 2016 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1. Introduction Much emphasis is now given to development and application of fully closed and controlled plant factories, aimed at supplying various vegetables safely and constantly. Many plant factories have been equipped and operated in suburban areas and downtowns for supplying the safe and high quality vegetables directory to the markets [1, 2]. However, one of the most important problems of the plant factories to be solved is the production costs due to the investment and equipment in the factories and the daily operations. A mathematical model is proposed in the research to estimate the production costs of the vegetables due to the investment, equipment and operations, based on the production conditions of the vegetables and their production volumes in the plants. The proposed model are verified by comparing the estimated production costs by the proposed model with ones of the real plant equipped in Osaka Prefecture University [3]. A production planning method is proposed to select a suitable production plans of the vegetables to be cultivated in the daily operations of the plant factories referring to the forecasting model developed to estimate the market prices of the vegetables. The market prices are forecasted by applying a GP (Genetic Programming) model representing the relationships between the market prices and the weather data. An electric power supply market is also disscussed to get the electric power for the plant factories, aimed at reducing the electricity costs, since the plant factory requires large amount of electric power for the lighting sources and the air conditioning equipment. Fig. 1. Vegetable market and electric power market Plant factory Vegetable market Electric power supplier Electric power market Vegetable supply Power supply Cash Flow Plant factory Vegetable market Fig. 1. Vegetable market and electric power market 2. Plant Factories and their Markets Plant factories are production systems which constantly produces safe and high quality vegetables rapidly and constantly. However, the production costs in the plant factories are higher than the ones of the conventional farms, due to initial investment costs including plant constructions, air conditioning equipment, lighting devices and produciton equipment and also running costs for electirc power supply. Therefore, two markets are considerd in the research to reduce the operarion costs and to increase the selling prices of the produced vegetables. Figure 1 shows the vegetable markets and the electirc suppliers related with the plant factories. 1. Introduction The vegetable markets are the markets dealing with the vegetables produced by both the conventional farms and the plant factories, and the market prices are determined based on the supplies and the demands in the markets. The market prices of the leaf lettuces dynamically change day by day due to various environmental conditions, and the selling prices are increased if the delivery timing is fit to the date with high prices of the lettuces. Therefore, a suitable production plans are required to deliver the lettuces to the high price markets. As regards the electric power supply, two types of supply systems are considered. They are, conventional electric power suppliers and electric power markets. The plant factories get most of the required electirc powers from the conventinal suppliers and a part of the electirc powers from the markets to decrease the procurement costs. The following three issues will be discussed in the paper to manage the plant factories based on the vegetable markets and the electirc power markets. (1) Production cost analysis of plant factories, (2) Production planning of plant fatories based on vegetable market prices, and (3) Purchasing electirc powers from electirc power markets 3. Production Cost Analysis of Plant Factories 3. Production Cost Analysis of Plant Factories The production costs of the vegetables produced in the plant factories are classified in to following items. 2 (1) Construction costs of plant factories, (1) Construction costs of plant factories, (2) Machinery costs of plant factories, such as pallets to seed vegetables, conveyers to transport the pallets, and devices to pick up the grown-up vegetables, (3) Lighting source costs to give required photon flux density of the vegetables, (4) Air-conditioning device costs to keep the environments in the plant factories, such as temperature and humidity, (5) Running costs for the electricity required to operate the plant factories, and (6) Other running costs. (2) Machinery costs of plant factories, such as pallets to seed vegetables, conveyers to transport the pallets, and devices to pick up the grown-up vegetables, (2) Machinery costs of plant factories, such as pallets to seed vegetables, conveyers to transport the pallets, and devices to pick up the grown-up vegetables, p p p p g p g (3) Lighting source costs to give required photon flux density of the vegetables, (3) Lighting source costs to give required photon flux density of the vegetables, (4) Air-conditioning device costs to keep the environments in the plant factories, such as temperature and humidity, (4) Air-conditioning device costs to keep the environments in the plant factories such as temperature and humidity, (5) Running costs for the electricity required to operate the plant factories, and The individual cost items are investigated based on the documents and data in the web-sites to estimate the production costs of one piece of the vegetables. The cost items considered here include such items as the construction costs of the plant factory buildings for an unit area, the conveyer costs for an unit length, the lighting device costs for the fluorescent lights and/or LED lights required to cultivate the vegetables, the air-conditioning costs to remove an unit kWh heat, and the power purchasing costs for an unit kWh. The cost items are analyzed and a set of formulas are developed to estimate the production costs for the individual pieces of the vegetables. By applying the formulas, the production costs for the leaf lettuces are estimated for the cases of the fluorescent lights and the LED lights. For an example, Eq. 3. Production Cost Analysis of Plant Factories (1) gives the photon flux requirement of the lighting sources to produce the vegetables, which is one of the most important formulas for designing the plant factories. L all p V S PFD PF      (1) (1) L all p V S PFD PF      where, , PF mol s-1] : Total photon flux required in the plant factories. PFD mol m-2 s-1] : Photon flux density required to the lighting sources to cultivate the vegetables. PF mol s-1] : Total photon flux required in the plant factories. PFD mol m-2 s-1] : Photon flux density required to the lighting sources to cultivate the vegetables. PFD mol m-2 s-1] : Photon flux density required to the lighting sources to cultivate the vegetables. g p [m2] : Areas which the individual vegetables occupy Vall : Total number of vegetables in the plant factories. Vall : Total number of vegetables in the plant factories L : Efficiency of the lighting sources. L : Efficiency of the lighting sources. The numbers of the lighting sources are estimated by applying Eq. (1), and the capacity and the electric powers of both the lighting sources and the air-conditioning systems are estimated based on the numbers and the electric powers of the lighting sources. The basic specification of the pant factories considered here is summarized in Table 1, based on the experimental plant factory in Osaka Prefecture University. Table 2 shows the specification of the leaf lettuces produced in the experimental plant factories. Whole plans of the plant factories for both fluorescent lights and the LED lights have been designed based on the specifications, and the production costs are estimated for individual pieces of the lettuces. 3 Table 1 Specification of a plant factory Daily production 5,000 [pieces] Capacity 25,000 [pieces] Lighting sources Fluorescent lights or LED Efficiency of light 80 [%] Table 2 Specification of a leaf lettuce Cultivation period 25 [days] Photon flux density 150 [mol m2 s-1] Table 3 Summary of production costs for plant factories (Yen/Piece) Plant factories equipped with fluores- cent lights Plant factories equipped with LED Lighting source 9.88 58.9 Air conditioning 14.2 2.19 Construction 11.0 11.0 Equipment 2.78 2.78 Seeds, water, etc. 6.10 6.10 Electric power 88.4 28.4 Total Cost 132 109 Table 3 Summary of production costs for plant factories (Yen/Piece) The estimated costs are summarized in Table 3 for the plant factories equipped with the fluorescent lights and ones with LEDs. The estimated costs are roughly same with the production costs of the existing plant factories. The differences of the cost items between two light sources are also roughly same as the existing plant factories. where, In the case of ones equipped with LED lights, the lightning device cost is high, but the air- conditioning cost and the electricity costs are low, due to high efficiency and low energy consumption of the LED lights against the fluorescent lights. 4. Production Planning and Purchasing of Electric Power 4.1 Estimation of Wholesale Market Prices 4. Production Planning and Purchasing of Electric Power 4.1 Estimation of Wholesale Market Prices A suitable production plans for the Plant factories are required to produce and to sup- ply the vegetables in suitable timing to the markets. A production planning method is discussed in the section based on the market price estimation of the lettuces. The market prices of lettuces are changing day by day due to surrounding conditions such as seasons, whether, and climate [4]. Many research works have been carried out to forecast the vegetable market prices by applying the ARIMA model of the time series analysis and the Neural Network model [5-6]. However, it is required to estab- lish long-term forecasting methods for the production planning, due to the long lead time in the plant factories. Therefore, the correlations between the wholesale market prices in Tokyo and the various parameters of the weather such as temperature, hu- midity, and so on, were firstly analyzed, and it was found that the lowest temperature 4 Fig. 2. Relationships betweeen real markett prices and fo orecasted pricees Fig. 2. Relationships betweeen real markett prices and fo orecasted pricees has highe system ha time serie forecast t training d month fo nomial fo est correlation as been devel es data of both the market pr data of the pr or 8 years. Equ or forecasting ns with the le loped to forec h the market p rocess are gen rices in the r uation (2) sho the prices. ettuce prices. cast the marke prices and the nerated by ap real markets a ows the obtain A genetic pro et prices of th lowest tempe pplying the GP and the lowes ned formula re ogramming (G he lettuces bas erature. The fo P method bas st temperature epresented by GP) based sed on the ormulas to sed on the e in every y the poly- n k nL k EP    0 n LT (2) n k nL k EP    0 n LT (2) (2) where, where, EP: Mark LT: Lowe kn: Const ket prices of le est temperatur tants eaf lettuces re where, EP: Mark LT: Lowe kn: Const ket prices of le est temperatur tants eaf lettuces re EP: Market prices of leeaf lettuces kn: Constants The form training d tionships tion (2) o deliver th mulas are appli data for 8 yea between the obtained here he vegetables t ied to the fore ars. 4. Production Planning and Purchasing of Electric Power 4.1 Estimation of Wholesale Market Prices Figure 2 s real wholesal is applied to to the markets ecast of the ma shows the eva ale market pric the productio s at suitable ti arket prices fo aluation result ces and the fo on planning o ming. or 3 years othe ts, which give orecasted pric of the plant fa er than the e the rela- ces. Equa- actories to 4.2 Production Planninng Based on M Market Prices STEP 1: Forecasting of lowest temperature and wholesale market prices STEP 1: Forecasting of lowest temperature and wholesale market prices The lowest temperature of 25 days after are firstly forecasted based on the weather forecast and the wholesale market prices of the lettuces are also estimated by applying Eq. (2) . The cultivation period in the plant factories is set to be 25 days, and the pro- duction planning requires the forecast of the market prices of 25 days after, in order to select the cultivation operations in the next day. 4.2 Production Planninng Based on M Market Prices 4.2 Production Planninng Based on M Market Prices A produc wholesale daily ope ction planning e market pric erations. g method is p es. This meth proposed base hod selects a s ed on the for suitable veget recasting syste able to be cul em of the ltivated in Two types of plant facttories are connsidered in the present reseaarch. They aree, (1) Factory A producces only leaf leettuces, and (2) Fact ble c tory B has the called . e capability off producing bboth leaf lettucces and anothher vegeta- The following items arre assumed forr the ease of pproduction plaanning. The following items arre assumed forr the ease of pproduction plaanning. 5 5 (a) The factory B has the equipment to produce two vegetables and the production costs are higher than ones of the factory A, (a) The factory B has the equipment to produce two vegetables and the production costs are higher than ones of the factory A, g y (b) Both the leaf lettuces and the vegetable have same cultivation period of 25 days, (b) Both the leaf lettuces and the vegetable have same cultivation period of 25 days, (c) The vegetable  can be sold by the price same as the production costs, and (d) The retail vegetable market prices are higher than the wholesale market prices in either 3, 3.5 or 4 times. (d) The retail vegetable market prices are higher than the wholesale market prices in either 3, 3.5 or 4 times. (1) Plant factory A The production planning system for the plant factory A selects the one of the follow- ing tow operations for the next day. ing tow operations for the next day. g p y a) Cultivating lettuces, if the forecasted price is higher than the production costs, or a) Cultivating lettuces, if the forecasted price is higher than the production costs, or b) No cultivation, if the forecasted price is lower than the production costs. ) ) g , p g p , b) No cultivation, if the forecasted price is lower than the production costs. c) (2) Plant factory B The production planning system of factory B selects one the following two operations for the next day. a) Cultivating lettuces, if the forecasted price is higher than the production costs, or a) Cultivating lettuces, if the forecasted price is higher than the production costs, or b) Cultivating vegetable if the forecasted price is lower than the production costs a) Cultivating lettuces, if the forecasted price is higher than the production costs, or b) Cultivating vegetable , if the forecasted price is lower than the production costs. b) Cultivating vegetable , if the forecasted price is lower than the production costs. In the plant factory B, the vegetable  is cultivated to avoid the loss of the running cost, for the cases that the forecasted lettuce price is lower than the production costs. However, the production cost of the plant factory B is higher than one of the plant factory A, due to the additional production equipment for two types of vegetables. 4.3 Purchasing of electric power from market STEP2: Selection of vegetables to be cultivated in the nest day STEP2: Selection of vegetables to be cultivated in the nest day The following procedure is proposed to carry out production planning. STEP 1: Forecasting of lowest temperature and wholesale market prices 5. Simulation Results One year simulations of both the factories A and B have been carried out by applying the forecasted prices and the real prices of the lettuces from Jan. to Dec., 2010. As regards the electric power market, a statistical simulation model is proposed, which include 10 suppliers and 20 customers including the plant factory. The daily supplies and demands of the individual suppliers and customers are selected randomly in the simulations. Figure 5 shows the simulation results about the profits of the plant factories A and B. The profits of both the factories depend on the market price of the lettuce. When the market price is low, the combined production of the factory B reduces the loss by producing the vegetable . The simple production of the factory A gets the higher profit in the case of high market price. The combined production in factory B is not so efficient, if the market price is high and stable. The production volumes in factory B is summarized in Fig. 6. The percentage of the lettuce is high in the winter seasons, since the market price is high due to low temperature, as shown in Fig. 3. The simula- tions have been carried out for five times due to the statistical characters of the market price forecasting. The individual lines in Fig. 6 show the different simulation results, but all the tendency are almost same. 4.3 Purchasing of electric power from market 4.3 Purchasing of electric power from market As regards the electric power supply, two suppliers are considered in the research. One is the conventional electric power suppliers, which provide the electricity by the fixed prices based on the long term contracts. Another is electric power markets, in which the amount of power supply and the prices are determined based on the daily supplies and demands, such as Nord Pool in EU and JEPX (Japan Electric Power eXchange) in Japan [7]. Figures 3 and 4 show a structure of the electric power mar- kets and the relationships between the balanced prices and the amount of the electric power supplied to the markets, respectively. When the vegetables to be cultivated in the next day are fixed, the total electric power requirement is estimated and the demands to the electric power market are determined. The demand of the plant factory is sent to the market, and the market determines the amount of electric power and its price in the next day. 6 Fig. 3. Electric Power Market Fig. 4. Market mechanisms Electric Power Supplies for Market Electric Power Market Customers for Market (Plant Factories) Supplies Demands Results Results Price P (Yen/kWh) Electric Power Q (kWh) Supply Demand Balanced Point Fig. 3. Electric Power Market Electric Power Supplies for Market Electric Power Market Customers for Market (Plant Factories) Supplies Demands Results Results Fig. 4. Market mechanisms Price P (Yen/kWh) Electric Power Q (kWh) Supply Demand Balanced Point Fig. 3. Electric Power Market Electric Power Supplies for Market Electric Power Market Customers for Market (Plant Factories) Supplies Demands Results Results Electric Power Supplies for Market Price P (Yen/kWh) Supplies Balanced Point Fig. 4. Market mechanisms Fig. 3. Electric Power Market 5. Conclusions A plant factories and its production planning system are discussed in the paper. The followings are concluded. (1) The production cost items are considered to plan and to estimate the production costs of individual piece of leaf lettuces, based on the investigations of the exper- imental plant factories in Osaka Prefecture University and related documents. (2) A genetic programming (GP) based method is proposed to forecast the future pric- es of the lettuces, since the long lead time of 25 days are required for cultivating the lettuces. (3) A production planning system for the plant factories is proposed to select a suita- ble vegetable to be cultivated in every day operations, aimed at increasing the profits and decreasing the operation costs. 7 7 -30 -20 -10 0 10 20 30 40 3倍 3.5倍 4倍 Profit of plant factories (Million Yen) 3 3.5 4 Retail Market Price / Wholesale Market Price Plant Factory A Plant Factory B Fig. 5. Profit of plant factory 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 1 61 121 181 241 301 Production Ratio of Lettuce (%) 日数(日) Days (2010 Jan. to 2010 Dec. Fig. 6. Production volumes of lettuce in plant factory B -30 -20 -10 0 10 20 30 40 3倍 3.5倍 4倍 Profit of plant factories (Million Yen) 3 3.5 4 Retail Market Price / Wholesale Market Price Plant Factory A Plant Factory B Fig. 5. Profit of plant factory 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 1 61 121 181 241 301 Production Ratio of Lettuce (%) 日数(日) Days (2010 Jan. to 2010 Dec. Fig. 6. Production volumes of lettuce in plant factory B Rreferences Rreferences [1] Takatsuji. M., 2007, Fully Controlled and Closed Plant Factory, Ohmsha Ltd., pp63-65 (in Japamese) [1] Takatsuji. M., 2007, Fully Controlled and Closed Plant Factory, Ohmsha Ltd., pp63-65 (in Japamese) pp p [2] Outline of Facilities (R&D Center for the Plant Factory), (http://www.plant- factory.21c.osakafu-u.ac.jp/english/index.html) [3] Takatsuji. M. ,Mori.Y, 2011, LED Plant factory, Nikkan Kogyo Shimbun Ltd., pp10-27 (in Japanese) [4] Tokyo Metropolitan Central Wholesale Market (http://www.shijou.metro.tokyo. jp/torihiki/) (in Japanese) [5] Shukla M. and Jharkharia S., 2011, Applicability of ARIMA Models in Whole- sale Vegetable Market, Proc. of the 2011 International Conference on Industrial Engineering and Operations Management, pp1125-1130. [6] Nasira, G.M. and Hemageetha, A., 2012, Forecasting Model for Vegetable Price Using Back Propagation Neural Network, Int. J. of Computational Intelligence and Informatics, Vol.2, No.2, pp110-115. pp [7] Japan Electric Power eXchange (http://www.jepx.org/english/aboutus/index. html) 8
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A Tribute to Prof. Keiichiro Fukumoto
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ARKIVOC 2003 (viii) 1-7 Keiichiro Fukumoto ARKIVOC 2003 (viii) 1-7 alkaloids such as the morphine-, aporphine-, proaporphine-, protoberberine-, hasbanan-, benzophenanthridine-, and sendaverine types, the phenethylisoquinoline type, Erythrina-, Amaryllidaceae- and Ipecac- alkaloids, were totally synthesized. His books, “The Chemistry of the Isoquinoline Alkaloids, vols. 1 and 2”, published along with the late Professor Kametani in 1968 and 1974, respectively, contributed tremendously to this field. The principle of ‘Retro Mass Spectral Synthesis’ that he proposed together with the late Professor Kametani in 1974 was especially brilliant and fruitful. Various biologically active natural products such as steroids, diterpenes, triterpenes, protoberberine-, phthalideisoquinoline-, quinazolinocarboline-, spiro-benzylisoquinoline- alkaloids, Ipecac alkaloids, and indole-, and diterpene alkaloids, were elegantly synthesized. In particular, the syntheses of estrone, (+)- estradiol, alnusenone and friedelin using the intramolecular Diels–Alder reaction of benzocyclobutene, based on the principle, are outstanding. Applications utilizing pericyclic reactions of o-quinodimethane developed by his group are regarded as leading research in synthetic chemistry. As an extension of pericyclic reactions, he and his associates studied the development of new cascade reactions, forming plural bonds in a stereo- and regioselective manner in one procedure. The intramolecular double-Michael reaction and the intramolecular Michael–aldol reactions, elaborated by them, are useful tools for the synthesis of polycyclic natural products. It was established that both cascade reactions could be performed by treatment with lithium amides or treatment with various combinations of Lewis acids and amines. They showed many advantages, owing to the characteristic features of the intramolecular reaction and cascade reaction. The intramolecular double-Michael reaction is applicable not only to carbocyclic compounds but also to heterocyclic compounds. The alkaloids epilupinine and tylophorine, were effectively synthesized by the intramolecular aza- double- Michael reaction. Syntheses of terpenoids, pentalenene, pentalenic acid, 8,14-cedranediol and atisirene, were carried out in highly stereoselective manners by employing the intramolecular double-Michael reaction. The asymmetric synthesis of the Aconitium alkaloid atisine, accomplished by this methodology, is a beautiful example. Unique polycyclic ring systems fused to cyclobutane were constructed efficiently by the intramolecular Michael–aldol reaction. The method has recently been further extended to other new types of cascade reactions by his associates. In every respect, Professor Fukumoto has been a brilliant and productive chemist. Furthermore, he is an outstanding human being and an excellent educator. We have always been stimulated to do our best for research by his encouragement. He was a good baseball player when he was a high- school student. ARKIVOC 2003 (viii) 1-7 ARKIVOC 2003 (viii) 1-7 Issue in Honor of Prof. Keiichiro Fukumoto Professor Keiichiro Fukumoto A Tribute This Special Issue of Arkivoc is to celebrate the 70th anniversary of Professor Keiichiro Fukumoto’s birth Professor Keiichiro Fukumoto A Tribute Professor Keiichiro Fukumoto A Tribute This Special Issue of Arkivoc is to celebrate the 70th anniversary of Professor Keiichiro Fukumoto’s birth Professor Keiichiro Fukumoto was born in Shodo Island, Kagawa prefecture, on February 10, 1934. He graduated from the Pharmaceutical Institute, Osaka University in 1956 and obtained his Ph.D. degree from Osaka University in 1964, studying the synthesis of isoquinoline alkaloids, cularine, and related ones, under the guidance of the late Professor Tetsuji Kametani. He was appointed Assistant Professor of the Pharmaceutical Institute, Tohoku University, in 1959, and promoted to Associate Professor in 1972 and Full Professor in 1981. In 1997 he retired from Tohoku University and became Emeritus Professor. From 1964, he spent one year at the University of Alberta with Professor S. Masamune and one year at the University of Sussex, with Professor A. I. Scott, as a postdoctoral fellow. Professor Fukumoto has won a number of awards such as The Pharmaceutical Society of Japan (The PSJ) Award for Young Scientists in 1976, The PSJ Award in 1997, the Academic Award of The Society of Synthetic Organic Chemistry, Japan, in 1993, and Medal of Honor with Purple Ribbon in 2000. He has served the chemical community in many ways: he has acted for a long period as the Editor of Heterocycles, after Professor Kametani passed away in 1988. Professor Fukumoto’s research interests have ranged widely over synthetic chemistry; in developments of novel synthetic methodologies and syntheses of biologically active compounds. He has synthesized over 200 natural products and published 633 scientific papers. Selected papers are listed according to categories at the end of this Tribute. I give here a brief description of his selected research activities. His research career as an organic chemist started in 1955, and his first project was the synthesis of isoquinoline- and indole alkaloids. He obtained his Ph.D. with the accomplishment of the total synthesis of cularine, as mentioned above. Until the early 1970s, his main interest was the synthesis of isoquinoline alkaloids by biomimetic approaches utilizing phenol oxidation, the modified Pschorr reaction, and so on. A number of isoquinoline ISSN 1551-7012 ©ARKAT USA, Inc ©ARKAT USA, Inc Page 1 Issue in Honor of Prof. ARKIVOC 2003 (viii) 1-7 With an attitude similar to sports, he has been at the forefront of research in new areas and has tackled the most difficult problems with the deepest insight. We all wish Professor Fukumoto a healthy and happy life with Mrs Fukumoto. Masataka Ihara Sendai January 2003 ©ARKAT USA, Inc Page 2 Page 2 Page 2 ISSN 1551-7012 ISSN 1551-7012 ARKIVOC 2003 (viii) 1-7 Issue in Honor of Prof. Keiichiro Fukumoto Quinoline Alkaloids 1. Double Enamine Annelation of 3,4-Dihydro-1-methyl-β-carboline and Isoquinoline Derivatives with 6-Methyl-2-pyrone-3,5-dicarboxylates and its Application for the Synthesis of (±)-Camptothecin, Ihara, M.; Noguchi, K., Ohsawa, T.; Fukumoto, K.; Kametani T. J. Org. Chem. 1983, 48, 3150. 1. Double Enamine Annelation of 3,4-Dihydro-1-methyl-β-carboline and Isoquinoline Derivatives with 6-Methyl-2-pyrone-3,5-dicarboxylates and its Application for the Synthesis of (±)-Camptothecin, Ihara, M.; Noguchi, K., Ohsawa, T.; Fukumoto, K.; Kametani T. J. Org. Chem. 1983, 48, 3150. 2. Total Synthesis of Hydrocinchonidine and Hydrocinchonine from an Indole Derivative via Oxidation with Singlet Oxygen, Ihara, M.; Taniguchi, N.; Noguchi, K.; Fukumoto, K.; Kametani, T. J. Chem. Soc. Chem. Commun. 1986, 573. Indole Alkaloids 1. Asymmetric Total Synthesis of Tacamonine (Pseudovincamone I) via Radical Cyclization, Ihara, M.; Setsu, F.; Shohda, M. (nee Hosoda); Taniguchi, N; Tokunaga, Y.; Fukumoto, K. J. Org. Chem. 1994, 59, 5317. 1. Asymmetric Total Synthesis of Tacamonine (Pseudovincamone I) via Radical Cyclization, Ihara, M.; Setsu, F.; Shohda, M. (nee Hosoda); Taniguchi, N; Tokunaga, Y.; Fukumoto, K. J. Org. Chem. 1994, 59, 5317. 2. Stereoselective Construction of the Diterpene Part of Indole Alkaloids, Radarins, by Way of Intramolecular Diels–Alder Reaction, Ihara, M.; Katsumata, A.; Egashira, M.; Suzuki, S.; Tokunaga, Y.; Fukumoto, K. J. Org. Chem. 1995, 60, 5560. 2. Stereoselective Construction of the Diterpene Part of Indole Alkaloids, Radarins, by Way of Intramolecular Diels–Alder Reaction, Ihara, M.; Katsumata, A.; Egashira, M.; Suzuki, S.; Tokunaga, Y.; Fukumoto, K. J. Org. Chem. 1995, 60, 5560. Isoquinoline Alkaloids 1. A Total Synthesis of (±)-Cularine, Kametani, T.; Fukumoto, K. J. Chem. Soc. (C) 1963, 4289. 1. A Total Synthesis of (±)-Cularine, Kametani, T.; Fukumoto, K. J. Chem. Soc. (C) 1963, 4289. 2. Synthesis of Morphinandienone Alkaloids by Phenol Oxidation and the Pschorr Reaction, Kametani, T.; Fukumoto, K. J. Heterocyclic Chem. 1971, 8, 341. 2. Synthesis of Morphinandienone Alkaloids by Phenol Oxidation and the Pschorr Reaction, Kametani, T.; Fukumoto, K. J. Heterocyclic Chem. 1971, 8, 341. 3. Photochemical Synthesis of Isoquinoline Alkaloids, Kametani, T.; Fukumoto, K. Acc. Chem. Res. 1972, 5, 212. 3. Photochemical Synthesis of Isoquinoline Alkaloids, Kametani, T.; Fukumoto, K. Acc. Chem. Res. 1972, 5, 212. 4. Application of Phenolic Oxidation to the Total Syntheses of the Isoquinoline and Related Alkaloids; Biogenetic Type Syntheses, Kametani, T; Fukumoto, K. Synthesis 1972, 657. 4. Application of Phenolic Oxidation to the Total Syntheses of the Isoquinoline and Related Alkaloids; Biogenetic Type Syntheses, Kametani, T; Fukumoto, K. Synthesis 1972, 657. Terpenes p 1. Convenient and Stereoselective Route to Basic Frameworks for Synthesis of Unsymmetrical Pentacyclic Triterpenes Kametani, T.; Hirai, Y.; Shiratori, Y.; Fukumoto, K.; Satoh, F. J. Am. Chem. Soc. 1978, 100, 554. 2. A Stereoselective Total Synthesis of (±)-∆9(12)Capnellene via the Intramolecular Diels–Alder Approach, Ihara, M.; Suzuki, T.; Katogi, M.; Taniguchi, N.; Fukumoto K. J. Chem. Soc., Chem. Commun. 1991, 646. 3. The First Enantioselective Total Synthesis of (+)-Laurene, H. Nemoto, Nagamochi, M.; Fukumoto, K. J. Chem. Soc., Chem. Commun. 1992, 1695. 3. The First Enantioselective Total Synthesis of (+)-Laurene, H. Nemoto, Nagamochi, M.; Fukumoto, K. J. Chem. Soc., Chem. Commun. 1992, 1695. 4. Pd2+-Promoted Cyclization in Linear Triquinane Synthesis. Total Synthesis of (±)-Hirsutene, Toyota, M.; Nishikawa, Y.; Motoki, K.; Yoshida, N.; Fukumoto, K. Tetrahedron Lett. 1993, 34, 6099. 5. Intramolecular Michael Reaction Using Trialkylsilyl Trifluoromethanesulfonates and Tertiary Amine Systems: Total Synthesis of (±)-Ricciocarpin A, Ihara, M.; Suzuki, S.; Taniguchi, N.; Fukumoto, K. J. Chem. Soc., Chem. Commun. 1993, 755. 6. A Remarkable Substituent Effect on the Enantioselectivity of Tandem Asymmetric Epoxidation and Enantiospecific Ring Expansion of Cyclopropylidene Alcohols: A New Enantiocontrolled Synthesis of (-)-Debromoaplysin and (-)-Aplysin, Nemoto, H; Nagamochi, M.; Ishibashi, H.; Fukumoto, K. J. Org. Chem. 1994, 59, 74. 7. A Simple Total Synthesis of (±)-Spirojatamol and (±)-Erythrodiene via Intramolecular 1,3- Dipolar Cycloaddition, Tokunaga, Y.; Yagihashi, M.; Ihara, M.; Fukumoto, K. J. Chem. Soc., Chem. Commun. 1995, 955. 7. A Simple Total Synthesis of (±)-Spirojatamol and (±)-Erythrodiene via Intramolecular 1,3- Dipolar Cycloaddition, Tokunaga, Y.; Yagihashi, M.; Ihara, M.; Fukumoto, K. J. Chem. Soc., Chem. Commun. 1995, 955. 8. Stereoselective Construction of Copaborneol and Longiborneol Frameworks by Intramolecular Double Michael Reaction, Ihara, M.; Makita, K.; Fujiwara, Y.; Tokunaga, Y.; Fukumoto, K. J. Org. Chem. 1996, 61, 6416. 8. Stereoselective Construction of Copaborneol and Longiborneol Frameworks by Intramolecular Double Michael Reaction, Ihara, M.; Makita, K.; Fujiwara, Y.; Tokunaga, Y.; Fukumoto, K. J. Org. Chem. 1996, 61, 6416. 9. Total Synthesis of (±)-Methyl Atis-16-en-19-oate via Homoallyl–Homoallyl Radical Rearrangement, Toyota, M.; Wada, T.; Fukumoto, K.; Ihara, M. J. Am. Chem. Soc. 1998, 120, 4961. Aconitium Alkaloids 1. A Facile Regiospecific and Stereocontrolled Synthesis of a Diterpene Alkaloid Intermediate from Benzocyclobutene, Kametani, T.; Kato, Y.; Honda, T.; Fukumoto, K. J. Am. Chem. Soc. 1976, 98, 8185. 2. Stereoselective Total Synthesis of (±)-Atisine via Intramolecular Double Michael Reaction, Ihara, M.; Suzuki, M.; Fukumoto, K.; Kametani, T. J. Am. Chem. Soc. 1988, 110, 1963. 2. Stereoselective Total Synthesis of (±)-Atisine via Intramolecular Double Michael Reaction, Ihara, M.; Suzuki, M.; Fukumoto, K.; Kametani, T. J. Am. Chem. Soc. 1988, 110, 1963. 3. Asymmetric Total Synthesis of Atisine via Intramolecular Double Michael Reaction, Ihara, M. ; Suzuki, M.; Fukumoto, K.; Kabuto, C. J. Am. Chem. Soc. 1990, 112, 1164. 3. Asymmetric Total Synthesis of Atisine via Intramolecular Double Michael Reaction, Ihara, M. ; Suzuki, M.; Fukumoto, K.; Kabuto, C. J. Am. Chem. Soc. 1990, 112, 1164. ISSN 1551-7012 ©ARKAT USA, Inc Page 3 Page 3 ISSN 1551-7012 ARKIVOC 2003 (viii) 1-7 Issue in Honor of Prof. Keiichiro Fukumoto Steroids A Novel Strategy for the Enantioselective Synthesis of the Steroidal Framework Using Cascade Ring Expansion Reactions of Small Ring System; Asymmetric Total Synthesis of (+)-Equilenin, Nemoto, H.; Yoshida, M.; Fukumoto, K.; Ihara, M. Tetrahedron Lett. 1999, 40, 907. Synthetic Methodologies 1. A Facile Synthesis of Quinazoline System by Condensation of Iminoketene with Imines; A Total Synthesis of Evodiamine and Rutecarpine by Retro Mass-Spectral Synthesis, Kametani, T.; Higa, T.; Loc, C. V.; Ihara, M.; Koizumi, M.; Fukumoto, K. J. Am. Chem. Soc. 1976, 98, 6186. 1. A Facile Synthesis of Quinazoline System by Condensation of Iminoketene with Imines; A Total Synthesis of Evodiamine and Rutecarpine by Retro Mass-Spectral Synthesis, Kametani, T.; Higa, T.; Loc, C. V.; Ihara, M.; Koizumi, M.; Fukumoto, K. J. Am. Chem. Soc. 1976, 98, 6186. 2. Total Synthesis of Natural Products by Retro Mass Spectral Synthesis, Kametani, T.; Fukumoto, K. Acc. Chem. Res. 1976, 9, 319. 2. Total Synthesis of Natural Products by Retro Mass Spectral Synthesis, Kametani, T.; Fukumoto, K. Acc. Chem. Res. 1976, 9, 319. 3. Total Synthesis of Natural Products by Retro Mass Spectral Synthesis, Kametani, T.; Fukumoto, K. J. Synth. Org. Chem. Japan 1976, 34, 934. 3. Total Synthesis of Natural Products by Retro Mass Spectral Synthesis, Kametani, T.; Fukumoto, K. J. Synth. Org. Chem. Japan 1976, 34, 934. 4. Synthesis of β-Lactam Antibiotics by the Sulfeno–Cycloamination, Ihara, M.; Haga, Y.; Yonekura, M.; Ohsawa, T.; Fukumoto, K.; Kametani, T. J. Am. Chem. Soc. 1983, 105, 7345. 4. Synthesis of β-Lactam Antibiotics by the Sulfeno–Cycloamination, Ihara, M.; Haga, Y.; Yonekura, M.; Ohsawa, T.; Fukumoto, K.; Kametani, T. J. Am. Chem. Soc. 1983, 105, 7345. 5. Tandem Electrocyclic-Sigmatropic Reaction of Benzocyclobutenes. An Expedient Route to 4,4-Disubstituted Isochromanones, Shishido, K.; Shitara, E.; Fukumoto, K.; Kametani, T. J. Am. Chem. Soc. 1985, 107, 5810. 5. Tandem Electrocyclic-Sigmatropic Reaction of Benzocyclobutenes. An Expedient Route to 4,4-Disubstituted Isochromanones, Shishido, K.; Shitara, E.; Fukumoto, K.; Kametani, T. J. Am. Chem. Soc. 1985, 107, 5810. 6. Enantioselective Construction of a Quaternary Asymmetric Carbon Center: A Versatile Synthesis of α-Alkyl α-Amino Acids, Ihara, M.; Takahashi, M.; Niitsuma, N.; Taniguchi, N.; Yasui, K.; Fukumoto, K. J. Org. Chem. 1989, 54, 5413. 7. Novel Construction of Polycyclic Systems Fused to Cyclobutane by Tandem Intramolecular Michael–Aldol Reaction, Ihara, M.; Ohnishi, M.; Takano, M.; Makita, K.; Taniguchi, N.; Fukumoto, K. J. Am. Chem. Soc. 1992, 114, 4408. 8. Synthesis of Polycyclic Cyclobutane Derivatives by Tandem Intramolecular Michael–Aldol Reaction under Two Complementary Conditions, TBDMSOTf–Et3N and TMSI–(TMS)2NH, Ihara, M.; Taniguchi, T.; Makita, K.; Takano, M.; Ohnishi, M.; Taniguchi, N.; Fukumoto, K.; Kabuto, C. J. Am. Chem. Soc. 1993, 115, 8107. 9. Steroids 1. A Formal Regio- and Stereoselective Total Synthesis of Estrone. A Convenient Synthesis of D-Homoestrone, Kametani, T.; Nemoto, H.; Ichikawa, H.; Shiroyama, K.; Fukumoto, K. J. Am. Chem. Soc. 1976, 98, 3378. 1. A Formal Regio- and Stereoselective Total Synthesis of Estrone. A Convenient Synthesis of D-Homoestrone, Kametani, T.; Nemoto, H.; Ichikawa, H.; Shiroyama, K.; Fukumoto, K. J. Am. Chem. Soc. 1976, 98, 3378. 2. Asymmetric Total Synthesis of Estradiol by an Intramolecular Cycloaddition of Benzocyclobutene Derivative, Kametani, T.; Matsumoto, H.; Nemoto, H.; Fukumoto, K. J. Am. Chem. Soc. 1978, 100, 6218. 2. Asymmetric Total Synthesis of Estradiol by an Intramolecular Cycloaddition of Benzocyclobutene Derivative, Kametani, T.; Matsumoto, H.; Nemoto, H.; Fukumoto, K. J. Am. Chem. Soc. 1978, 100, 6218. 3. Chiral Synthesis of Androsterone through Intramolecular Diels–Alder Reaction, Ihara, M.; Sudow, I.; Fukumoto, K.; Kametani, T. J. Org. Chem. 1985, 50, 144. 3. Chiral Synthesis of Androsterone through Intramolecular Diels–Alder Reaction, Ihara, M.; Sudow, I.; Fukumoto, K.; Kametani, T. J. Org. Chem. 1985, 50, 144. ©ARKAT USA, Inc ISSN 1551-7012 Page 4 Page 4 ARKIVOC 2003 (viii) 1-7 Issue in Honor of Prof. Keiichiro Fukumoto 4. A Novel Strategy for the Stereoselective Total Synthesis of C-17 Spiro Steroids. Total Synthesis of 19-Norcanrenone, a Formal Total Synthesis of 19-Norspironolactone, Nemoto, H.; Fujita, S.; Nagai, M.; Fukumoto, K.; Kametani, T. J. Am. Chem. Soc. 1988, 110, 2931. 5. A Novel Synthetic Approach to Steroids via Intramolecular 1,3-Dipolar Cycloaddition. A Highly Stereocontrolled Synthesis of Testosterone, Ihara, M.; Tokunaga, Y.; Fukumoto, K. J. Org. Chem. 1990, 55, 4497. 5. A Novel Synthetic Approach to Steroids via Intramolecular 1,3-Dipolar Cycloaddition. A Highly Stereocontrolled Synthesis of Testosterone, Ihara, M.; Tokunaga, Y.; Fukumoto, K. J. Org. Chem. 1990, 55, 4497. 6. First Enantioselective Total Synthesis of (+)-Cortisone, Nemoto, H.; Matsuhashi, N.; Imaizumi, M.; Nagai, M.; Fukumoto, K. J. Org. Chem. 1990, 55, 5625. 6. First Enantioselective Total Synthesis of (+)-Cortisone, Nemoto, H.; Matsuhashi, N.; Imaizumi, M.; Nagai, M.; Fukumoto, K. J. Org. Chem. 1990, 55, 5625. 7. A Novel Strategy for the Enantioselective Synthesis of the Steroidal Framework Using Cascade Ring Expansion Reactions of Small Ring System; Asymmetric Total Synthesis of (+)-Equilenin, Nemoto, H.; Yoshida, M.; Fukumoto, K.; Ihara, M. Tetrahedron Lett. 1999, 40, 907. 7. Synthetic Methodologies Syntheses of Polycyclic Natural Products Employing the Intramolecular Double Michael Reaction, Ihara, M.; Fukumoto, K. Angew. Chem. Int. Ed. Engl. 1993, 32, 1010. ISSN 1551-7012 Page 5 ©ARKAT USA, Inc ARKIVOC 2003 (viii) 1-7 Issue in Honor of Prof. Keiichiro Fukumoto 10. Stereoselective Formation of Three Carbon–Carbon Bonds by Cascade Reaction with Enolate Anion: Synthesis of Tricyclo[6.2.2.01,6]dodecane and Tricyclo[5.3.1.03,8]undecane Derivatives, Ihara, M.; Makita, K.; Tokunaga, Y.; Fukumoto, K. J. Org. Chem. 1994, 59, 6008. 11. 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Facile Construction of Bicyclo[6.4.0]dodecane System by Intramolecular Michael Addition of Sulfonyl Carbanion, Ihara, M.; Suzuki, S.; Tokunaga, Y.; Takeshita, H.; Fukumoto, K. Chem. Commun. 1996, 1801. 16. 1,3-Dipolar Cycloaddition of 1-Carboxynitrone: Different Stereoselectivity Caused by Salt Effect, Tokunaga, Y.; Ihara, M.; Fukumoto, K. Tetrahedron Lett. 1996, 37, 6157. 17. Allenylcyclobutanol, a Versatile Initiator for the Palladium-Catalyzed Cascade Reaction: A Novel Route to Bicyclo[5.3.0]- and -[6.3.0] Frameworks, Nemoto, H.; Yoshida, M.; Fukumoto, K. J. Org. Chem. 1997, 62, 6450. 17. Allenylcyclobutanol, a Versatile Initiator for the Palladium-Catalyzed Cascade Reaction: A Novel Route to Bicyclo[5.3.0]- and -[6.3.0] Frameworks, Nemoto, H.; Yoshida, M.; Fukumoto, K. J. Org. Chem. 1997, 62, 6450. 18. A Novel Palladium-Mediated Cascade Reaction Triggered by Strain Release of the Cyclobutane System. A New General Route to Benzo- and Naphthohydrindans, Nemoto, H.; Miyata, J.; Yoshida, M. ; Raku, N.; Fukumoto, K. J. Org. Chem. 1997, 62, 7850. 18. A Novel Palladium-Mediated Cascade Reaction Triggered by Strain Release of the Cyclobutane System. A New General Route to Benzo- and Naphthohydrindans, Nemoto, H.; Miyata J ; Yoshida M ; Raku N ; Fukumoto K J Org Chem 1997 62 7850 Syntheses of Mycalamides, Toyota, M.; Hirota, M.; Nishikawa, Y.; Fukumoto, K.; Ihara, M. J. Org. Chem. 1998, 63, 5895. Medicinal Chemistry 1. Highly Stereocontrolled Synthesis of the Key Intermediate of 1β-Methylcarbapenem Antibiotic via Intramolecular Nitrone 1,3-Dipolar Cycloaddition, Ihara, M.; Takahashi, T.; Fukumoto, K.; Kametani, T. J. Chem. Soc., Chem. Commun. 1988, 9. 1. Highly Stereocontrolled Synthesis of the Key Intermediate of 1β-Methylcarbapenem Antibiotic via Intramolecular Nitrone 1,3-Dipolar Cycloaddition, Ihara, M.; Takahashi, T.; Fukumoto, K.; Kametani, T. J. Chem. Soc., Chem. Commun. 1988, 9. 2. An Efficient Synthesis of the Naphthalene Moiety of Neocarzinostatin Chromophore, Shishido, K.; Yamashita, A.; Hiroya, K.; Fukumoto, K. Tetrahedron Lett. 1989, 30, 111. 2. An Efficient Synthesis of the Naphthalene Moiety of Neocarzinostatin Chromophore, Shishido, K.; Yamashita, A.; Hiroya, K.; Fukumoto, K. Tetrahedron Lett. 1989, 30, 111. 3. A Novel o-Quinodimethane Strategy for an Active Metabolite of Vitamin D3. A Total Synthesis of 25-Hydroxy Windaus–Grundmann Ketone, Nemoto, H.; Ando, M.; Fukumoto, K. Tetrahedron Lett. 1990, 31, 6205. 4. Palladium-Catalyzed Intramolecular Allylic Alkylation Reaction in Marine Natural Product Synthesis: Enantioselective Synthesis of (+)-Methyl Pederate, a Key Intermediate in ISSN 1551-7012 ©ARKAT USA, Inc Page 6 Issue in Honor of Prof. Keiichiro Fukumoto ARKIVOC 2003 (viii) 1-7 Syntheses of Mycalamides, Toyota, M.; Hirota, M.; Nishikawa, Y.; Fukumoto, K.; Ihara, M. J. Org. Chem. 1998, 63, 5895. ARKIVOC 2003 (viii) 1-7 ARKIVOC 2003 (viii) 1-7 ARKIVOC 2003 (viii) 1-7 Issue in Honor of Prof. Keiichiro Fukumoto Issue in Honor of Prof. Keiichiro Fukumoto Syntheses of Mycalamides, Toyota, M.; Hirota, M.; Nishikawa, Y.; Fukumoto, K.; Ihara, M. J. Org. Chem. 1998, 63, 5895. ©ARKAT USA, Inc ISSN 1551-7012 Page 7 Page 7
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Development of a scale prototype of isokinetic dynamometer
Ingeniare. Revista chilena de ingeniería
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Ingeniare. Revista chilena de ingeniería, vol. 23 Nº 2, 2015, pp. 196-207 Ingeniare. Revista chilena de ingeniería, vol. 23 Nº 2, 2015, pp. 196-207 ABSTRACT In recent decades, scientific advances in strength training and physical therapy enabled the development of machines, which can monitor and perform the exercise with versatility and multifunctionality, such as the isokinetic dynamometers (ID). Nowadays it can be identified some problems with ID, including: a) high cost, b) isokinetic impact, c) posture on the dynamometer do not simulate the posture on sports, d) misalignment of the articular anatomic center of rotation to the center of rotation of the dynamometer. The objective of the present research is to develop a prototype of a new ID conception that should give theoretical and practical support to performing solutions that satisfy the problems related above. The prototype of a new ID conception is deployed by PRODIP methodology encompassing the phases of planning design, informational design, conceptual design and preliminary design. Comparing the simulated results with the theory, for each exercise mode, it can be concluded that the prototype can be used to support possible innovations for the problems mentioned above. Keywords: Isokinetic dynamometer, physiotherapy, control, mechatronics, product development. Daniel Alejandro Ponce Saldías1    Daniel Martins1    Carlos Martin1 Fabíola Da Silva Rosa2    Carlos Rodrigo de Mello Roesler2    Ari Digiácomo Ocampo Moré2 Daniel Alejandro Ponce Saldías1    Daniel Martins1    Carlos Martin1 Fabíola Da Silva Rosa2    Carlos Rodrigo de Mello Roesler2    Ari Digiácomo Ocampo Moré2 Recibido 3 de enero de 2014, aceptado 10 de julio de 2014 Received: January 3, 2014    Accepted: July 10, 2014 p g ; g ; g 2 Laboratório de Engenharia Biomecânica LEBm. Universidade Federal de Santa Catarina. /UFSC. Campus Universit João David Ferreira Lima, Trindad. Florianópolis, Santa Catarina, Brasil. CEP: 88040-900. 1 Departamento de Engenharia Mecânica. Universidade Federal de Santa Catarina / UFSC. Campus Universitário Reitor João David Ferreira Lima, Trindade. Florianópolis, Santa Catarina, Brasil. CEP: 88040-900. E-mail: danielpo25@gmail.com; danielemc@gmail.com; cam@grucon.ufsc.br p E-mail: fahbysr@gmail.com; rroesler@hu.ufsc.br; arimore@terra.com.br 1 Departamento de Engenharia Mecânica. Universidade Federal de Santa Catarina / UFSC. Campus Universitário Reitor João David Ferreira Lima, Trindade. Florianópolis, Santa Catarina, Brasil. CEP: 88040-900. E-mail: danielpo25@gmail.com; danielemc@gmail.com; cam@grucon.ufsc.br 2 Laboratório de Engenharia Biomecânica LEBm. Universidade Federal de Santa Catarina. /UFSC. Campus Universitário Reitor João David Ferreira Lima, Trindad. Florianópolis, Santa Catarina, Brasil. CEP: 88040-900. E-mail: fahbysr@gmail.com; rroesler@hu.ufsc.br; arimore@terra.com.br 1 Departamento de Engenharia Mecânica. Universidade Federal de Santa Catarina / UFSC. Campus Universitário R David Ferreira Lima, Trindade. Florianópolis, Santa Catarina, Brasil. CEP: 88040-900. p g ; g ; g 2 Laboratório de Engenharia Biomecânica LEBm. Universidade Federal de Santa Catarina. /UFSC. Campus Universitário Reitor João David Ferreira Lima, Trindad. Florianópolis, Santa Catarina, Brasil. CEP: 88040-900. E-mail: fahbysr@gmail.com; rroesler@hu.ufsc.br; arimore@terra.com.br Desarrollo de un prototipo a escala de dinamómetro isocinético Daniel Alejandro Ponce Saldías1    Daniel Martins1    Carlos Martin1 Da Silva Rosa2    Carlos Rodrigo de Mello Roesler2    Ari Digiácomo Ocampo Moré2 RESUMEN En las últimas décadas los avances científicos relacionados al entrenamiento de fuerza y a la fisioterapia, han permitido desarrollar máquinas que pueden monitorear y realizar ejercicios tan versátiles y multifuncionales como los dinamómetros isocinéticos (DI). Hoy en día pueden identificarse algunos problemas con los DI, que incluyen: A) alto costo, b) choque isocinético, c) la postura de los DI no simula la postura deportiva, d) desalineamiento del centro de rotación anatómico con el centro de rotación del dinamómetro. El objetivo del presente estudio es desarrollar un prototipo de una nueva concepción de DI que de soporte, teórico y práctico, para la implementación de soluciones que satisfagan los problemas mencionados. El prototipo de la nueva concepción de DI es desarrollado mediante la metodología PRODIP, abarcando las fases de planeamiento de diseño, diseño informacional, diseño conceptual y diseño preliminar. Comparando los resultados experimentales con la teoría, para cada modo de ejercicio, puede concluirse que el prototipo puede ser usado como soporte para posibles innovaciones basadas en los problemas indicados anteriormente. Palabras clave: Dinamómetro isocinético, fisioterapia, control, mecatrónica, desarrollo de producto. Ponce, Martins, Martin, Da Silva, De Mello and Ocampo: Development of a scale prototype of isokinetic dynamometer INTRODUCTION The ID is an electromechanical equipment that performs controlled exercise at specific velocities, torques and positions, using a servo motor and a closed loop control system. On the last decades the knowledge on strength training and physiotherapy have evolved resulting in significant improvement on physical performance and muscular recovery from injury. At the same time the robotic automatization was developed and applied, mostly because of microelectronic advances and significant increasing on the quality of testing and measuring instruments used on research by professionals from sports and rehabilitation [1]. There upon, it becomes possible to develop complex models of mechanical systems for special equipment for physical exercise. A group of machines stand up: the isokinetic dynamometers, machines that monitor and perform exercises, also know as robotic dynamometers (Figure 1), adapted from Wimpenny [2]. Nowadays it can be identified some problems with ID, including: a) high cost, b) isokinetic impact, c) posture on the dynamometer do not simulate the real posture on sports, d) misalignment of the articular anatomic center of rotation related to the center of rotation of the dynamometer. This work originates from the need for improvement of ID, considering the problems associated with these devices. Thus, this research aims to develop a prototype of a new ID conception that should give support to performing solutions to satisfy the problems related above. In order to achieve this, it was performed a critical introduction to isokinetic dynamometers, provided by the explanation of its operation and utilities. The ID are still not widespread due to high cost, limiting its use to advanced medical centers, sports studies and physical therapy [1, 3-5]. Operation of Isokinetic Dynamometers An ID responds very fast and accurately to movements, variations of speed and torque of the patient or athlete using a system of closed loop control. A functional diagram of the dynamometer is shown in Figure 2 (adapted from Ponce [6]) and consists of two interfaces (machine-operator and patient-machine) and four subsystems: command- control, drive, mechanisms, and measurement. These subsystems are explained next: Mechanisms Subsystem: executes the patient-machine interface. It is composed by the mechanical and ergonomics components that allow the patient or athlete to perform the exercise and evaluation in a comfortable posture, enabling the isolated work of a single muscle group of interest. – Command-control subsystem: performs the machine-operator interface and the control of the driver subsystem. The operator can give instructions through a software manager to set each mode of exercise to be performed by the patient or athlete. This subsystem receives the simultaneous information from the measurement system and processes it for maintaining the dynamic characteristics and safety of the exercise. Here, the exercise of the patient or athlete is also monitored, reports are printed and evaluations are given. – Command-control subsystem: performs the machine-operator interface and the control of the driver subsystem. The operator can give instructions through a software manager to set each mode of exercise to be performed by the patient or athlete. This subsystem receives the simultaneous information from the measurement system and processes it for maintaining the dynamic characteristics and safety of the exercise. Here, the exercise of the patient or athlete is also monitored, reports are printed and evaluations are given. MATERIALS AND METHODS Figure 1. Models of isokinetic dynamometers. To perform a critical introduction of ID, the authors have searched the literature related to ID’s, physiotherapy, training and evaluation of force, with selected papers up to December 2013, from reviewing journals in the areas of biomechanics, sports medicine and sports science, bioengineering, physiotherapy, orthopedics and sciences of motion, listed in the references section [1-19, 26], as well as patents of equipment for exercise and physical therapy [20-24]. In order to find applicable studies, the following keywords, in English and Portuguese were used: isokinetic, isokinetic evaluation, isokinetic dynamometry, isokinetic dynamometers, strength evaluation, rehabilitation, and muscular performance. Figure 1. Models of isokinetic dynamometers. Commonly, the ID is used for clinical and sport evaluation, but rarely for the purpose of strength training. These devices allow operating in two main Functions: Evaluation Function and Exercise Function. These Functions can be applied in high performance athletes and injured patients. The Evaluation Function is an indicator of the physical condition of the athlete and monitors the progress of injured tissues in the clinical patient. Also, the evaluation is very important for diagnostic and treat specific deficiencies preventively [5]. The Exercise Function means training for the athlete and physical therapy for the clinical patient. The new ID conception was deployed by PRODIP methodology [19], encompassing the phases of planning design, informational design, conceptual design and preliminary design including the implementation of the prototype. This design methodology allows to prototype a system that suits the user requirements, enabling for example, experiment with different torque sensors and algorithms to perform isokinetic mode, passive mode, isometric mode and isotonic mode, thanks to the modularity and ease of system tests designed. 197 Ingeniare. Revista chilena de ingeniería, vol. 23 Nº 2, 2015 This prototype also allows proposing and testing solutions to problems on the existing equipment. This prototype also allows proposing and testing solutions to problems on the existing equipment. a function of the force performed by the patient or athlete, resulting in a smooth man-machine movement. The motor is connected to the mechanisms subsystem through a mechanical reductor. The driver or power amplifier is the component that manages and provide to motor the voltage and electric current needed, using a reference signal coming from the control-command. Measurement subsystems, which are basically three: (a) Torque measurement system: Strain gages are usually used in the effector. In modern ID torque information is obtained by measuring the motor electric current; (b) Speed sensor: It is usually used a tachogenerator. In modern models is used an encoder; (c) Position sensor: A resistive sensor is used often, but modern models use an encoder. Isometric Mode A wide range of exercises and physical evaluations can be performed by ID as shown in Figure 3. Evaluation Function uses the basic modes. Exercise function uses basic and advanced modes. A clinical advantage of the Exercise Function is that it offers multiple options of training strategies and exercises [26]. Based on immobility or null displacement, isometric mode consists in to performing the maximum voluntary muscle activation against an invincible resistance [13]. When exercising, isometric muscle contractions can occur in many constant angular positions, within ranges of movements predetermined by the physiotherapist or trainer. Figure 3. The two main Functions of ID with their respective modes. Based on [26]. Some trainers qualify isometric mode as a training method that ease the growth of muscle volume, focused on the special training of muscle hypertrophy. From the physiotherapist point of view, isometric mode has defined therapeutic applications, as members stabilization, start the motor control of the patient, specific muscular strengthening, muscle stiffness remotion. Also the contraction/relaxation work and maintenance/relaxation of the muscle is allowed [2]. Functions of Isokinetic Dynamometers Functions of Isokinetic Dynamometers Drive subsystem: it consists of a motor and its driver. The motor (hydraulic or electromechanical) provides a resistance load, as The ID can provide two main Functions: Evaluation Function and Exercise Function [6, 26]. Both Figure 2. Functional diagram of the ID. Figure 2. Functional diagram of the ID. 198 once, Martins, Martin, Da Silva, De Mello and Ocampo: Development of a scale prototype of isokinetic dynamometer Table 1. Basic modes and dynamic characteristics. Based on [26]. Basic modes Isometric Isotonic Passive Isokinetic Position θ constant variable variable variable Speed &θ null variable constant constant Acceleration &&θ null variable null null Torque τ variable constant variable variable Functions are applied in healthy athletes and in patients with injuries. Evaluation Function serves as an indicator of the physical state of the athlete, and monitors the evolution of injured tissues on patient to help identifying different pathologies [7-9]. Exercise Function means training to the athlete and physiotherapy to the patient. There is evidence of improvement on muscle strength in different populations after isokinetic training [10-12]. Isotonic Mode During this exercise, speed and acceleration varies while the torque is kept constant. The speed and acceleration depend on the ability of the patient/ athlete and the safety limits of the magnitudes programmed on the isokinetic dynamometer [14]. In therapeutic use, the execution of this exercise not only allows a gain in muscle strength, but also a progressive neuromuscular response [2]. The isotonic mode can be set for specific magnitudes of torque according to the necessities of the patient or athlete. In strength training, it is equivalent to perform exercises on gym machines with constant loads. Figure 3. The two main Functions of ID with their respective modes. Based on [26]. The advanced mode “protocol” consists of an individual configuration organized by the physiotherapist or trainer. It involves basic modes with specific magnitudes of movements, speed and strength through the manager software. Many “protocols” could be stored in memory and screened (by name, date, etc.) to be repeated or modified. Some exercises of the “protocol” can be sequentially linked to create customized exercises through the “sequential” advanced mode. Passive Mode It is also known as continuous passive movement (CPM). This mode is used in patients in postoperative or in patients who had muscle or physical injuries and are looking for rehabilitation. Thus, the passive mode is classified as therapeutic. During this exercise, The dynamic characteristics of the basic modes shown in Figure 3, are presented in Table 1 and explained below. 199 Ingeniare. Revista chilena de ingeniería, vol. 23 Nº 2, 2015 usually performed in different places, therefore the continuous monitoring is impaired. the patient’s articulation is submitted to a movement with a low and constant speed. The evaluation position is different from the training position: the isokinetic dynamometer usually does not copy the performed movement of some real sport training (Figure 4), therefore during the evaluation, the athlete keeps a different posture from training posture. There are no curves of isokinetic tests for exercises that copy specifics sports movements (with closed kinematic chain, involving kinetic energy in several articulations), nor interpretation methods for these curves. According to Terreri [16], due to the fact of the isokinetic equipment not performing the specific movement of a determined sport modality, the effort performed does not involve the kinetic energy in several articulations, but of a single articulation, while the rest of the body remains without displacement. The benefits of the passive mode are: increase of soft tissue elasticity, short familiarization time with the equipment and improve muscle motor control [14]. Isokinetic Mode The isokinetic mode consists of performing muscular contractions, concentric and eccentric with a predefined speed that remains constant during the main part of movement [13], except on initial and final stages. The concentric contraction causes a shortening of the muscle during contraction, provoking an approximation of the joint segments [2]. The eccentric contraction causes an elongation of the muscle during contraction, causing a distancing of the joint segments [2]. The isokinetic term must be reserved, therefore, to designate a type of muscular action that accompanies a constant angular movement on one articulation [15]. The isokinetic contraction can only be done by special electromechanical equipment, with a control system, which the isokinetic machine responds in form of resistance directly proportional to the force exerted by the person. Figure 4. Difference between the positions of assessment and training. The indications for performing this kind of evaluation and exercise, refers to the agonist/antagonist muscular balance (muscle performing the movement/muscle opposing the movement), and the difference between the muscular groups from one side compared to their contralateral side (muscles on the left side compared to the right side). The clinical applications of this mode are, for example: Resistance Exercises, Submaximal and Maximal Resistance, Resistance Training or Endurance Training and Motion Control of the Patient in addition to physical assessment. Figure 4. Difference between the positions of assessment and training. – Isokinetic impact [13, 26]: When a high acceleration is performed on the ID before it reaches the constant speed stage of exercise, an impact (deceleration step) happens at the moment of reaching the isokinetic speed, because the mechanism “brakes” in order to perform a high deceleration and thus keeping the constant speed condition. It is produced an “overshoot” that can be read as a torque oscillation (not due to the patient) that may confuse the operator at the moment of interpreting the reported curve [6, 13]. – Isokinetic impact [13, 26]: When a high acceleration is performed on the ID before it reaches the constant speed stage of exercise, an impact (deceleration step) happens at the moment of reaching the isokinetic speed, because the mechanism “brakes” in order to perform a high deceleration and thus keeping the constant speed condition. It is produced an “overshoot” that can be read as a torque oscillation (not due to the patient) that may confuse the operator at the moment of interpreting the reported curve [6, 13]. Conceptual Design The conceptual design phase results in generation of design choices, where the new ID conception is obtained. This conception is chosen by comparison and evaluation of several other product conceptions, observing which of all conceptions best accomplishes design specifications. The product conceptions are obtained by a Morphological Matrix [19] knowing the functions that the product should offer and the principles of solution that meet each of these functions. Functions that the product should offer are shown below, as F1, F2, etc. The Morphological Matrix, including the new ID conception, is shown in Table 3. In [6] a detailed explanation of this table is developed. – Other problems that were manifested on interviews with experts in the field during the execution of this work were [26]: appearance of looseness in bearings with short time of use, low robustness of the structure, patient/ athlete accommodation on the equipment bench, electronic problems due the environment humidity and ergonomic problems at shoulder and hip. In this context, the development of a new design is proposed. This proposal consists in a mechatronic system for algorithm and architecture test, that serves for the development of equipment technology applied to the performing of therapeutic and sports exercises. F1. Information processing; F1. Information processing; F1.1. Control and selection/adjustment of exercise; F2. To generate exercises (mechanical magnitudes); F2.1. To generate torque and angular speed; F2.2. To reduce angular speed and to increase torque; F2.3. Engagement and alignment of the effector; F2.4. Ergonomic effector (engagement); F3. To convert signal into power; F4. To measure motor torque; F5. To measure kinematics magnitudes; F5.1. To measure angular speed; F5.2. To measure angular position. This new design must allow to perform the functions available on ID, but in order to know better the technologies that will allow improving the current systems. F2.4. Ergonomic effector (engagement); Isokinetic Mode – Isokinetic impact [13, 26]: When a high acceleration is performed on the ID before it reaches the constant speed stage of exercise, an impact (deceleration step) happens at the moment of reaching the isokinetic speed, because the mechanism “brakes” in order to perform a high deceleration and thus keeping the constant speed condition. It is produced an “overshoot” that can be read as a torque oscillation (not due to the patient) that may confuse the operator at the moment of interpreting the reported curve [6, 13]. Problems related to Isokinetic Dynamometers Nowadays, some problems related to ID are known, which consideration is pivotal for the future improvement of these equipments. The mains problems are [2, 6, 13, 16-18, 26]: Misalignment of the articular anatomic center of rotation in relation to the center of rotation of the dynamometer: authors such Wimpenny [2] indicate protocols with specific instructions to avoid this kind of problem. According to Aquino [1], studies that uses the isokinetic dynamometry Misalignment of the articular anatomic center of rotation in relation to the center of rotation of the dynamometer: authors such Wimpenny [2] indicate protocols with specific instructions to avoid this kind of problem. According to Aquino [1], studies that uses the isokinetic dynamometry – High cost: the isokinetic allows performing evaluation, training and physiotherapy, but these equipments are less known due the high cost, limiting its use to medical centers and studies; then the evaluation and training are – High cost: the isokinetic allows performing evaluation, training and physiotherapy, but these equipments are less known due the high cost, limiting its use to medical centers and studies; then the evaluation and training are 200 once, Martins, Martin, Da Silva, De Mello and Ocampo: Development of a scale prototype of isokinetic dynamometer priorities have raised design specifications. In order to illustrate it, the main design specifications are shown in the left column of the Table 2. In the right column are the target values: it consists in conditions (magnitude, demands or desirable conditions) that must be accomplish by the design specifications. with several purposes often presents conflicting results, that is because differences at the subject positioning related to the evaluated articulations. The alignment process must be strict, since alignment errors may turn into low reliability and low repeatability reading data for the same patient or athlete [17-18]. Planning Design Here it is defined the problem, the research, the objectives and constraints of this work. DESIGN OF ISOKINETIC DYNAMOMETER F3. To convert signal into power; F4. To measure motor torque; F5. To measure kinematics magnitudes; To model, simulate and prototype the new conception it is used the process of product development PRODIP [19] until the preliminary design stage. Thus, the structure has four major stages: F5.1. To measure angular speed; F5.2. To measure angular position. The developed solution is shown in Figure 5. As an application example, it is allocated for hand exercises. This conception allows evaluating algorithms and hardware architectures with small external torques, applied by the patient’s hand. This is much cost effective than a large ID for leg exercises for example. It is chosen a hand coupling because the torque needed is smaller (in comparison to the torque needed for a leg coupling) and therefore, the motor needed is smaller and safer when the new algorithms are tested. On the programmer-machine interface, different control algorithms are generated for each exercise mode. The speeds, angles and torques, involved in the execution of the exercise are saved and displayed Informational Design The user needs are defined based on the problems related to the ID mentioned above. Then, these user needs were transformed into user requirements using technical language suitable for expressing quality attributes of the product. After that, user requirements are evolved, assigning dimensions to them, emerging design requirements. Finally, the set of design requirements associated with service The speeds, angles and torques, involved in the execution of the exercise are saved and displayed 201 Ingeniare. Revista chilena de ingeniería, vol. 23 Nº 2, 2015 Table 2. Main Design Specifications for the development of an experimental ID. Specification list Target values Functionality System must acquire angular speed Dynamic zone: 0-300o/s Resolution: 1º/s System must acquire angular position Dynamic zone: 0-360o/s Resolution: 1o System must acquire torque Resolution:0.01Nm Data storage 1 Mbyte Time response of the System <50ms Four modes of exercises: Passive, Isokinetic, Isotonic and Isometric Demands Passive mode Constant angular speed Demands Continuous movement Demands Isokinetic mode Constant angular speed Demands Continuous movement Demands Isotonic mode Constant torque Demands Continuous movement Demands Isometric mode Constant position Demands Continuous movement Demands Generalities Ergonomic Wishes Security Demand Strong Wishes Table 3. Morphological Matrix. Ingeniare. Revista chilena de ingeniería, vol. 23 Nº 2, 2015 Table 2. Main Design Specifications for the development of an experimental ID. Specification list Target values Functionality System must acquire angular speed Dynamic zone: 0-300o/s Resolution: 1º/s System must acquire angular position Dynamic zone: 0-360o/s Resolution: 1o System must acquire torque Resolution:0.01Nm Data storage 1 Mbyte Time response of the System <50ms Four modes of exercises: Passive, Isokinetic, Isotonic and Isometric Demands Passive mode Constant angular speed Demands Continuous movement Demands Isokinetic mode Constant angular speed Demands Continuous movement Demands Isotonic mode Constant torque Demands Continuous movement Demands Isometric mode Constant position Demands Continuous movement Demands Generalities Ergonomic Wishes Security Demand Strong Wishes Table 2. Main Design Specifications for the development of an experimental ID. Table 3. Morphological Matrix. Table 3. Morphological Matrix. Table 3. Morphological Matrix. 202 once, Martins, Martin, Da Silva, De Mello and Ocampo: Development of a scale prototype of isokinetic dynamometer Figure 5. Prototype of the new conception with connectivity diagram and signal flow. by the data acquisition software StampPlotLite Inc. of Pallarax. On machine-operator interface is also selected the exercise and adjusted operating and safety parameters as torque, speed and angular range limit. Preliminary Design This section involves four steps, which result in a model of the plant for the new ID conception and the design of the simulation. The steps are: Physical sketch of system: Sketch that allows visualizing the interactions between system components. The sketch is shown in Figure 6, and it is composed by the electric system of the DC motor, the mechanical system of the DC motor and the effector system. In the electrical system of the motor: ua is the armature voltage, ui is the induced voltage, Ra is the electrical resistance, La is the motor inductance. In the mechanical part of the Dc motor: ω is the angular speed of the rotor, Bm is the bearing friction, and Jm is the rotor inertia. In the effector system Bs is the friction due the coupling and reductor, Js is the mechanical system inertia, and θ is the angular position of the end-effector. The parameters used to perform the simulation and control of the system are shown in Table 4. – Physical sketch of system: Sketch that allows visualizing the interactions between system components. The sketch is shown in Figure 6, and it is composed by the electric system of the DC motor, the mechanical system of the DC motor and the effector system. In the electrical system of the motor: ua is the armature voltage, ui is the induced voltage, Ra is the electrical resistance, La is the motor inductance. In the mechanical part of the Dc motor: ω is the angular speed of the rotor, Bm is the bearing friction, and Jm is the rotor inertia. In the effector system Bs is the friction due the coupling and reductor, Js is the mechanical system inertia, and θ is the angular position of the end-effector. The parameters used to perform the simulation and control of the system are shown in Table 4. The program developed in the BS2 microcontroller allows controlling each exercise mode, checking the conditions of: constant velocity for isokinetic and passive mode; constant torque for isotonic mode; and constant position to isometric mode (Table 1). Also continuously checks the limit magnitude of torque, speed and position (security limit setting) that trigger the signal to stop the exercise. Informational Design All this information is compiled in Basic Stamp® microcontroller (BS2) by the acquisition board and control Pallarax Inc. through a RS-232 channel communication. Also in Figure 5 is shown a diagram of connectivity and signal flow of the new conception. The implementation of this conception is available in [6]. The control algorithm of the exercise is implemented from a computer to the BS2 microcontroller. By following a functional sequence, the motor is triggered generating torque and angular velocity. In parallel, a person performs the exercise by exerting a load on the effector. Variations in physical magnitudes are measured by the sensors and read by BS2. The BS2 receives signals from two sensors: torquemeter Teldix 2125 and accelerometer MEMSIC that measures acceleration, angular position and speed. Figure 5. Prototype of the new conception with connectivity diagram and signal flow. Preliminary Design Model parameters for simulation. magnitude in its electrical analogy. Therefore, the model can be solved as an electrical system. magnitude in its electrical analogy. Therefore, the model can be solved as an electrical system. After that, the solution can return to its original mechanical analogy, in order to obtain the model of the plant of the new ID conception, as shown in equation (1). In [6] and [25] is developed a detailed analysis of this method. After that, the solution can return to its original mechanical analogy, in order to obtain the model of the plant of the new ID conception, as shown in equation (1). In [6] and [25] is developed a detailed analysis of this method. J&&θm + Bθm +C ⋅sen θm n ⎛ ⎝⎜ ⎞ ⎠⎟= n⋅τ m (1) (1) Where θm is effector angular position &θm is the motor shaft velocity, &&θm is the motor shaft acceleration, J&&θm is the torque component due to the acceleration, B &θm is the torque component due to the friction, C ⋅sen(θm / n) is the torque component due to the gravity, n is the mechanical reducer factor and tm is the total motor torque. Where θm is effector angular position &θm is the motor shaft velocity, &&θm is the motor shaft acceleration, J&&θm is the torque component due to the acceleration, B &θm is the torque component due to the friction, C ⋅sen(θm / n) is the torque component due to the gravity, n is the mechanical reducer factor and tm is the total motor torque. – Simulation: Numerical simulation were performed to obtain time and frequency responses of the system. For each basic mode different PID controls were implemented. Preliminary Design The BS2 determines in which situation to increase or decrease the torque and speed of the effector by sending a duty cycle signal to the motor driver, who delivers a PWM signal to the high dynamic DC motor (maximum torque 0.15Nm, at 0.5 rev/s). Modeling by generalized circuit: Performed with dynamic systems tools based on power flows [25] applied in each functional element of the system, such as inertia (kinetic energy storage), elasticity, rigidity (potential energy storage) and damping (energy sinks) (Figure 7). Kv and Km are the constants of velocity and torque of the motor. The motor has to overcome a total torque tm=t1+t2+tg as indicated in Figure 7, which does not considers the patient’s torque. A historical data with positions, speeds and torques related to time, is stored on the microcontroller memory BS2 and is available for the user, in order to analysis the exercise and improve the algorithm and PID control. For instance the PID control is used to improve the dynamics of the exercise system. For each exercise mode, different PID gains are settings by the programmer on the microcontroller. 203 Ingeniare. Revista chilena de ingeniería, vol. 23 Nº 2, 2015 Figure 6. Physical sketch of new ID conception. Figure 6. Physical sketch of new ID conception. Figure 6. Physical sketch of new ID conception. Figure 6. Physical sketch of new ID conception. Table 4. Model parameters for simulation. ua Armature voltage= 0 to12 V uR Resistance voltage → V La Armature inductance=72 µH Ra Armature resistance= 9 Ω ui Induced voltage → V uLa Coil voltage → V w Angular velocity→ rad/s ia Armature current= 0 to 5 A Bm Friction coefficient of motor bearings= 0.0047 kg∙m2∙(rad∙s)-1 BS Friction coefficient of the mechanical system=0.005 kg∙m2∙(rad∙s)-1 Js Inertia of the mechanical system= 0.0026 kg∙m2. Jm Inertia of the motor shaft= 0.0006 kg∙m2 θ Angular position of the end effector m Mass effector → kg l Length of the effector= 0.08m km Constant of velocity of the motor= 0.03557 Nm/A kv Constant of torque of the motor= 28.1135 V.s/rad &θ Angular velocity of the end effector→ rad/s &&θ Angular acceleration of the end effector → rad/s2 τm Motor torque → N τBm Torque due to the friction in bearings→ N τJm Torque due to the shaft inertia→ N τBs Torque by mechanic system friction→ N τJs Torque due to the shaft friction→ N Table 4. – Block diagram: Each functional element is converted to its transfer function as impedance or admittance, transforming each mechanical RESULTS The results of the simulations are shown in Figure 8, where the curves in red characterize the main behavior of each basic mode. Comparing Figure 8 with Table 1, it can be seen that the results are as expected. For isometric mode the position is kept with a constant value (previously set) for variations of the patient torque. In the isotonic mode the motor torque remains constant at variations – Block diagram: Each functional element is converted to its transfer function as impedance or admittance, transforming each mechanical 204 once, Martins, Martin, Da Silva, De Mello and Ocampo: Development of a scale prototype of isokinetic dynamometer CONCLUSIONS The design methodology used allowed to develop a project design system that met the needs of the user, Figure 7. Generalized circuit model of the new ID conception. of position, velocity and patient torque. The passive mode has a constant speed (and positive) in the up movement and a constant speed (and negative) in the down movement. A similar behavior is observed Figure 8. Results of new ID conception simulation. Figure 7. Generalized circuit model of the new ID conception. Figure 7. Generalized circuit model of the new ID conception. Figure 8. Results of new ID conception simulation. Figure 8. Results of new ID conception simulation. CONCLUSIONS of position, velocity and patient torque. The passive mode has a constant speed (and positive) in the up movement and a constant speed (and negative) in the down movement. A similar behavior is observed in the isokinetic mode, where the velocity curve remains constant (and positive) in movement upwards and constant (and negative) in movement downwards. The difference between passive mode and isokinetic mode, from a mechanical point of view is that: in the passive mode the motor torque is slightly higher than the patient’s torque, so the resultant movement follows the motor direction. In the isokinetic mode, the patient torque is higher than the motor torque, so the resultant movement follows the direction of the patient torque. The design methodology used allowed to develop a project design system that met the needs of the user, and should give theoretical and practical support would allow to create solutions to satisfy the needs. The methodology allowed to experiment with different algorithms in order to perform isokinetic modes, passive, isotonic and isometric, thanks to the ease of testing of the system designed. In accordance with the objectives of the work was acquired knowledge to be applied in future studies of control projects of man-machine interface. 205 Ingeniare. Revista chilena de ingeniería, vol. 23 Nº 2, 2015 dynamometer to assess shoulder function in tennis players”. Sports Biomech. Vol. 4, Issue 1, pp. 101-11. 2005. The use of the new ID concept, gives training and practice in motor control, modeling, and knowledge about behavior of the mechanical system under active biomechanical loads, allowing the creation of new solutions such as control algorithms for systems with interaction man-machine. [6] D. Ponce. “Estudo introdutório e desenvolvimento experimental de sistemas automatizados para exercícios terapêuticos e esportivos”. EDUFSC Universidade Federal de Santa Catarina Nº 1, pp. 33-184. Florianópolis, Brasil. 2009. Future projects can be aimed to create possible innovations, such as an ID that enables performing exercises that simulate sports positions, to reduce purchase costs, improve the control algorithm and develop systems for align the anatomical rotation center with the dynamometer rotation center. [7] [7] T.A. Blackburn, W.G. Eiland and W.D. Banly. “An introduction to the plica”. J. Orthop Sports Phys. Ther. Vol. 3, pp. 171-76. 1982. [8] B. Hoke. “The relationship between isokinetic testing and dynamic patellofemoral compression”. J. Orthop. Sports Phys. Ther. Vol. 4, pp. 150-155. 1983. ACKNOWLEDGMENT [10] The authors would like to thank the CAPES- Coordenacão de Aperfeiçõamento de Pessoal de Nível Superior and CNPq – Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil, that provided financial support for this research. D. Connelly and A. Vandervoort. “Effects of isokinetic strength training on concentric and eccentric torque development in the ankle dorsiflexors of older adults”. J. Gerontol A. Biol. Sci. Med. Sci. Vol. 55, pp. B465-72. 2000. [11] CONCLUSIONS The PRODIP method and specifically the modeling process proposed in the Preliminary Design, presented as a sequential and unique method that proved to be useful to solve complex mechatronic systems. [9] K.E. Wilk, M.A. Keirns and J.R. Andrews. “Anterior cruciate ligament reconstruction rehabilitation: a six month follow up of isokinetic testing in recreational athletes”. Isokinet Exerc. Sci. Vol. 1, pp. 2-9. 1991. REFERENCES [11] R. Alaca, B. Yilmaz, A. Goktepe and H. Mohur. “Efficacy of isokinetic exercise on functional capacity and pain in patellofemoral pain syndrome”. J. Gerontol A Biol. Sci. Med. Sci. Vol. 81, pp. 807-813. 2002. [1] C.F. Aquino, D.V. Vaz, R.S. Brício, P.L.P. Silva, J.M. Ocarino and S.T. Fonseca. “Isokinetic dynamometry in sports and rehabilitation sciences”. Revista Brasileira de Ciência e Movimento. Vol. 15 Nº 1, pp. 93-100. 2007. [12] [12] H. Heitkamp, T. Horstmann, F. Mayer and J. Weller. “Gin in strength and muscular balance after balance training”. Int. J. Sports Med. Vol. 22, pp. 285-290. 2001. [2] P. Wimpenny. “Isokinetics Explained: Definitions”, pp. 1-2. December, 2010. Date of visit: December 18, 2013. URL: http:// www.isokinetics.info/isokinetics/definitions/ what-is-isokinetic.html [13] J. González e E. Gorostiga. “Fundamentos de treinamento da força, aplicação ao alto rendimento esportivo“. Artmed. Nº 2. Porto Alegre, Brasil. Vol. 1, pp. 33-188. 1997. [3] [3] J.L. Keating and T.A. Matyas. “The influence of subject and test design on dynamometric measurements of extremity muscles”. Physical Therapy. Vol. 76, pp. 866-89. 1996. [14] G. Chattanooga. “Clinical Desk Reference”. Chattanooga Group, KIN-COM. 1st edition. Tennessee, United States. Vol. 1, pp. 10-96. 1995. [4] [4] L. Ozçakar, F. Inanici, B. Kaymak, G. Abali, A Cetin and Z. Hasçelik. “Quantification of the weakness and fatigue in thoracic outlet syndrome with isokinetic measurements”. Br. J. Sports Med. Vol. 39, pp. 178-81. 2005. [15] G. Shinzato, J. Vasconcelos, C. Ogawa e I. Sampaio. “Protocolo de avaliação funcional de joelho em patologias ortopédicas”. Acta Fisiátrica. Vol. 3, pp. 30-36. 1996. [5] H. Tunstall, D.R. Mullineaux and T. Vernon. “Criterion validity of an isokinetic [16] A. Terreri, G. Júlia and M. Marco. “Avaliação isocinética no joelho do atleta”. Revista 206 once, Martins, Martin, Da Silva, De Mello and Ocampo: Development of a scale prototype of isokinetic dynamometer [22] R. Ruggles. “Isokinetic exercise device with speed control”. Google Patents. Patent number U.S.: 4374588. United States. 1983. brasileira de medicina do esporte. Vol. 7, pp. 37-45. 2001. [17] [17] T. Houweling and M. Hamzeh. “Does knee joint alignment with the axis of the isokinetic dynamometer affect peak torque?”. Isokinet Exerc. Sci. Vol. 18, pp. 217-21. 2010. [23] E.M. Mattox, E.D. Mask and J.D. Beeding. “Isokinetic exerciser”. Google Patents. Patent number U.S.: 4385760. United States. 1983. [18] [18] [18] D. Scoramuzza, R. Axtell and R. Thiel. REFERENCES “Effect of knee joint alignment on isokinetic torque production during leg extension and flexion exercise”. Med. Sci. Sports Exerc. Vol. 22, pp. 190-198. 2010. [24] S.M. Sagedahl, J.W. German and R.M. Wolfe. “Isokinetic exercise apparatus and method”. Google Patents. Patent number U.S.: 4592545. United States. 1986. [19] N. Back, A. Ogliari, A. Dias and J. Silva. “Projeto integrado de produtos”. Manole. 1st edition. São Paulo, Brasil. Vol. 1, pp. 22-124. 2008. [25] K. Janschek. “Mechatronic Systems Design: Methods, Models, Concepts”. Springer. 1. Berlin, Germany. 2012. ISBN: 9783642175305. [20] M. Boettcher. “Exercise and monitoring machine”. Google Patents. 21. Patent number U.S.: 4565368. United States. 1983. [26] [26] D. Ponce, C.A. Martin, M.C. Andrade and D. Martins. “Conceitualização e análise crítica dos dinamômetros isocinéticos”. Revista Brasileira de Biomecânica. Vol. 12 Nº 23, pp. 65-74. Junio 2011. ISSN: 1518-8191. [21] J. Perrine. “Isokinetic exercise process and apparatus”. Google Patents. Patent number U.S.: 3465592. United States. 1969. 207
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The Approaching New Grand Solar Minimum and Little Ice Age Climate Conditions
Natural science
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Abstract By about 2030-2040, the Sun will experience a new grand solar minimum. This is evident from multiple studies of quite different characteristics: the phasing of sunspot cycles, the cyclic obser- vations of North Atlantic behaviour over the past millennium, the cyclic pattern of cosmogenic ra- dionuclides in natural terrestrial archives, the motions of the Sun with respect to the centre of mass, the planetary spin-orbit coupling, the planetary conjunction history and the general plane- tary-solar-terrestrial interaction. During the previous grand solar minima—i.e. the Spörer Mini- mum (ca 1440-1460), the Maunder Minimum (ca 1687-1703) and the Dalton Minimum (ca 1809- 1821)—the climatic conditions deteriorated into Little Ice Age periods. Keywords Solar Variability, Grand Solar Minima, Little Ice Ages, The 2030-2040 Solar Minimum Natural Science, 2015, 7, 510-518 Published Online November 2015 in SciRes. http://www.scirp.org/journal/ns http://dx.doi.org/10.4236/ns.2015.711052 Natural Science, 2015, 7, 510-518 Published Online November 2015 in SciRes. http://www.scirp.org/journal/ns http://dx.doi.org/10.4236/ns.2015.711052 The Approaching New Grand Solar Minimum and Little Ice Age Climate Conditions Nils-Axel Mörner Paleogeophysics & Geodynamics, Stockholm, Sweden Nils-Axel Mörner Paleogeophysics & Geodynamics, Stockholm, Sweden Received 7 October 2015; accepted 16 November 2015; published 19 November 2015 Copyright © 2015 by author and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC B http://creativecommons.org/licenses/by/4.0/ Copyright © 2015 by author and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ Copyright © 2015 by author and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons org/licenses/by/4 0/ Copyright © 2015 by author and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). h // i /li /b /4 0/ How to cite this paper: Mörner, N.-A. (2015) The Approaching New Grand Solar Minimum and Little Ice Age Climate Condi- tions. Natural Science, 7, 510-518. http://dx.doi.org/10.4236/ns.2015.711052 1. Introduction The solar activity exhibits a fairly regular alternation between solar maxima and solar minima (e.g. [1] [2]). The grand solar minima known as the Spörer Minimum (ca 1440-1460), the Maunder Minimum (ca 1687-1703) and the Dalton Minimum (ca 1809-1821) are all well known, not least because they correspond quite well with cold periods known as “Little Ice Ages” [3]-[5]. In the period 1997-2003, I chaired an INTAS project on Geomagnetism & Climate; we concluded that we, in the middle of the 21st century, had to be back in a new solar minimum with Little Ice Age climatic conditions [6] [7]. Several authors have made similar observations and claims [5]-[31]. How to cite this paper: Mörner, N.-A. (2015) The Approaching New Grand Solar Minimum and Little Ice Age Climate Condi- tions. Natural Science, 7, 510-518. http://dx.doi.org/10.4236/ns.2015.711052 N.-A. Mörner Several of the papers in the Special Issue of PRP [1] addressed the question of an approaching new Solar Minimum. The conclusions [21] were quite straightforward: Obviously we are on our way into a new grand so- lar minimum. This sheds serious doubts on the issue of a continued, even accelerated, warming as proposed by the IPCC project. This quite innocent—and very true—conclusion [21] made the publisher take the quite remarkable step to close down the entire scientific journal [32]. This closing down gave rise to turbulence and objections within the scientific community [31] [33]-[39]. It even became the incitement to a new book [2]. In this paper, I will review some of the leading facts for the proposition of an approaching Grand Solar Minimum and a related climatic deterioration of Little Ice Age type, in analogy with what happened during the last three solar minima, viz. the Dalton, Maunder and Spörer Minima. 2. The Claim of Priority With respect to all the authors having claimed that we are approaching a solar minimum and/or a new Little Ice Age [1] [2] [5]-[31], there is hard, even meaningless, to try to identify a paper of priority (maybe [8]). Appar- ently, several authors had the same idea based on a verity of different data; heliomagnetic cyclicity, atmospheric production of radionuclides, planetary beat, history and cyclicity of various terrestrial variables. What we can say, however, is that paper [25] by no means is a candidate for such a priority although this was claimed by media and some blogs [40]. There are many “pioneer” papers before that paper. I am well aware of the saying: the true pioneers never (or at least seldom) get credit for the discovery. We leave this discussion with references to a number of preceding papers [1] [5] [6]-[21]. I am well aware of the saying: the true pioneers never (or at least seldom) get credit for the disco We leave this discussion with references to a number of preceding papers [1] [5] [6]-[21]. y g p ( ) g f y We leave this discussion with references to a number of preceding papers [1] [5] [6]-[21]. We leave this discussion with references to a number of preceding papers [1 3. Predicting Solar Variability and Climate During the Spörer, Maunder and Dalton Minima, Earth’s rate of rotation speeded-up, the main Gulf Stream flow was directed SE:wards and Arctic water penetrated far down into the North Atlantic [5]. At the New Grand Solar Minimum similar conditions are likely to re-occur. Figure 2. During the Spörer, Maunder and Dalton Minima, Earth’s rate of rotation speeded-up, the main Gulf Stream flow was directed SE:wards and Arctic water penetrated far down into the North Atlantic [5]. At the New Grand Solar Minimum similar conditions are likely to re-occur. The analogy with the past three grand solar minima seems quite firm. At all those solar minima the North At- lantic circulation strongly deformed as illustrated in Figure 2 [5]. The analogy with the past three grand solar minima seems quite firm. At all those solar minima the North At- lantic circulation strongly deformed as illustrated in Figure 2 [5]. herefore, the same is supposed to re-occur at the New Grand Solar Minimum at 2030-2040 [5] [11]. Therefore, the same is supposed to re-occur at the New Grand Solar Minimum at 2030-2040 [5] [11]. This was quite a revolutionary conclusion [5] [11] as it totally contradicted the scenario by the IPCC of a con- tinually increasing warming over the next centuries [45]. s quite a revolutionary conclusion [5] [11] as it totally contradicted the scenario by the IPCC of a con reasing warming over the next centuries [45]. The date of the New Solar Minimum has been assigned at around 2040 by Mörner et al. [6], at 2030-2040 by Velasco Herrera [27], and at around 2040 by Abdussamatov [23], implying a fairly congruent picture despite different ways of transferring past signals into future predictions. y g p g p In conclusion, we assign an age of the approaching New Grand Solar Minimum of about 2030-2040. This im- plies that we must expect cold conditions [11]—not warm conditions [45]—at the middle of the present century [11]. Figure 3 gives the cyclic phasing of the Gleisberg and De Vries cycles over the past 600 years with a predic- tion also for the 21st century [6] [7] [9] [11]. This implies that we by mid 21st century must expect cold climatic conditions and extensive ice expansion in the Arctic [11]—in total disagreement with the widely spread IPCC scenario of an open Arctic by 2050. 3. Predicting Solar Variability and Climate The validity of physical laws and driving forcing functions is revealed in the appearance in time; i.e., the evolu- tionary history of cosmos, the planetary system, the Earth-Moon system and the terrestrial processes. The base is a chronology set by dating or time-series with a stratigraphic order. In such records we observe the appearance and re-appearance of events and changes, which often form patterns. In these patterns, we may recognize forcing functions and cyclic patterns. In there cognitions of patterns and cycles, we not only make order of the past, but may also assess predictions of future changes [41]. The cyclic alternations between solar maxima and minima were found to correlate with periods ng-up and slowing-down in the Earth’s rate of rotation as illustrated in Figure 1. At the Spörer, Maunder and Dalton Grand Solar Minima the circulation pattern of the North Atlantic changed dramatically [5] as illustrated in Figure 2 [5] [11]. At the approaching New Grand Solar Minimum similar conditions are likely to re-occur [5] [11] [17] [28] [42]; i.e. a return to Little Ice Age conditions. A return to cold climate conditions at around 2040 is also predicted in cyclicity of Barents Sea fish catch and the interchanges of Atlantic and Barents Sea water masses [43] [44]. Figure 1. Relations among solar cycles, Earth’s rate of rotation and the observed changes in the ocean circulation in the North Atlantic (from [5] [11]). Figure 1. Relations among solar cycles, Earth’s rate of rotation and the observed changes in the ocean circulation in the North Atlantic (from [5] [11]). 511 N.-A. Mörner Figure 2. During the Spörer, Maunder and Dalton Minima, Earth’s rate of rotation speeded-up, the main Gulf Stream flow was directed SE:wards and Arctic water penetrated far down into the North Atlantic [5]. At the New Grand Solar Minimum similar conditions are likely to re-occur. Figure 2. During the Spörer, Maunder and Dalton Minima, Earth’s rate of rotation speeded-up, the main Gulf Stream flow was directed SE:wards and Arctic water penetrated far down into the North Atlantic [5]. At the New Grand Solar Minimum similar conditions are likely to re-occur. Figure 2. During the Spörer, Maunder and Dalton Minima, Earth’s rate of rotation speeded-up, the main Gulf Stream flow was directed SE:wards and Arctic water penetrated far down into the North Atlantic [5]. At the New Grand Solar Minimum similar conditions are likely to re-occur. Figure 2. Wilson [19] showed that solar variability is primarily caused by a spin-orbital coupling between Venus, Earth and Jupiter (VEJ). This is illustrated in Figure 5 [19] [28]. 3. Predicting Solar Variability and Climate Cyclic phasing of the combined “Gleisberg” and De Vries Cycles over the last 600 years giving a new Solar Minimum at about 2040-2050 with cold climate conditions and major ice expansion in the Arctic (from [9] [11]). Vertical scale gives temperature in centigrade. Figure 4. Atlantic warm (A) and cold (B) periods (above), and solar variability (below) with the Spörer (S), Maunder (M), Dalton (D) and Future (F) grand solar minima of Figure 2 character marked (from [41]). The “solar irradiance” curve [46] records the changes in Solar Wind, not irradiance, and must hence be relabelled “a curve of the changes in Solar Wind activity” (therefore their vertical scale of irradiance is put in brackets and quotation marks). Figure 3. Cyclic phasing of the combined “Gleisberg” and De Vries Cycles over the last 600 years giving a new Solar Minimum at about 2040-2050 with cold climate conditions and major ice expansion in the Arctic (from [9] [11]). Vertical scale gives temperature in centigrade. Figure 3. Cyclic phasing of the combined “Gleisberg” and De Vries Cycles over the last 600 years giving a new Solar Minimum at about 2040-2050 with cold climate conditions and major ice expansion in the Arctic (from [9] [11]). Vertical scale gives temperature in centigrade. Figure 4. Atlantic warm (A) and cold (B) periods (above), and solar variability (below) with the Spörer (S), Maunder (M), Dalton (D) and Future (F) grand solar minima of Figure 2 character marked (from [41]). The “solar irradiance” curve [46] records the changes in Solar Wind, not irradiance, and must hence be relabelled “a curve of the changes in Solar Wind activity” (therefore their vertical scale of irradiance is put in brackets and quotation marks). Figure 4. Atlantic warm (A) and cold (B) periods (above), and solar variability (below) with the Spörer (S), Maunder (M), Dalton (D) and Future (F) grand solar minima of Figure 2 character marked (from [41]). The “solar irradiance” curve [46] records the changes in Solar Wind, not irradiance, and must hence be relabelled “a curve of the changes in Solar Wind activity” (therefore their vertical scale of irradiance is put in brackets and quotation marks). Figure 5. Wilson’s VEJ tidal-torqueing model [19], providing an 11.07 yr beat period on the Sun. It forms the base for Salvador’s [20] mathematical model of the sunspot cycles. Figure 5. 3. Predicting Solar Variability and Climate Solar maxima occur in the mid 16th century, in the early and late 18th cen- tury and in the early and late 20th century. Solar minima occur in mid 15th century, in late 18th century and in early and late 19th century, indicating a new solar minimum in the med 21st century. y y g y Another way of estimating the changes is to use the cyclic pattern of the cosmogenic radionuclides (i.e. 14C and 10Be) of the last 800 years with an extrapolation into the 21st century [11]. This is illustrated in Figure 4 [11] [44]. It suggests that there will be a new minimum around 2040. Hansen [46] observed that the sea level changes in the Kattegatt-Baltic region were dominated by lunar tidal forces. He documented the observed changes for the last 300 years and predicted them for the coming 200 years. Major low levels (in the order of 3 - 4 cm) occur at 1809 (i.e. in the Dalton Minimum) and will re-occur in 2034 according to Hansen [47], fitting very well with the notion of a New Grand Solar Minimum around 2030-2040. It also opens new perspectives on the ~60 year cycle [29] [39]. Wilson [19] showed that solar variability is primarily caused by a spin-orbital coupling between Venus, Earth and Jupiter (VEJ). This is illustrated in Figure 5 [19] [28]. 512 N.-A. Mörner 513 Figure 3. Cyclic phasing of the combined “Gleisberg” and De Vries Cycles over the last 600 years giving a new Solar Minimum at about 2040-2050 with cold climate conditions and major ice expansion in the Arctic (from [9] [11]). Vertical scale gives temperature in centigrade. Figure 4. Atlantic warm (A) and cold (B) periods (above), and solar variability (below) with the Spörer (S), Maunder (M), Dalton (D) and Future (F) grand solar minima of Figure 2 character marked (from [41]). The “solar irradiance” curve [46] records the changes in Solar Wind, not irradiance, and must hence be relabelled “a curve of the changes in Solar Wind activity” (therefore their vertical scale of irradiance is put in brackets and quotation marks). Figure 5. Wilson’s VEJ tidal-torqueing model [19], providing an 11.07 yr beat period on the Sun. It forms the base for Salvador’s [20] mathematical model of the sunspot cycles. Figure 3. 3. Predicting Solar Variability and Climate Wilson’s VEJ tidal-torqueing model [19], providing an 11.07 yr beat period on the Sun. It forms the base for Salvador’s [20] mathematical model of the sunspot cycles. Figure 5. Wilson’s VEJ tidal-torqueing model [19], providing an 11.07 yr beat period on the Sun. It forms the base for Salvador’s [20] mathematical model of the sunspot cycles. 513 N.-A. Mörner Salvador [20] presented a mathematical model of the sunspot cycles based on Wilson’s tidal-torque model of Figure 5. The model had an 85% correlation with the sunspot numbers observed for 1749-2013, and made “a reasonable representation of the sunspot cycles for the past 1000 yr”. Therefore, it justified an extrapolation for the next century, as shown in Figure 6. y g The Figure 6 prediction gives an extended low up to 2160 with the lowest values reached within the period 2028-2042; i.e. just where we expect the New Grand Solar Minimum to occur [1] [2]. In 2015, Salvador extended his analysis over the last 4000 years [28], comparing his model with the observed 10Be variations [48], as illustrated in Figure 7. Figure 6. A comparison of monthly sunspot numbers from 1987 to 2013 (in blue) with the absolute value of the correlation model (in red), derived using data up to 2013 and the extended forecast to 2100 [20]. Figure 7. Comparison between planetary beat according to the VEJ SOC Solar System Resonance Model of Salvador [20] based on the VEJ theory of Wilson [19], and the dTSI as calculated from terrestrial 10Be variations [48]. The agreement is striking over 4000 years. We take this as a confirm- ation of the planetary-solar-terrestrial interaction [28]. Figure 6. A comparison of monthly sunspot numbers from 1987 to 2013 (in blue) with the absolut value of the correlation model (in red), derived using data up to 2013 and the extended forecast to 2100 [20]. Figure 7. Comparison between planetary beat according to the VEJ SOC Solar System Resonance Model of Salvador [20] based on the VEJ theory of Wilson [19], and the dTSI as calculated from terrestrial 10Be variations [48]. The agreement is striking over 4000 years. We take this as a confirm- ation of the planetary-solar-terrestrial interaction [28]. 514 N.-A. Mörner 4. Discussion The Earth experienced speeding-up periods at grand solar minima and slowing-down periods at grand solar maxima [5] [17] [29] [49]. This controls the main ocean surface circulation. In the North Atlantic this is seen in the beat and directional shifts of the Gulf Stream [5] [9] [11] as illustrated in Figure 1. It is also recorded in the rises and falls in sea level in the Indian Ocean (Figure 2 of [29]). The link between solar variability and terrestri- al responses must go via the interaction of the Solar Wind with the Earth’s magnetospher [5] [17] [42] [44] [49] as illustrated in Figure 8. The origin of solar variability may be generated in the solar core [50], at the tachocline [51] or at the surface [52] [53]. The driving forces are likely to be spin-orbital effects from the planets as proposed by Wilson [19] and established by Salvador [20] [28] as illustrated in Figure 6 and Figure 7. Another important factor must be the constant motions of the planetary bary-centra inside and outside the Sun providing an “excenter-wheel motor” [30], fully capable to affect the radioactive interior, the tachocline and the surface in ways generation cyclically occurrence in the solar variability. References [1] Mörner, N.-A., Tattersall, R. and Solheim, J.-E. (2913) Pattern in Solar Variability, Their Planetary Origin and Terre- strial Impact. Pattern Recognition in Physics, 1, 203-204. www.pattern-recogn-phys.net/special_issue2.html 2] Mörner, N.-A. (2015) Planetary Influence on the Sun and the Earth, and a Modern Book-Burning. Nova S lishers, New York. [3] Eddy, J.A. (1976) The Maunder Minimum. Science, 192, 1189-1202. http://dx.doi.org/10.1126/science.192.4245.1189 [4] Lamb, H.H. (1979) Climate Variation and Changes in the Wind and Ocean Circulation. Quaternary Research, 11, 1- 20. http://dx.doi.org/10.1016/0033-5894(79)90067-X [5] Mörner, N.-A. (2010) Solar Minima, Earth’s Rotation and Little Ice Ages in the Past and in the Future. The North At- lantic-European Case. Global Planetary Change, 72, 282-293. http://dx.doi.org/10.1016/j.gloplacha.2010.01.004 [6] Mörner, N.-A., Nevanlinna, H., Dergachev, V., Shumilov, O., Raspopov, O., Abrahamsen, N., Pilipenko, O., Trubikhin, V. and Gooskova, E. (2013) Geomagnetism and Climate V: General Conclusions. EGS-AGU-EGU, Nice, Abstracts CL2.08. [7] Mörner, N.A., Nenanlinna, H. and Shumilov, O. (2013) Past Climate Changes, Origin And Predictions. EGS-AGU- EGU, Nice, Abstracts CL2.07. [8] Landscheidt, T. (2003) New Little Ice Age Instead of Global Warming. Energy and Environment, 14, 327-350. http://dx.doi.org/10.1260/095830503765184646 [9] Mörner, N.-A. (2006) 2500 Years of Observations, Deductions, Models and Geoethics. Bollettino della Società Ge- ologica Italiana, 125, 259-264. [10] Charvátová, I. (2009) Long-Term Predictive Assessments of Solar and Geomagnetic Activities Made on the Basis of the Close Similarity between the Solar Inertial Motions in the Intervals 1840-1905 and 1980-2045. New Astronomy, 14, 25-30. http://dx.doi.org/10.1016/j.newast.2008.04.005 [11] Mörner, N.-A. (2011) Arctic Environment by the Middle of This Century. Energy & Environment, 22, 207-218. http://dx.doi.org/10.1260/0958-305X.22.3.207 [12] D’Aleo, J. (2011) Chapter 10: Solar Changes and Climate. In: Easterbrook, D.J., Ed., Evidence-Based Climate Science, Elsevier, Amsterdam, 253-276. http://dx.doi.org/10.1016/B978-0-12-385956-3.10010-5 [13] Archibald, D. (2011) Chapter 11: The Current Solar Minimum and Its Consequences for Climate. In: Easterbrook, D.J., Ed., Evidence-Based Climate Science, Elsevier, Amsterdam, 277-287. http://dx.doi.org/10.1016/B978-0-12-385956-3.10011-7 [14] Casey, J.L. (2011) Cold Sun: A Dangerous “Hibernation” of the Sun Has Begun. Trafford Publishing, Bloomington. [15] Scafetta, N. (2012) Multi-Scale Harmonic Model for Solar and Climate Cyclical Variation throughout the Holocene Based on Jupiter-Saturn Tidal Frequencies plus the 11-Year Solar Dynamo Cycle. Journal of Atmospheric and Solar- Terrestrial Physics, 80, 296-311. http://dx.doi.org/10.1016/j.jastp.2012.02.016 [16] Cionco, R.G. and Compagnucci, R.H. (2012) Dynamical Characterization of the Last Prolonged Solar Minima. Ad- vances in Space Research, 50, 1434-1444. http://dx.doi.org/10.1016/j.asr.2012.07.013 [17] Mörner, N.-A. (2013) Planetary Beat and Solar-Terrestrial Responses. Pattern Recognition in Physics, 1, 107-116. http://dx.doi.org/10.5194/prp-1-107-2013 [18] Solheim, J.-E. 5. Conclusions Mörner Not only is it a question about facts [36] [37] but also a respect to geoethical principles [37] [38]. 5. Conclusions The phasing of the solar cycles gives a clear message for the middle of the century: there will be a New Grand Solar Minimum [1]. This is also the case when we consider the cyclic relations between Earth’s rotation, ocean circulation and Arctic climate [5] [17] [29] as illustrated in Figure 1 and Figure 4. During the last three grand solar minima—the Spörer, Maunder and Dalton Minima—global climate expe- rienced Little Ice Age conditions [3]-[5]. Arctic water penetrated to the south all the way down to Mid Portugal, and Europe experienced severe climatic conditions as recorded in Figure 1 and Figure 2. The Arctic ice cover expanded significantly [11]. By 2030-2040 we will be in a New Grand Solar Minimum (cf. Figure 3, Figure 4), which by analogy to past minima must be assumed to lead to a significant climatic deterioration with ice expansion in the Arctic [5] [11]. By 2030-2040 we will be in a New Grand Solar Minimum (cf. Figure 3, Figure 4), which by analogy to past minima must be assumed to lead to a significant climatic deterioration with ice expansion in the Arctic [5] [11]. The mathematical model by Salvador [20] [28] seems to provide an excellent tool for the prediction of future sunspot variations (Figure 5, Figure 6). The mathematical model by Salvador [20] [28] seems to provide an excellent tool for the prediction of future sunspot variations (Figure 5, Figure 6). We now seem to be in possession of quite convergent data, indicating that we by 2030-2040 will be in a New Grand Solar Minimum. This precludes a continual warming as claimed by the IPCC project [45]. Instead of this, we are likely to face a new Little Ice Age (Figure 2). So, when Hunter [54] talked about “Thermageddon: countdown to 2030”, exactly the opposite is likely to by the true climatic shift [34] [55]; viz. a significant cooling, maybe even a Little Ice Age, and by no means any disastrous warming. Consequently, by 2030-2040, it seems most likely that climate reality—a cooling associated with a grand so- lar minimum—will imply a long nose to the IPCC concept [45] and the idea of a thermageddon countdown [54]. 515 Figure 8. Planetary beat processes and the spectrum of terrestrial variables affected [17] [28] [42] [44]. N.-A. References (2013) Signals from the Planets, via the Sun to the Earth. Pattern Recognition in Physics, 1, 177-184. http://dx.doi.org/10.5194/prp-1-177-2013 [19] Wilson, I.R.G. (2013) The Venus-Earth-Jupiter Spin-Orbit Coupling. Pattern Recognition in Physics, 1, 147-158. http://dx.doi.org/10.5194/prp-1-147-2013 [20] Salvador, R. (2013) A Mathematical Model of the Sunspot Cycle for the Past 1000 yr. Pattern Recognition in Physics, 1, 117-122. http://dx.doi.org/10.5194/prp-1-117-2013 [21] Mörner, N.-A., Tattersall, R., Solheim, J.-E., Charvatova, I., Scafetta, N., Jelbring, H., Wilson, J.R., Salvador, R., Willson, R.C., Hejda, P., Soon, W., Velasco Herrera, V.M., Humlum, O., Archibald, D., Yndestad, H., Easterbrook, D.J., Casey, J., Gregori, G. and Henriksson, G. (2013) General Conclusions regarding the Planetary-Solar-Terrestrial Interaction. Pattern Recognition in Physics, 1, 205-206. http://dx.doi.org/10.5194/prp-1-205-2013 [22] Charvátová, I. and Hejda, P. (2014) Responses of the Basic Cycles of 178.7 and 2402 yr in Solar-Terrestrial Phenom- ena during the Holocene. Pattern Recognition in Physics, 2, 21-26. http://dx.doi.org/10.5194/prp-2-21-2014 [23] Abdussamatov, H.J. (20114) Long-Term Negative Average Annual Energy Balance of the Earth Leads to the Little Ice [23] Abdussamatov, H.J. (20114) Long-Term Negative Average Annual Energy Balance of the Earth Leads to the Little Ice 516 N.-A. Mörner Age. Proceedings of the XVIII Conference on Solar and Solar-Terrestrial Physics 2014, Pulkovo, 20-25 October 2014, 3-6. [24] Georgieva, K., Nagovitsyn, Y. and Kirov, B. (2014) Solar Magnetic Field and Terrestrial Climate. Proceedings of the XVIII Conference on Solar and Solar-Terrestrial Physics 2014, Pulkovo, 20-25 October 2014, 99-104. [25] Shepherd, S.I., Zharkov, S.I. and Zharkova, V.V. (2014) Prediction of Solar Activity from Solar Background Magnetic Field Variations in Cycles 21-23. Astrophysics Journal, 795, 46. http://dx.doi.org/10.1088/0004-637X/795/1/46 [26] Cionco, R.G. and Soon, W. (2015) A Phenomenological Study of the Timing of Solar Activity Minima of the Last Millennium through Modelling the Sun-Planets Interaction. New Astronomy, 34, 164-171. [26] Cionco, R.G. and Soon, W. (2015) A Phenomenological Study of the Timing of Solar Activity Minima of the Last Millennium through Modelling the Sun-Planets Interaction. New Astronomy, 34, 164-171. http://dx.doi.org/10.1016/j.newast.2014.07.001 [27] Velasco Herrera, V.M., Mendoza, B. and Velasco Herrera, G. (2015) Reconstruction and Prediction of the Total Solar Irradiance; from the Medieval Warm Period to the 21st Century. New Astronomy, 34, 221-233. http://dx.doi.org/10.1016/j.newast.2014.07.009 [28] Mörner, N.-A. (2015) Chapter 2: Planetary-Solar-Terrestrial Interaction: A Review. In: Mörner, N.-A., Ed., Planetary Influence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 5-32. [29] Mörner, N.-A. (2015) Chapter 4: Multiple Planetary Influences on the Earth. References In: Mörner, N.-A., Ed., Planetary Influ- ence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 39-49. [30] Mörner, N.-A. (2015) Chapter 9: Concluding Remarks. In: Mörner, N.-A., Ed., Planetary Influence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 109-120. [31] Mörner, N.-A., Monckton, C., Gregori, G., Tattersall, R., Solheim, J.-E., Scafetta, N., Charvatova, I., Jelbring, H., Wilson, J.R., Salvador, R., Willson, R.C., Hansen, J.M., Humlum, O., Karlén, W., Nemec, N., Kalenda, P., Archibald, D., Velasco Herrera, V.M., Grandpierre, A. and Easterbrook, D.J. (2015) Chapter 17: Conclusions and Perspectives. In: Mörner, N.-A., Ed., Planetary Influence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Pub- lishers, Hauppauge, 187-190. [32] Mörner, N.-A. (2015) Chapter 10: An Unbelievable Decision. In: Mörner, N.-A., Ed., Planetary Influence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 123-126. [33] Mörner, N.-A. (2015) Chapter 11: Introduction to Section B. In: Mörner, N.-A., Ed., Planetary Influence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 129-134. [34] Monckton of Brenchley, C. (2015) Chapter 12: The Thermaggedon Cult Strikes Again. In: Mörner, N.-A., Ed., Plane- tary Influence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 135-138. [35] Gregori, G. (2015) Chapter 13: Science: An Historical Perspective. In: Mörner, N.-A., Ed., Planetary Influence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 139-146. [36] Monckton of Brenchley, C. (2015) Chapter 14: What Is Science? In: Mörner, N.-A., Ed., Planetary Influence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 149-165. [37] Mörner, N.-A. (2015) Chapter 15: Climate Fundamentalism. In: Mörner, N.-A., Ed., Planetary Influence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 167-174. [38] Mörner, N.-A. (2015) When Reason Sleeps, Monster Take Wings. In: Mörner, N.-A., Ed., Planetary Influence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 177-184. [39] Mörner, N.-A. (2015) Chapter 18: Concluding Editorial Remarks. In: Mörner, N.-A., Ed., Planetary Influence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 191-193. [40] The Telegraph July 11 2015 [39] Mörner, N.-A. (2015) Chapter 18: Concluding Editorial Remarks. References In: Mörner, N.-A., Ed., Planetary I Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 191-193. [40] The Telegraph, July 11, 2015. http://www.telegraph.co.uk/news/science/11733369/Earth-heading-for-mini-ice-age-within-15-years.html?WT.mc_id= e_DM30959&WT.tsrc=email&etype=Edi_FPM_New&utm_source=email&utm_medium=Edi_FPM_New_2015_07_1 1&utm_campaign=DM30959 [40] The Telegraph, July 11, 2015. http://www.telegraph.co.uk/news/science/11733369/Earth-heading-for-mini-ice-age-within-15-years.html?WT.mc_id= e_DM30959&WT.tsrc=email&etype=Edi_FPM_New&utm_source=email&utm_medium=Edi_FPM_New_2015_07_1 1& i DM30959 [41] Mörner, N.-A. (2015) An Editorial Note on the Desperate Search for a Stable Point. In: Mörner, N.-A., Ed., Planetary Influence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 121. [42] Mörner, N.-A. (2012) Planetary Beat, Solar Wind and Terrestrial Climate. In: Escaropa, C.D. and Berós Cruz, A.F., Eds., Solar Wind: Emission, Technology and Impacts, Nova Science Publishers, Hauppauge, 47-66. [43] Klyashtorin, L.B., Borisov, V. and Lyubushin, A. (2009) Cyclic Changes of Climate and Major Commercial Stocks of the Barents Sea. Marine Biology Research, 5, 4-17. http://dx.doi.org/10.1080/17451000802512283 [44] Mörner, N.-A. (2013) Solar Wind, Earth’s Rotation and Changes in Terrestrial Climate. Physical Review & Research International, 3, 117-136. 45] IPCC (2007) Climate Change. Cambridge University Press, Cambridge. [45] IPCC (2007) Climate Change. Cambridge University Press, Cambridge. [46] Hansen, J.M. (2015) Chapter 5: Sea-Level Effects on NOA and AMO: Synchronization and Amplitude Locking by the 517 N.-A. Mörner Lunar Nodal Oscillation in the North Sea and the Baltic Embayment. In: Mörner, N.-A., Ed., Planetary Influence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 51-77. [47] Bard, E., Raisbeck, G., Yiou, F. and Jouzel, J. (2000) Solar Irradiance during the Last 1200 Years Based on Cos- mogenic Nuclides. Tellus, 52B, 985-992. http://dx.doi.org/10.1034/j.1600-0889.2000.d01-7.x [48] Steinhilber, F., Abreu, J.A., Beer, J., et al. (2012) 9,400 Years of Cosmic Radiation and Solar Activity from Ice Cores and Tree Rings. Proceedings of the National Academy of Sciences of the United States of America, 109, 5967-5971. http://dx.doi.org/10.1073/pnas.1118965109 [49] Mörner, N.-A. (1996) Global Change and Interaction of Earth Rotation, Ocean Circulation and Paleoclimate. Brazilian Academy of Sciences, 68, 77-94. [50] Grandpierre, A. (2015) Chapter 8: The Origin of Solar Activity: Local Thermonuclear Runways in Hot Bubbles and Their Triggers. In: Mörner, N.-A., Ed., Planetary Influence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 91-108. [51] Abreu, J.A., Beer, J., Ferriz-Mas, A., McCracken, K.G. and Steinhilber, F. (2012) Is There a Planetary Influence on Solar Activity? Astronomy & Astrophysics, A88, 1-9. http://dx.doi.org/10.1051/0004-6361/201219997 [52] Jelbring, H. (2013) Energy Transfer in the Solar System. Pattern Recognition in Physics, 1, 165-176. http://dx.doi.org/10.5194/prp-1-165-2013 [53] Mörner, N.-A. References (2015) Chapter 7: The Bölling/Alleröd—Younger Dryas Oscillations. In: Mörner, N.-A., Ed., Planetary Influence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 79-89. [54] Hunter, R. (2003) Thermageddon: Countdown to 2030. Arcade Publishing, New York, 288 p. [55] Mörner, N.-A. (2015) Editorial Note on Thermageddon. In: Mörner, N.-A., Ed., Planetary Influence on the Sun and the Earth, and a Modern Book-Burning, Nova Science Publishers, Hauppauge, 138.
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Evaluation of cognitive function in the Dog Aging Project: associations with baseline canine characteristics
Scientific reports
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Sarah Yarborough 1*, Annette Fitzpatrick 1,2, Stephen M. Schwartz 1,3 & Dog Aging Project Consortium * Sarah Yarborough 1*, Annette Fitzpatrick 1,2, Stephen M. Schwartz 1,3 & Dog Aging Project Consortium * Canine cognitive dysfunction (CCD) is a neurodegenerative disease in aging dogs. It has been described previously in relatively small cohorts of dogs using multiple different rating scales. This study aimed to use a minimally modified CCD rating scale developed by previous researchers to describe the prevalence of CCD more thoroughly in a large, nationwide cohort of companion dogs participating in the Dog Aging Project (DAP) (n = 15,019). Associations between various canine characteristics, predicted lifespan quartiles, and CCD were examined using univariable and multivariable logistic regression models and receiver operating curve (ROC) analysis. When controlling for all other characteristics, the odds of CCD increased 52% with each additional year of age. Among dogs of the same age, health status, breed type, and sterilization status, odds of CCD were 6.47 times higher in dogs who were not active compared to those who were very active. When controlling for age, breed type, activity level, and other comorbidities, dogs with a history of neurological, eye, or ear disorders had higher odds of CCD. Lifespan quartile analysis showed excellent discriminating ability between CCD positive and negative dogs. Weight-based lifespan quartile estimation could therefore serve as a tool to inform CCD screening by veterinarians. Neurodegenerative diseases associated with aging have become increasingly prevalent among the aging American population. In 2020, approximately 5.8 million Americans were living with Alzheimer’s disease (AD). It is the sixth leading cause of death in the ­country1. Despite this, the complex pathological pathways that lead to the development of AD are still not fully understood and can be difficult to study in vivo in early phases of disease progression. While transgenic animal models have been extensively used to study the pathophysiology of AD, limitations have been identified that have prompted investigation into non-transgenic animal ­models2.h i The clinical and histological presentation of human AD and Canine Cognitive Dysfunction (CCD) in dogs have many similarities. As with humans, canine cognitive function declines throughout the course of the dog’s ­lifespan3. Clinical signs of this decline appear to be related to learning and memory deficits, loss of spatial aware- ness, altered social interactions, and disrupted sleeping ­patterns4–6. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | (2022) 12:13316 OPEN Sarah Yarborough 1*, Annette Fitzpatrick 1,2, Stephen M. Schwartz 1,3 & Dog Aging Project Consortium * Methods S d Study setting. Data were obtained from the DAP, a nationwide longitudinal study on aging initiated in 2018. One goal of the DAP is to better define aging in dogs based on comorbidities, frailty, and ­inflammaging17. Companion dog owners interested in enrolling their dogs complete multiple surveys throughout their involve- ment in the DAP. For the purposes of this report, these included the baseline Health and Life Experience Survey (HLES) and the CSLB survey completed during the first-year enrollment in DAP. The HLES is an extensive online survey that is distributed to all dog owners upon baseline enrollment, with sections on dog and owner household demographic characteristics, dog physical activity, and health status. The CSLB survey is a 13-item questionnaire based on Salvin et al.’s CCDR scale that aims to assess ­CCD4. It is described in further detail below. Study design. This study analyzed baseline associations between selected characteristics collected through the HLES and dog cognitive characteristics reported in the CSLB. HLES data were provided by participants immediately upon enrollment, and CSLB data were provided by participants when that survey was adminis- tered. Dates of collection for HLES data used in the study ranged from December 23, 2019, to December 11, 2020. Dates of collection for CSLB data used in the study ranged from October 30, 2020, to December 31, 2020. The median time between completion of the two surveys was 5 weeks (range: 0–50.3 weeks). Study subjects. All dogs whose owners did not complete both the baseline HLES and CSLB surveys prior to December 31, 2020 were excluded from study. Further, all dogs whose owners indicated that they were not able to confidently report their dog’s age, as assessed in the HLES, were also excluded. We began with a study sample of 27,542 dogs whose owners had completed the baseline HLES; 20,096 of those dogs’ owners had also completed the CSLB survey. Of those 20,096 dogs, 15,019 owners reported being certain of their dog’s age. The final sample for this study consisted of 15,019 dogs. Data sources. The validated CCDR instrument, used by Salvin et al.4 for the identification of CCD, was minimally modified and renamed the CSLB (Canine Social and Learned Behavior Survey) for this study. Methods S d In contrast to the original CCDR: (1) the instrument name was modified so as not to suggest dementia or cognitive dysfunction (identified in the term CCD) to the owners of enrolled dogs; and (2) it was programmed as an on- line digital tool with American English spelling. The CSLB thus consisted of 13 questions that assessed behaviors such as getting stuck behind objects, pacing, and failing to recognize familiar people. Responses for each ques- tion were scored based on frequency of the behavior (1 = never, 2 = once a month, 3 = once a week, 4 = once a day, 5 = more than once a day). In addition, some questions asked owners to compare the severity of their dog’s current behavior to the severity of the behavior 6 months prior. Numeric scores were summed from each owner’s responses. Certain questions had multipliers based on their clinical severity, as identified in CCD diagnosed ­dogs4. A minimum score of 16 and a maximum score of 80 were possible, with higher values indicating more severe levels of cognitive dysfunction. Age, sex, sterilization status, breed group, geographic region, physical activity level, comorbidities, and primary formal dog activity were assessed through the HLES. y p y g y g For owner-reported purebred dogs, breed was elicited and assigned to a breed group based on the eight groups defined by the American Kennel Club: herding, hound, toy, non-sporting, sporting, terrier, working, and miscel- laneous/Foundation Stock ­Service18. Primary formal dog activity refers to a dog’s main job or activity with the owner and was characterized as one of the following: companion animal or pet, obedience, show, breeding, agility, hunting, working, field trials, search and rescue, service dog, or assistance/therapy dog. Physical activity over the past year was classified as not active, moderately active, or very active. Major comorbidities considered were owner-reported in the HLES, including any history of cancer, endocrine disorders, kidney disorders, immune disorders, trauma, toxin consumption, infectious disease, hematologic disorders, neurological disorders, ortho- pedic disorders, reproductive disorders, liver disorders, gastrointestinal disorders, respiratory disorders, cardiac disorders, skin disorders, oral disorders, eye disorders, or ear disorders. Geographic region was determined based on the owner’s primary state of residence and categorized as West, Southwest, Midwest, Southeast, or Northeast. It is worth noting that the HLES collected hundreds of data points on participants’ behavior, lifestyle, diet and overall health. Sarah Yarborough 1*, Annette Fitzpatrick 1,2, Stephen M. Schwartz 1,3 & Dog Aging Project Consortium * Further, the presentation of human AD and CCD share certain neuropathological features such as amyloid-β plaque ­deposition2,7–10.hf p g y β p q p The observed parallels between CCD and human AD suggest that dogs exhibiting CCD may offer researchers a valuable animal model in which to study characteristics of neurodegenerative diseases that are relevant to, but challenging to study in, human ­populations11. Further, dogs with CCD could serve as candidates for AD preventa- tive and/or therapeutic ­strategies2. Finally, CCD is also a major health concern of dog owners and veterinarians. An increased understanding of CCD may help to advance treatment of cognitive disease in dogs. These potential benefits highlight the importance of accurate diagnosis of CCD in companion dogs. i A wide range of structured interviews and theory-driven scales for evaluating CCD have been developed within the last 20 ­years4,12–14. Studies of these scales have shown a correspondingly wide range of prevalence esti- mates that nonetheless appear to indicate an increased prevalence of cognitive impairment as a dog ­ages7,14,15. In order to gain a better understanding of CCD in aging dogs, researchers recently have developed assessment tools that are able to distinguish cognitively impaired aged dogs from those who are experiencing healthy ­aging11,14. 1Department of Epidemiology, University of Washington, Seattle, WA, USA. 2Department of Family Medicine, University of Washington, Seattle, WA, USA. 3Epidemiology Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. *A list of authors and their affiliations appears at the end of the paper. *email: sarahy24@ uw.edu Scientific Reports | (2022) 12:13316 | https://doi.org/10.1038/s41598-022-15837-9 www.nature.com/scientificreports/ These scales provide veterinarians and researchers with tools to better identify the prevalence of CCD in aging dogs but have only been used to describe cognitive function in relatively small populations (< 1000 dogs)11,14. The purpose of this study was to describe the range of cognitive function scores and prevalence of CCD in a very large, nationwide sample of companion dogs participating in the Dog Aging Project (DAP) (final n = 15,019). The DAP utilized the Canine Social and Learned Behavior (CSLB) survey, a minimally modified version of the validated canine cognitive dysfunction rating scale (CCDR) developed by Salvin et al.4. Further, we examined associations between weight-based lifespan quartile and CCD. Classifying a dog’s lifespan into quartiles and quantifying the predictive ability of the CCDR in each quartile potentially allows for preventative healthcare measures to be taken by owners and veterinarians. Sarah Yarborough 1*, Annette Fitzpatrick 1,2, Stephen M. Schwartz 1,3 & Dog Aging Project Consortium * The results could eventually lead to more timely detection and treatment of ­CCD16. Results f h Of the 15,019 dogs included in analyses, 19.5% were classified as being in their 4th quartile of life, 24.4% were classified as being in their 3rd quartile of life, 27.0% were in their 2nd quartile of life, and 29.1% of dogs in their 1st quartile of life. A total of 1.4% of dogs were classified as having CCD using the binary cut-off of ≥ 50. There were similar distributions of sex, geographic region, primary role, and breed type among each lifespan quartile (Table 1). Sterilization status, history of each of 17 categories of health problems reported in HLES, purebred breed group, and activity level were substantially positively associated with weight-based projected lifespan quartile. Dogs in a higher weight-based projected lifespan quartile were more likely to be sterilized, more likely to have history of the above comorbidities, and were less active. Associations between various canine characteristics and binary CCD assignment were assessed using univari- able and multivariable logistic regression models (Table 2). When assessing age alone, the odds of being diag- nosed with CCD increased almost 70% with each additional year of age (OR = 1.68, 95% CI 1.60, 1.77) (Fig. 1). nosed with CCD increased almost 70% with each additional year of age (OR = 1.68, 95% CI 1.60, 1.77) (Fig. 1). A directed acyclic graph (DAG) was constructed to highlight relevant covariates to include in the multivari- able logistic regression model assessing the association between age and CCD (Fig. 2). Age, sterilization status, history of 15 categories of health problems, breed type, and activity level were selected for the final logistic regression model (Table 2). When controlling for all other characteristics, the odds of CCD increased 52% with each additional year of age (OR = 1.52, 95% CI 1.44, 1.61). Interestingly, among dogs of a given age, health status, breed type, and activity level, those who were intact had a higher odds of CCD, although this finding was not statistically significant (OR = 1.21, 95% CI 0.51–2.51). Although attenuated, an inverse association between activity level and CCD was still found. Among dogs of the same age, health status, breed type, and sterilization status, odds of CCD were 6.47 times higher in dogs who were not active compared to those who were very active (OR = 6.47, 95% CI 2.93–17.23). Results f h When controlling for age, breed type, activity level, and other comorbidities, dogs with a history of neurological, eye, or ear disorders had higher odds of CCD (OR = 1.84, 95% CI 1.26, 2.65; OR = 2.16, 95% CI 1.57, 2.98; OR = 1.96, 95% 1.42, 2.70, respectively).i p y Adjusted and unadjusted logistic regression models were then fit using the weight-based projected lifespan quartiles. Adjusted models included all covariates from the multivariable logistic regression model assessing the association between age and CCD (Table 2). These weight-based projected lifespan quartile models were evalu- ated for their ability to accurately classify CCD at two different diagnostic thresholds: ≥ 50, as cited in previous literature, and > 37, which captures the upper 4th quartile of CSLB scores. In total, four predictive models were assessed by evaluating the area under the curve (AUC) of an ROC curve (Fig. 3). The diagnostic threshold of CSLB score ≥ 50 had the highest predictive capacity. The adjusted model that controlled for health status, breed type, sterilization status, and activity level provided a slightly improved predictive capacity over the unadjusted model (AUC = 0.892, 95% CI 0.854–0.930 vs. AUC = 0.862, 95% CI 0.831–0.893). The threshold of CSLB score > 37 had lower predictive capacity for both the adjusted and unadjusted models (AUC = 0.701, 95% CI 0.682–0.721 vs. AUC = 0.644, 95% CI 0.624–0.665). www.nature.com/scientificreports/ mined a priori to be associated with CCD, informed by a directed acyclic graph (DAG) (Fig. 2), were included in a multivariable logistic regression model. Predicted lifespan quartile was then estimated for each subject. Projected mean life expectancies were calcu- lated on a separate data set collected by Urfer et al. consisting of private U.S. veterinary hospital medical records for companion ­dogs19. Mean life expectancies were then assigned to each dog in the DAP data set as a function of their weight or projected weight class, sterilization status, and ­sex16,19. Each dog was assigned a predicted lifespan quartile that was equivalent to the proportion of their current life lived to their projected lifespan. Lifes- pan quartile was then evaluated for its ability to predict CCD using adjusted and unadjusted logistic regression models. The fitted models were used to construct a receiver operating curve (ROC) for two possible diagnostic thresholds (CSLB score ≥ 50, representative of previously defined thresholds, and > 37, which represented the highest quartile of scores in the data). Finally, area under the curve (AUC) was calculated in order to summarize predictive capacity of the fitted models. All statistical analyses were conducted in RStudio (Version 1.3)20. All methods and experimental protocols were carried out in accordance with relevant guidelines and regulations. The University of Washington Institutional Review Board (IRB) reviewed the human subjects involvement for this study and determined that the limited dog owner demographic and lifestyle and anecdotal health informa- tion collected in the HLES is human subjects research that meets the qualifications for Exempt Status (Category 2). Given the Exempt status, the IRB did not require informed consent for completion of the surveys, but we nevertheless designed an informed consent form and process that meets the regulatory requirements laid down in U.S. Code of Federal Regulations, Title 45 Department of Health and Human Services Part 46, Protection of Human Subjects. Methods S d Although these variables were not considered to be within the scope of this study, future analyses intend to explore these associations further. Data analyses. The primary outcome of interest was total CSLB score. Additionally, canine cognitive func- tion was treated as a binary measure such that dogs with a CSLB score ≥ 50 were classified as having ­CCD4. Logistic regression models were fit to assess univariable associations between CCD and age, sex, sterilization status, breed group, geographic region, major comorbidities, and activity variables. Covariates that were deter- Scientific Reports | (2022) 12:13316 | https://doi.org/10.1038/s41598-022-15837-9 https://doi.org/10.1038/s41598-022-15837-9 www.nature.com/scientificreports/ Discussion Results from this study indicate a positive association between age and CCD in companion dogs, even adjusting for other characteristics in a multivariable logistic regression model. There also existed associations between CCD and decreased activity level, as well as CCD and history of an eye, ear, or neurological disorder. The association between age and CCD aligns with the progressive nature of CCD and with previous dog research findings, which have shown an exponential increase in CCD prevalence with increasing ­age4. g g An inverse association was seen in dogs whose owners indicated higher dog activity levels over the past year. Previous studies with rodent models have demonstrated that exercise can have protective effects against the development of biological markers and subsequent behavioral deficits characteristic of AD, and numerous observational human studies have consistently shown inverse associations between exercise and ­AD21–25. Scientific Reports | (2022) 12:13316 | Discussion These observations may reflect a variety of biologic mechanisms, including a reduction of pro-inflammatory cytokines Scientific Reports | (2022) 12:13316 | https://doi.org/10.1038/s41598-022-15837-9 www.nature.com/scientificreports/ Characteristic Life stage quartile First (n = 4368) n, % Second (n = 4050) n, % Third (n = 3676) n, % Fourth (n = 2925) n, % Sex Female 2207 (50.5) 1987 (49.1) 1865 (50.7) 1425 (48.7) Male 2161 (49.5) 2063 (50.9) 1811 (49.3) 1500 (51.3) Sterilization status Intact 834 (19.1) 319 (7.9) 191 (5.2) 116 (4.0) Desexed 3534 (80.9) 3731 (92.1) 3485 (94.8) 2809 (96.0) Any comorbidity history 2817 (64.5) 3184 (78.6) 3246 (88.3) 2764 (94.5) Cancer 24 (0.5) 98 (2.4) 270 (7.3) 456 (15.6) Endocrine disorder 4 (0.1) 50 (1.2) 155 (4.2) 228 (7.8) Kidney disorder 163 (3.7) 240 (5.9) 296 (8.1) 434 (14.8) Immune disorder 7 (0.2) 34 (0.8) 36 (1.0) 44 (1.5) Trauma 732 (16.8) 1070 (26.4) 1191 (32.4) 1029 (35.2) Toxin consumption 376 (8.6) 400 (9.9) 470 (12.8) 382 (13.1) Infectious disease 1170 (26.8) 1093 (27.0) 949 (25.8) 791 (27.0) Hematologic disorder 13 (0.3) 12 (0.3) 12 (0.3) 36 (1.2) Neurological disorder 34 (0.8) 108 (2.7) 166 (4.5) 356 (12.2) Orthopedic disorder 218 (5.0) 489 (12.7) 822 (22.4) 1196 (40.9) Reproductive disorder 114 (2.6) 117 (2.9) 98 (2.7) 70 (2.4) Liver disorder 33 (0.8) 80 (2.0) 164 (4.5) 252 (8.6) Gastrointestinal disorder 506 (11.6) 579 (14.3) 572 (15.6) 548 (18.7) Respiratory disorder 51 (1.2) 62 (1.5) 121 (3.3) 231 (7.9) Cardiac disorder 49 (1.1) 104 (2.6) 269 (7.3) 411 (14.1) Skin disorder 767 (17.6) 1125 (27.8) 1232 (33.5) 1100 (37.6) Oral disorder 340 (7.8) 803 (19.8) 1316 (35.8) 1410 (48.2) Eye disorder 261 (6.0) 345 (8.5) 493 (13.4) 822 (28.1) Ear disorder 328 (7.5) 380 (9.4) 463 (12.6) 751 (25.7) Geographic region Midwest 982 (22.5) 871 (21.5) 762 (20.7) 597 (20.4) Northeast 813 (18.6) 715 (17.7) 698 (19.0) 548 (18.7) Southeast 770 (17.6) 751 (18.5) 658 (17.9) 548 (18.7) Southwest 380 (8.7) 362 (8.9) 365 (9.9) 286 (9.8) West 1395 (31.9) 1326 (32.7) 1178 (32.0) 925 (31.6) Missing 28 (0.6) 25 (0.6) 15 (0.4) 21 (0.7) Primary role Companion/pet 4169 (95.4) 3850 (95.1) 3498 (95.2) 2802 (95.8) Agility 9 (0.2) 16 (0.4) 15 (0.4) 6 (0.2) Assistance or therapy 22 (0.5) 32 (0.8) 32 (0.9) 20 (0.7) Breeding 4 (0.1) 5 (0.1) 2 (0.05) 7 (0.2) Field trials 2 (0.1) 2 (0.1) 2 (0.05) 0 (0) Hunting 6 (0.1) 7 (0.2) 7 (0.2) 4 (0.1) Obedience 39 (0.9) 22 (0.5) 14 (0.4) 15 (0.5) Search and rescue 10 (0.2) 7 (0.2) 3 (0.1) 4 (0.1) Service 42 (1.0) 42 (1.0) 33 (0.9) 17 (0.6) Show 10 (0.2) 6 (0.1) 7 (0.2) 3 (0.1) Working 9 (0.2) 14 (0.3) 11 (0.3) 7 (0.2) Other 46 (1.1) 47 (1.2) 52 (1.4) 40 (1.4) Breed type Purebred 2259 (51.7) 2376 (58.7) 2205 (60.0) 1733 (59.2) Mixed 1784 (48.3) 1667 (41.3) 1466 (40.0) 1189 (40.8) Purebred breed group Herding 475 (10.9) 452 (11.2) 331 (9.0) 255 (8.7) Hound 175 (4) 180 (4.4) 206 (5.6) 162 (5.5) Non-sporting 269 (6.2) 232 (5.7) 198 (5.4) 156 (5.3) Sporting 900 (20.6) 788 (19.5) 684 (18.6) 542 (18.5) Continued https://doi.org/10.1038/s41598-022-15837-9 Scientific Reports | (2022) 12:13316 | www.nature.com/scientificreports/ Table 1. Discussion Impairments in visual acuity and visual fields have been observed more frequently in AD patients than in similarly aged individuals without ­AD27,28. Further, the presence of ophthalmic conditions, such as cataracts and age-related macular degeneration, has been found to be associated with an increased risk of all-cause dementia and ­AD29. Amyloid deposits, which have been linked to the development of AD and have been associated with the accel- eration of AD progression, have been found in the lens of the eye in some individuals with age-related cataracts, potentially indicating common neuropathological pathways leading to AD and ­cataracts30. Age-related macular degeneration has also been associated with an increased risk of all-cause dementia and ­AD30. Like the potential relationship between cataracts and AD, amyloid deposits have been detected in the small fatty protein deposits that accumulate under the eye of individuals with age-related macular ­degeneration31. In addition to numerous studies identifying hearing loss as a possible risk factor for cognitive decline and all-cause dementia, a recent study found that individuals with dual sensory impairment (combined visual and hearing impairments) had a substantially increased risk of AD, possibly as a result of reduced neural resources needed for cognitive ­performance31–34. Further analysis of those with combined impairments may be used to investigate the possibility of this increased risk in our cohort. g p y Alternatively, the associations we observed between sensory impairment and CCD could be due at least in part to misclassification. The CSLB survey, which is closely derived from Salvin et al.’s CCDR, is intended to distin- guish between behaviors associated with CCD as opposed to those that are part of normal dog ­aging4. However, some behaviors assessed by the rating scale may not arise solely from cognitive ­impairment4. Questions such as, “How often does your dog walk into walls or doors?” and “How often does your dog have difficulty finding food dropped on the floor?” were included in the CCDR because increased frequency of these behaviors is seen in dogs with CCD. However, sensory impairments could also cause increased frequency of these behaviors, resulting in high scores on the instrument. g g We observed a dog’s estimated lifespan quartile, which is a function of their age, weight, sex, and steriliza- tion status, to have excellent discriminating ability between CCD positive and negative dogs (CSLB score ≥ 50). Discussion Selected canine demographic characteristics by weight-based projected lifespan quartile (n = 15,019), Dog Aging Project 2020–2021. Characteristic Life stage quartile First (n = 4368) n, % Second (n = 4050) n, % Third (n = 3676) n, % Fourth (n = 2925) n, % Terrier 151 (3.5) 179 (4.4) 204 (5.5) 176 (6.0) Toy 223 (5.1) 215 (5.3) 310 (8.4) 242 (8.3) Working 319 (7.3) 272 (6.7) 227 (6.2) 168 (5.7) Miscellaneous/FSS 32 (0.7) 26 (0.6) 19 (0.5) 17 (0.6) Non-AKC 34 (0.8) 32 (0.8) 26 (0.7) 15 (0.5) Activity level Very active 1552 (35.5) 919 (22.7) 528 (14.4) 236 (8.1) Moderately active 2696 (61.7) 2828 (69.8) 2630 (71.5) 1914 (65.4) Not active 120 (2.7) 303 (7.5) 518 (14.1) 775 (26.5) Table 1. Selected canine demographic characteristics by weight-based projected lifespan quartile (n = 15,019), Dog Aging Project 2020–2021. in the brain that otherwise contribute to neural damage and death, and an increase in neural ­plasticity24,25. The reduced odds of CCD among more active dogs in our cohort may be a result of these same mechanisms, but it is important to note that this correlation could also exist simply because of dogs exhibiting less activity due to their cognitive decline. Additionally, the presence of a third, unmeasured variable (such as owner interaction) could be amplifying this association.h in the brain that otherwise contribute to neural damage and death, and an increase in neural ­plasticity24,25. The reduced odds of CCD among more active dogs in our cohort may be a result of these same mechanisms, but it is important to note that this correlation could also exist simply because of dogs exhibiting less activity due to their cognitive decline. Additionally, the presence of a third, unmeasured variable (such as owner interaction) could be amplifying this association.h p y g The association we observed between dogs who have ever had a neurological disorder and their prevalence of CCD is expected. Dementia, a neurological disorder that could have been indicated by an owner in their HLES survey, is characterized by a decline in cognitive function. Seizure disorders, which also could have been indicated by the owner, have been found to occur more commonly in human AD patients than in comparable individuals without ­AD26. Human studies have demonstrated potential links between eye and ear disorders and AD. Discussion This improved only slightly in the model adjusted for age, sterilization status, history of 15 categories of health problems, breed type, and activity level. We also considered a lower diagnostic threshold for CCD assignment by using the top quartile of CSLB scores as a cut-off. However, that classification yielded much lower discrimi- nating ability between CCD positive and negative dogs for lifespan quartile. Although the diagnostic threshold of 50 had high predictive capacity, it is important to note that this threshold between normal aging and CCD was chosen by previous researchers, and measures of sensitivity and specificity would be difficult to ­obtain4. It would therefore be beneficial to examine the validity of this diagnostic tool more closely in future studies. The CCDR scale (on which the CSLB was based) was chosen for this study due, in part, to its high reported diag- nostic accuracy (98.9%) and its high re-test ­reliability4. However, it only identified CCD in 1.4% of our total canine population (mean age = 6.9, SD = 4.2). Although this aligns well with previously reported prevalence of veterinary-diagnosed dementia, higher CCD prevalence has been reported in older dog cohorts (8 + years) using the CCDR 14. Scientific Reports | (2022) 12:13316 | Discussion This could be due to several reasons, including reliance on self-reported owner data as opposed Scientific Reports | (2022) 12:13316 | https://doi.org/10.1038/s41598-022-15837-9 www.nature.com/scientificreports/ Characteristic (n) Univariable analysis Multivariable analysis OR 95% CI OR 95% CI Age (integer years) 1.68 1.60–1.77 1.52 1.44–1.61 Sex Female (reference) (7484) 1.00 – Male (7535) 0.91 0.69–1.19 – – Sterilization status Desexed (reference) (13,559) 1.00 – 1.00 – Intact (1460) 0.36 0.16–0.67 1.21 0.51–2.51 Activity level Very active (reference) (3235) 1.00 – 1.00 – Moderately active (10,068) 4.80 2.29–12.33 2.19 1.00–5.83 Not active (1716) 40.46 19.42–103.51 6.47 2.93–17.23 Breed group Sporting (reference) (2914) 1.00 – Herding (1513) 1.07 0.55–1.99 Hound (723) 1.65 0.78–3.26 Terrier (710) 3.58 2.02–6.28 Toy (990) 3.80 2.29–6.38 – – Non-sporting (855) 3.49 2.03–6.00 Working (986) 0.76 0.31–1.67 Misc./FSS (94) 0.00 – Non-AKC (107) 1.01 0.05–4.80 Geographic region West (reference) (4824) 1.00 – Midwest (3212) 0.82 0.55–1.19 Northeast (2774) 0.90 0.61–1.32 – – Southeast (2727) 0.82 0.55–1.22 Southwest (1393) 0.81 0.47–1.32 History of cancer No (reference) (14,171) 1.00 – 1.00 – Yes (848) 4.09 2.85–5.72 1.35 0.90–1.97 History of kidney disorder No (reference) (13,886) 1.00 – 1.00 – Yes (1133) 3.53 2.52–4.87 1.21 0.82–1.76 History of endocrine disorder No (reference) (14,582) 1.00 – 1.00 – Yes (437) 3.74 2.30–5.80 1.09 0.63–1.79 History of immune disorder No (reference) (14,898) 1.00 – – – Yes (121) 1.16 0.19–3.67 History of trauma No (reference) (10,997) 1.00 – 1.00 – Yes (4022) 1.63 1.23–2.15 1.10 0.79–1.50 History of toxin consumption No (reference) (13,391) 1.00 – 1.00 – Yes (1628) 1.61 1.10–2.29 1.13 0.73–1.69 History of infectious disease No (reference) (11,016) 1.00 – – – Yes (4003) 0.81 0.58–1.11 History of hematologic disorder No (reference) (14,946) 1.00 – – – Yes (73) 5.16 1.79–11.71 History of neurological disorder No (reference) (14,355) 1.00 – 1.00 – Yes (664) 8.22 5.96–11.20 1.84 1.26–2.65 History of orthopedic disorder Continued https://doi.org/10.1038/s41598-022-15837-9 Scientific Reports | (2022) 12:13316 | www.nature.com/scientificreports/ Characteristic (n) Univariable analysis Multivariable analysis OR 95% CI OR 95% CI No (reference) (12,294) 1.00 – 1.00 – Yes (2725) 3.33 2.52–4.37 0.88 0.64–1.20 History of reproductive disorder No (reference) (14,620) 1.00 – – – Yes (399) 0.69 0.21–1.64 History of liver disorder No (reference) (14,490) 1.00 – 1.00 – Yes (529) 3.38 2.12–5.15 0.96 0.57–1.57 History of gastrointestinal disorder No (reference) (12,814) 1.00 – 1.00 – Yes (2205) 1.59 1.13–2.19 1.10 0.76–1.58 History of respiratory disorder No (reference) (14,554) 1.00 – 1.00 – Yes (465) 3.69 2.29–5.67 0.75 0.44–1.22 History of cardiac disorder No (reference) (14,186) 1.00 – 1.00 – Yes (833) 3.78 2.61–5.35 0.83 0.54–1.24 History of skin disorder No (reference) (10,795) 1.00 – 1.00 – Yes (4224) 1.68 1.27–2.21 0.84 0.61–1.15 History of oral disorder No (reference) (11,150) 1.00 – 1.00 – Yes (3869) 3.58 2.74–4.71 0.79 0.58–1.08 History of eye disorder No (reference) (13,098) 1.00 – 1.00 – Yes (1921) 8.94 6.81–11.78 2.16 1.57–2.98 History of ear disorder No (reference) (13,046) 1.00 – 1.00 – Yes (1973) 6.56 5.00–8.62 1.96 1.42–2.70 Breed Mixed (reference) (6106) 1.00 – 1.00 – Purebred (8913) 1.43 1.07–1.91 1.24 0.90–1.72 Primary activity Pet (reference) (14,319) 1.00 – Agility (46) 1.49 0.08–6.85 Assistance or therapy (106) 0.00 – Breeding (18) 0.00 – Field trials (6) 0.00 – Hunting (24) 0.00 – – – Obedience (90) 0.00 – Other (185) 0.73 0.12–2.30 Search and rescue (24) 0.00 – Service (134) 0.50 0.03–2.26 Show (26) 0.00 – Working (41) 0.00 – Table 2. Table 2.   Association between selected dog characteristics and Canine Cognitive Dysfunction, Dog Aging Project 2020–2021. Discussion ROC curve generated from predictive models relating selected dog characteristics to Canine Cognitive Dysfunction prevalence, Dog Aging Project 2020–2021. Figure 3. ROC curve generated from predictive models relating selected dog characteristics to Canine Cognitive Dysfunction prevalence, Dog Aging Project 2020–2021. behaviors and medical conditions that could have occurred from 6 months to many years prior could introduce either differential or non-differential misclassification, depending on whether the errors in recall were corre- lated with the dog’s cognitive status. Non-differential misclassification could arise from social desirability if, for example, owners indicated a higher level of physical activity for their dog, or a lower level of impairment on the CSLB survey. Such errors would tend to dampen associations between physical activity and CCD. Addition- ally, the reliance on owner-reported behavioral survey information in this study did not allow for any further exploration into the pathological presentations of CCD. Future studies on this cohort intend to explore these relationships further. behaviors and medical conditions that could have occurred from 6 months to many years prior could introduce either differential or non-differential misclassification, depending on whether the errors in recall were corre- lated with the dog’s cognitive status. Non-differential misclassification could arise from social desirability if, for example, owners indicated a higher level of physical activity for their dog, or a lower level of impairment on the CSLB survey. Such errors would tend to dampen associations between physical activity and CCD. Addition- ally, the reliance on owner-reported behavioral survey information in this study did not allow for any further exploration into the pathological presentations of CCD. Future studies on this cohort intend to explore these relationships further. p It is also important to recognize the drastic changes that have taken place in many households due to the COVID-19 pandemic, and the possibility that these changes influenced our results. Depending on when an owner completed their HLES and CSLB surveys, their dog’s activity level may have changed as a result of stay-at-home orders and/or the owner’s ability to work from home. Additionally, owners spending more time at home with their dog may have an impact on the dog’s health, and it may increase the likelihood of observing specific health behaviors that would affect a dog’s reported health status. These types of major lifestyle changes could have altered our data in ways that would be difficult to quantify. Discussion Association between selected dog characteristics and Canine Cognitive Dysfunction, Dog Aging Project 2020–2021. to veterinary assessment, as well as wider age ranges in our study. Further sensitivity analyses of specific CSLB questions may be appropriate in future studies.h to veterinary assessment, as well as wider age ranges in our study. Further sensitivity analyses of specific CSLB questions may be appropriate in future studies.h This study had several strengths, including a large sample size, standardized data collection methods, high participant response proportions within the cohort, and the inclusion of many potential confounders in analytic models. Important limitations did exist in this study that are worth addressing. In addition to potential concerns with the sensitivity and specificity of the diagnostic threshold chosen for CCD, and that this analysis considers https://doi.org/10.1038/s41598-022-15837-9 Scientific Reports | (2022) 12:13316 | www.nature.com/scientificreports/ Figure 1. Logistic Regression curve representing association between age and CCD status, Dog Aging Project, 2020–2021. Figure 1. Logistic Regression curve representing association between age and CCD status, Dog Aging Project, 2020–2021. Figure 1. Logistic Regression curve representing association between age and CCD status, Dog Aging Project, 2020–2021. Figure 2. Directed Acyclic Graph (DAG) representing associations between Canine Cognitive Dysfunction, age, and other characteristics of interest, Dog Aging Project, 2020–2021. Figure 2. Directed Acyclic Graph (DAG) representing associations between Canine Cognitive Dysfunction, age, and other characteristics of interest, Dog Aging Project, 2020–2021. only the first year of data and is not yet longitudinal, it is not possible to assess any prospective links between the various canine characteristics and CCD. While the HLES and CSLB surveys were administered to owners at different times (the maximum time interval between the completion of the HLES and CSLB surveys was 50.3 weeks), the median time between survey completion was only 5 weeks. 11,324 of the total 15,019 owners completed both surveys within 10 weeks. There was a low likelihood that this timeframe would result in any sort of meaningful changes in CCD status. Future studies using prospective DAP data will be able to explore potential risk factors for CCD as well as cognitive decline over time. g Another limitation is that all data used in this analysis were based on surveys completed by owners. Owner- eported information is potentially susceptible to multiple forms of bias. Questions requiring owners to recall https://doi.org/10.1038/s41598-022-15837-9 Scientific Reports | (2022) 12:13316 | www.nature.com/scientificreports/ Figure 3. Discussion Finally, there is the potential for unmeasured confounders that were not captured by our surveys, as well as survey data that were not considered for analysis, which could result in residual confounding potentially adding bias to our results.ii We identified a positive association between companion dog age and CCD. We also identified strong positive associations between history of a neurological, ear, or eye disorder as well as an association between decreased physical activity level and having a CSLB score of 50 or higher. Dogs were classified into their predicted quartile of life based on their age, weight, sex, and sterilization status. Using a binary diagnostic threshold of ≥ 50, a dog’s lifespan quartile showed excellent discriminating ability between CCD positive and negative dogs. This quartile estimation could potentially serve as a useful tool to inform whether a dog should be screened for CCD by their veterinarian. Finally, given increasing evidence of the parallels between canine and human cognitive disease, accurate CCD diagnosis in dogs may provide researchers with more suitable animal models in which to study aging in human populations. Additional studies that further explore factors that will provide a better understand- ing of canine cognitive function are needed. References Prevalence of behavioral changes associated with age-related cognitive impair- ment in dogs. J. Am. Vet. Med. Assoc. 218(11), 1787–1791. https://​doi.​org/​10.​2460/​javma.​2001.​218.​1787 (2001). 16. Creevy, K. E. et al. 2019 AAHA canine life stage guidelines. J. Am. Anim. Hosp. 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Fitzpatrick19,20, Unity Jeffery21, Erica C. Jonlin9,22, Matt Kaeberlein9, Elinor K. Karlsson23,24, Kathleen F. Kerr25, Jonathan M. Levine15, Jing Ma26, Robyn L. McClelland25, Daniel E. L. Promislow9,27, Audrey Ruple28, Stephen M. Schwartz20,29, Sandi Shrager30, Noah Snyder‑Mackler31,32,33, M. Katherine Tolbert14, Silvan R. Urfer9 & Benjamin S. Wilfond34,35 Kate E. Creevy4, Audrey Ruple5, Vanessa Wilkins4, Matt Kaeberlein6, Daniel Promislow7, Joshua M. Akey8, Brooke Benton9, Elhanan Borenstein10,11,12, Marta G. Castelhano13, Amanda E. Coleman14, Kate E. Creevy15, Kyle Crowder16,17, Matthew D. Dunbar17, Virginia R. Fajt18, Annette L. Fitzpatrick19,20, Unity Jeffery21, Erica C. Jonlin9,22, Matt Kaeberlein9, Elinor K. Karlsson23,24, Kathleen F. Kerr25, Jonathan M. Levine15, Jing Ma26, Robyn L. McClelland25, Daniel E. L. Promislow9,27, Audrey Ruple28, Stephen M. Schwartz20,29, Sandi Shrager30, Noah Snyder‑Mackler31,32,33, M. Katherine Tolbert14, Silvan R. Urfer9 & Benjamin S. Wilfond34,35 4Department of Small Animal Clinical Sciences, Texas A&M University College of Veterinary Medicine & Biomedical Sciences, College Station, TX, USA. 5Department of Population Health Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA. 6Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA, USA. 7Department of Biology, University of Washington, Seattle, WA, USA. 8Lewis‑Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA. 9Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA, USA. 10Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. 11Blavatnik School of Computer Science, Tel Aviv University, Tel Aviv, Israel. 12Santa Fe Institute, Santa Fe, NM, USA. 13Cornell Veterinary Biobank, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA. 14Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA, USA. 15Department of Small Animal Clinical Sciences, Texas A&M University College of Veterinary Medicine & Biomedical Sciences, College Station, TX, USA. 16Department of Sociology, University of Washington, Seattle, WA, USA. 17Center for Studies in Demography and Ecology, University of Washington, Seattle, WA, USA. 18Department of Veterinary Physiology and Pharmacology, Texas A&M University College of Veterinary Medicine & Biomedical Sciences, College Station, TX, USA. 19Department of Family Medicine, University of Washington, Seattle, WA, USA. 20Department of Epidemiology, University of Washington, Seattle, WA, USA. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. © The Author(s) 2022, corrected publication 2023 Acknowledgments Th A h Acknowledgments The Dog Aging Project thanks study participants, their dogs, and community veterinarians for their important contributions. g The Dog Aging Project thanks study participants, their dogs, and community veterinarians for their important contributions. Competing interests h The authors declare no competing interests. Author contributions S.Y. performed statistical analysis and contributed to the drafting and editing of the manuscript. A.F., S.M.S., K.C., and A.R. provided statistical advice and contributed to the drafting and editing of the manuscript. V.W., M.K., and D.P. provided scientific advice and contributed to the drafting and editing of the manuscript. The authors read and approved the final manuscript. Fundingh The Dog Aging Project is supported by U19 grant AG057377 from the National Institute on Aging, a part of the National Institutes of Health, and by private donations. References https://​doi.​org/​10.​1080/​01616​412.​1996.​11740​369 (1996).h p g ( ) 9. Hwang, P. H. et al. Ophthalmic conditions associated with dementia risk: The Cardiovascular Health Study. Alzheimer’s Dement https://​doi.​org/​10.​1002/​alz.​12313 (2021). p g 30. Wang, S., Mims, P. N., Roman, R. J. & Fan, F. Is Beta-Amyloid accumulation a cause or consequence of Alzheimer’s disease?. J. Alzheimer’s Parkinsonism Dement. 1(2), 007 (2016). 31. Loughrey, D. G., Kelly, M. E., Kelley, G. A., Brennan, S. & Lawlor, B. A. 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(A 12(1), e12054. https://​doi.​org/​10.​1002/​dad2.​12054 (2020). https://doi.org/10.1038/s41598-022-15837-9 Scientific Reports | (2022) 12:13316 | www.nature.com/scientificreports/ Additional information Correspondence and requests for materials should be addressed to S.Y. Medical School, Worcester, MA, USA. 24Broad Institute of MIT and Harvard, Cambridge, MA, USA. 25Department of Biostatistics, University of Washington, Seattle, WA, USA. 26Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. 27Department of Biology, University of Washington, Seattle, WA, USA. 28Department of Population Health Sciences, Virginia‑Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA. 29Epidemiology Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. 30Department of Biostatistics, Collaborative Health Studies Coordinating Center, University of Washington, Seattle, WA, USA. 31School of Life Sciences, Arizona State University, Tempe, AZ, USA. 32Center for Evolution and Medicine, Arizona State University, Tempe, AZ, USA. 33School for Human Evolution and Social Change, Arizona State University, Tempe, AZ, USA. 34Treuman Katz Center for Pediatric Bioethics, Seattle Children′s Research Institute, Seattle, WA, USA. 35Department of Pediatrics, Division of Bioethics and Palliative Care, University of Washington School of Medicine, Seattle, WA, USA. Dog Aging Project Consortium 21Department of Veterinary Pathobiology, Texas A&M University College of Veterinary Medicine & Biomedical Sciences, College Station, TX, USA. 22Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA. 23Bioinformatics and Integrative Biology, University of Massachusetts Chan https://doi.org/10.1038/s41598-022-15837-9 Scientific Reports | (2022) 12:13316 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ https://doi.org/10.1038/s41598-022-15837-9 Scientific Reports | (2022) 12:13316 |
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Leprosy and the elusive M. leprae: colonial and imperial medical exchanges in the nineteenth century
História, ciências, saúde-Manguinhos
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vol 10 (supplement 1):13- Leprosy and the elusive M. leprae: colonial and Imperial medical exchanges in the nineteenth century A lepra e o evasivo M. leprae: a troca de informações médicas nos períodos colonial e imperial do século XIX Jo Robertson Wellcome Unit for the History of Medicine Oxford 45 Banbury Road, Oxford, OX 2 6 PE jo.robertson@wuhmo.ox.ac.uk Leprosy and the elusive M. leprae: colonial and Imperial medical exchanges in the nineteenth century A lepra e o evasivo M. leprae: a troca de informações médicas nos períodos colonial e imperial do século XIX In the 1800s, humoral understandings of leprosy successively give way to disease models based on morbid anatomy, physiopathology, and bacteriology. Linkages between these disease models were reinforced by the ubiquitous seed/soil metaphor deployed both before and after the identification of M. leprae. While this metaphor provided a continuous link between medical descriptions, Henry Vandyke Carter’s On leprosy (1874) marks a convergence of different models of disease. Simultaneously, this metaphor can be traced in popular and medical debates in the late nineteenth century, accompanying fears of a resurgence of leprosy in Europe. Later the mapping of the genome ushers in a new model of disease but, ironically, while leprosy research draws its logic from a view of the world in which a seed and soil metaphor expresses many different aspects of the activity of the disease, the bacillus itself continues to be unreceptive to cultivation. KEYWORDS: leprosy, M. leprae, morbid anatomy, Henry Vandyke Carter, G. Armauer Hansen. KEYWORDS: leprosy, M. leprae, morbid anatomy, Henry Vandyke Carter, G. Armauer Hansen. ROBERTSON, J.: ‘A lepra e o evasivo M. leprae: a troca de informações médicas nos períodos colonial e imperial do século XIX’. História, Ciências, Saúde — Manguinhos, vol. 10 (suplemento 1): 13-40, 2003. No século XIX, abordagens humorais da lepra deram origem a sucessivos modelos da doença baseados na anatomia patológica, na fisiopatologia e na bacteriologia. As relações entre esses modelos da doença foram reforçadas pela onipresente metáfora ‘da semente e do solo’, difundida tanto antes quanto depois da identificação do M. leprae. À época em que a metáfora fornecia um elo de ligação contínuo entre as várias descrições médicas da doença, Henry Vandyke Carter publicava On leprosy (1874), estabelecendo uma convergência de seus diferentes modelos. Simultaneamente, a metáfora se fazia presente nos debates médicos e populares de fins do século XIX, juntamente com o medo do surgimento da lepra na Europa. Mais recentemente, o mapeamento do genoma humano determinou a formulação de um novo modelo para a doença. ROBERTSON, J.: ‘Leprosy and the elusive M. leprae: colonial and Imperial medical exchanges in the nineteenth century História, Ciências, Saúde — Manguinhos, vol. 10 (supplement 1): 13-40, 2003. ROBERTSON, J.: ‘Leprosy and the elusive M. leprae: colonial and Imperial medical exchanges in the nineteenth century História, Ciências, Saúde — Manguinhos, vol. 10 (supplement 1): 13-40, 2003. ROBERTSON, J.: ‘Leprosy and the elusive M. leprae: colonial and Imperial medical exchanges in the nineteenth century História, Ciências, Saúde — Manguinhos, vol. 10 (supplement 1): 13-40, 2003. ROBERTSON, J.: ‘Leprosy and the elusive M. leprae: colonial and Imperial medical exchanges in the nineteenth century História, Ciências, Saúde — Manguinhos, vol. 10 (supplement 1): 13-40, 2003. Jo Robertson Wellcome Unit for the History of Medicine Oxford 45 Banbury Road, Oxford, OX 2 6 PE jo.robertson@wuhmo.ox.ac.uk vol 10 (supplement 1):13- Leprosy and the elusive M. leprae: colonial and Imperial medical exchanges in the nineteenth century A lepra e o evasivo M. leprae: a troca de informações médicas nos períodos colonial e imperial do século XIX Jo Robertson Wellcome Unit for the History of Medicine Oxford 45 Banbury Road, Oxford, OX 2 6 PE jo.robertson@wuhmo.ox.ac.uk Mas, ironicamente, enquanto as pesquisas concernentes a ela se apóiam numa visão de mundo em que a metáfora da semente e do solo ainda expressa diferentes aspectos da ação da doença, o próprio bacilo permanece refratário a todos os esforços visando seu cultivo. PALAVRAS-CHAVE: lepra, M. leprae, anatomia patológica, Henry Vandyke Carter, G. Armauer Hansen. 13 2003 vol. 10 (supplement 1):13-40, 2003 JO ROBERTSON JO ROBERTSON The aetiological agent of leprosy is Mycobacterium leprae. It is a strongly acid-fast rod-shaped organism with parallel sides and rounded ends. It occurs in large numbers in the lesions of lepromatous leprosy, chiefly in masses within the lepra cells, often grouped together like bundles of cigars or arranged in a palisade. Most striking are the intracellular and extra-cellular masses, known as globi, which consist of clumps of bacilli in capsular material. Under the electron microscope, the bacillus appears to have a great variety of forms. The commonest is a slightly curved filament, containing irregular arrangements of dense material sometimes in the shape of rods. Short rod-shaped structures can also be seen (identical with the rod-shaped inclusions within the filaments) and also dense spherical forms. Some of the groups of bacilli can be seen to have a limiting membrane. (WHO web pages http:// www.who.int/lep/disease/disease.htm) T he World Health Organization (WHO) currently describes leprosy as a disease mainly affecting the skin, the peripheral nerves, the mucosa of the upper respiratory tract, and also the eyes, apart from some other structures. It is estimated that there are between one and two million people visibly and irreversibly disabled due to past and present leprosy. Treatment for leprosy only appeared in the late 1940s with the introduction of dapsone and its derivatives. Leprosy bacilli resistant to dapsone gradually appeared and became widespread, necessitating the identification and development of multi-drug therapy. In 1997, there were an estimated 1.2 million cases in the world, most of them concentrated in Southeast Asia, Africa, and the Americas. About 750,000 new cases are detected worldwide each year. T An examination of the ways in which the disease ‘leprosy’ is recognized and understood in any one historical moment demonstrates a dependence upon systems of medical knowledge available at the time. Interestingly, sometimes confusingly, and often imperceptibly, these systems of knowledge can be seen not to be self-contained — never hermetically sealed as discrete categories, distinct from those current in earlier or successive periods. História, Ciências, Saúde — Manguinhos, Rio de Janeiro História, Ciências, Saúde — Manguinhos, Rio de Janeiro vol 10 (supplement 1):13- Leprosy and the elusive M. leprae: colonial and Imperial medical exchanges in the nineteenth century A lepra e o evasivo M. leprae: a troca de informações médicas nos períodos colonial e imperial do século XIX Jo Robertson Wellcome Unit for the History of Medicine Oxford 45 Banbury Road, Oxford, OX 2 6 PE jo.robertson@wuhmo.ox.ac.uk The history of the deployment of medical categories has been well documented. They draw support from teleologies that determine an understanding of the body and also a sense of the place of the physician, and they possess their own logic. In addition, there may be an apparent eclecticism in the use of medical categories. Observation and description may draw on analogies and metaphors that seem most appropriate to the case in hand. Sometimes to do justice to the latest observation necessitates borrowing from earlier systems of understanding and combining them with more contemporary ones so that world views are brought together that are not always commensurate with each other. Other times, what needs to be said may necessitate a search for a whole new mode of expression. This 14 LEPROSY AND THE ELUSIVE LEPROSY AND THE ELUSIVE paper will examine the shifting medical categories and metaphoric continuities through which leprosy was thought of from the early 1800s in order to document the different ways in which leprosy, but most specifically the bacillus, has been thought of. As a work in progress, after the late 1800s, it will then take a very brief leap to the mapping of the genome. Intriguingly, there is still much to be learnt about leprosy. Even after M. leprae was shown, by G. Armauer Hansen1 in 1873, to be the bacillus consistently present in the nodes of leprosy patients, this entity would continue to puzzle histologists, pathologists, and clinicians. It is referred to as the aristocrat of diseases — the oldest, the most mysterious. Its effects depend more upon the reaction of the host than upon the action of the invader (Hastings, 1985, p. 32).2 Its stages and categories have been, for a long time, subject to debate,3 and how it enters the body and is transmitted to others is still unknown.4 Paradoxically, although it was the first bacillus to be identified, it has still not been cultivated in vitro. 5 The history of the disease is also shrouded in uncertainty that is heightened by the confusion and debate surrounding its naming. In the nineteenth century, naming and describing leprosy was a complicated and subtle process vulnerable to mistakes and misinterpretations, a trail of which had already been generated and which needed to be retraced every time attempts were made to document it comprehensively. vol 10 (supplement 1):13- Leprosy and the elusive M. leprae: colonial and Imperial medical exchanges in the nineteenth century A lepra e o evasivo M. leprae: a troca de informações médicas nos períodos colonial e imperial do século XIX Jo Robertson Wellcome Unit for the History of Medicine Oxford 45 Banbury Road, Oxford, OX 2 6 PE jo.robertson@wuhmo.ox.ac.uk Inevitably, at a material level, these mistakes and misinterpretations served to obscure and confuse diagnoses of leprosy, and, at the same time, they served to compound the already charged symbolic resonance of the disease and the corresponding force and power of its myriad representations. This paper will focus on a single and continuous metaphor that has resonated in many different registers and in separate, but overlapping, fields throughout the history of the disease in the English-speaking world. vol. 10 (supplement 1):13-40, 2003 The botanical metaphor In the 1800s, humoral and environmental explanations were drawn on to explain the occurrence of disease. Michael Worboys (2000, p. 31) identifies and explicates trajectories of medical knowledge from the mid-nineteenth century. These shift from a preoccupation with morbid anatomy or the end of disease, give way to an interest in physiology and patho-physiology, by 1875, and arrive at bacteriology and experimental pathology, with its subsequent focus on the causes of disease. Disease involved “structural and functional perturbations” so that the physician’s task was to intervene in positive ways which would “promote repair, … restore function, or … aid in the regeneration of damaged structures” (ibid, p. 33). Worboys (p. 193) also points out that significant continuities in the medical understanding of tuberculosis, for 15 JO ROBERTSON JO ROBERTSON example, were, in part, due to “a dominant seed and soil metaphor which allowed constitutional notions to be refashioned in terms of vulnerability of the human soil.” The same dominant seed/soil metaphor can be identified in the medical categories employed to understand leprosy. This metaphor can be observed operating and being reinflected both before and after M. leprae was identified. The notion of ‘soil’ is expressive of a wide range of possibilities. The hereditary predisposition of individual bodies, the lifestyle, dietary habits, practices, such as cultural customs, and moral behavior are all encompassed by the concept of a ‘soil’ that is conducive to the seeds of the disease. In addition, ‘racial types’, and certain groups of people with their specific customs, are seen as offering a milieu in which the seeds of the disease can flourish. Typical of early- to mid-nineteenth century descriptions of leprosy, Joseph Adams (1807), James Maxwell (1839), and Alexander Fiddes (1857) all demonstrate understandings of leprosy that seek to place it within a constitutional model of disease that depends upon the seed/ soil metaphor for expression of its complexities and variations. p p p Joseph Adams wrote his account from his experiences as a physician on Madeira, and his work was read before the London College and intended for publication in the Transactions of the London College. Careful observations of bodies bearing signs of what he understands as leprosy indicate that its effects produce disorders in structure, such as arresting growth to sexual maturity. História, Ciências, Saúde — Manguinhos, Rio de Janeiro The botanical metaphor Adams explains that leprosy seems to arise spontaneously, although climate, constitutional predisposition, and diet are considered responsible, either singly or in combination, for creating appropriate conditions for its appearance. If climate and diet are alone responsible, then the patient can be assisted. If there is a constitutional predisposition, then “the cure can only be permanent as long as the patient is removed from the exciting causes” (Adams, 1807, p. 269). The body acts as a predisposing soil carrying the seeds of the disease, but there is also the receptive soil of climate and/or diet in which the body as a seed, carrying a predisposition for the disease, may take root and flourish with leprosy. Body is both soil and seed. In addition, those who are predisposed to the disease usually contract it so early that they are unable to reproduce. Ironically, if a hereditary predisposition provides enabling conditions or receptive ‘soil’ for the seeds of the disease, then, infertility will be the result. Thirty-two years later, for James Maxwell, writing from his experiences in Jamaica, the signs of the disease, increasing in intensity and coming to a climax, and the accompanying fever are indicators of stages in the body’s attempt to eliminate the poison that arises in the blood. Initially it appears as “preliminary eruptions of blotches and scaly efflorescences” and then it shows itself as “an open ulcerated state” (Maxwell, 1839, pp. 235, 234). For some, symptoms are confined to the surface of the body, for others the nose and throat are attacked and the bones become 16 LEPROSY AND THE ELUSIVE LEPROSY AND THE ELUSIVE affected (ibid.). Its treatment should counteract and balance the natural progress of the disease: “These operations teach us to imitate nature by counter-irritants” so that suppressed symptoms are encouraged to develop and the affection is drawn from the face and throat. In this instance, the disease is again the seed lodged in the soil of the body. It may arise spontaneously in some individuals, but it also arises in those who have a hereditary predisposition to it. Some soils are more conducive to its growth than others. It may remain latent in those who have this predisposition, and, without the occurrence of external favorable circumstances, it may skip a generation. The botanical metaphor Happily, its virulence may be mitigated by breeding with healthy stock but “when it has been confined to a long race of ancestry, it becomes possessed with powers of great inveteracy” (ibid., p. 232). Conversely, improvements in hygiene and diet will, over generations, diminish its power, as instanced in its disappearance from Europe (ibid). Once again, in a paradoxical inversion of the botanical metaphor, poor soil allows the virulence of the strain to intensify, and good soil weakens its power. Maxwell is optimistic that eventually leprosy will be “disarmed” and vanish from the Western world in the same way that it has already done “from the shores of Europe” (p. 237). p (p 37) Like James Maxwell, Alexander Fiddes, publishing his observations of leprosy in Jamaica in 1857, understands the disease as typical of other blood diseases resulting from “morbid matter in the blood, and in the effusion of it on the solid textures” (Fiddes, 1857, pp. 1063, 1072, 1073). Repetitive febrile disturbances indicate natural attempts to expel poisonous matter from the system. This process of expulsion produces a disturbance and derangement in the skin and in the mucous membranes “ultimately effect[ing] a partial or complete disorganization of the texture.” There is the prospect of this process actually accomplishing the expulsion of the poison. Fiddes suggests that in some rare cases, “nature has proved adequate to expel the disease, and to remove the tubercles at the same time” and he gives one instance in which “the cure was accomplished through the intervention of an inflammatory condition. In this case, there can be no doubt that the morbid depositions were dissolved, and the poison eliminated by means of a congestive inflammation of the dermis” (ibid, pp. 1074-5). This understanding of the disease relies more on a sense of humoral balance, which, after a disturbance in the body, reasserts itself. Cultural differences, geography, latitude, and constitution are specified and rejected as immediate causes of leprosy, but they are considered as providing the soil that may modify the severity of the disease. Like James Maxwell, Alexander Fiddes, publishing his observations of leprosy in Jamaica in 1857, understands the disease as typical of other blood diseases resulting from “morbid matter in the blood, and in the effusion of it on the solid textures” (Fiddes, 1857, pp. 1063, 1072, 1073). Repetitive febrile disturbances indicate natural attempts to expel poisonous matter from the system. Cross-pollination: the Royal College of Physicians, Danielssen and Boeck, Vandyke Carter, and Hansen Cross-pollination: the Royal College of Physicians, Danielssen and Boeck, Vandyke Carter, and Hansen From the mid-nineteenth century, a significant collection of publications appeared, marking the point at which medical models of the disease, drawing upon morbid anatomy and descriptions of the effects of the disease on the structures of the body, give way to a more precise parasitic theory. With the shift from one disease model to another, it could be assumed that the soil/seed metaphor would be discarded, but as Worboys has already noted in his work on tuberculosis, the metaphor serves to bridge the transition to an understanding of the disease based on bacteriology. gy Conducted and published before the identification of the bacillus, The Report on Leprosy by the Royal College of Physicians (1867) originated from a suggestion to the College by James Walker, Governor-in-Chief of the Windward Isles, that reports be gathered on the “character and progress of the disease of leprosy” (p. a2). This grew out of concern that leprosy was “on the increase.” The survey was conducted by sending out a series of interrogatories to the colonies and related areas where leprosy may have been prevalent. There were seventeen questions. The botanical metaphor This process of expulsion produces a disturbance and derangement in the skin and in the mucous membranes “ultimately effect[ing] a partial or complete disorganization of the texture.” There is the prospect of this process actually accomplishing the expulsion of the poison. Fiddes suggests that in some rare cases, “nature has proved adequate to expel the disease, and to remove the tubercles at the same time” and he gives one instance in which “the cure was accomplished through the intervention of an inflammatory condition. In the early- to mid-nineteenth century, therefore, the seed/soil metaphor was deployed flexibly to express both the vulnerability of different bodies to leprosy, as well as the predisposing influence of external circumstances. 17 vol. 10 (supplement 1):13-40, 2003 JO ROBERTSON JO ROBERTSON História, Ciências, Saúde — Manguinhos, Rio de Janeiro Cross-pollination: the Royal College of Physicians, Danielssen and Boeck, Vandyke Carter, and Hansen In this instance, the ‘soil’ is the family, rather than the body. In addition, previously healthy soil can be altered by external influences so that it becomes receptive to the disease: “We have already said that leprosy may also be acquired. We speak of these cases where the malady declares itself in persons born of healthy parents, in whose families the disease had never resided, for a longer or shorter period, in countries where it is endemic, and who have lived under conditions liable to occasion its development” (p. lxviii). So residence in countries where the disease was endemic was considered to be conducive to its appearance in those whose family has never before shown a disposition to leprosy: colonial soil, by itself, was sufficient for the appearance of the disease in the bodies of previously untainted colonizers. p y Danielssen and Boeck (1847) appear again, with the work of Hansen, in Carter’s On leprosy and elephantiasis in 1874. In addition, Hansen’s ‘Preliminary contribution respecting the characteristics of leprosy’ in 1869 and his ‘Further contributions towards a knowledge of the characteristic features of leprosy (spedalskhed)’ (1870) were translated and published in the appendices. It is questionable what sort of circulation and direct impact Hansen’s early papers may have had with the British medical fraternity, until they appeared in Vandyke Carter’s 1874 publication. In addition to this intermeshing of research, Vandyke Carter visited Danielssen and Boeck in Norway in 1873. Without doubt, Vandyke Carter served to assimilate and disseminate knowledge about leprosy, from India to Europe and back again in this period. At this point, different models of disease converge, and morbid anatomy can be seen giving way to pathology. Carter marvels in the introduction that even what little is known of the pathology of the disease indicates how adaptable it is to a theory of chronic infection. He sets up opposing views for understanding the disease and accomplishes the succession of one view over the other. There is the earlier view in which “the local deposition of leprous matter in the shape of nodules or tubercles” was considered caused by a ‘dyscrasia’ of the blood. Danielssen and Boeck supported this, as did “standard English works.” Since then almost every observer of leprosy, has, it must be said, conformed in principle to the opinion so definitely put forth in Norway in 1848 (Carter, 1874, pp. 72-3). Cross-pollination: the Royal College of Physicians, Danielssen and Boeck, Vandyke Carter, and Hansen Respondents were asked if leprosy was known in their colony; to describe it as it occurred there; to enumerate its forms or outward manifestations; to give an opinion as to whether there were only varieties of the one disease or if there were distinct diseases; to describe the distinguishing characteristics of each form; to generalize about the age and the time of life at which the symptoms of the disease, its full development, and its most fatal stage became apparent; to generalize about its prevalence with respect to sex, race, and social group; to describe the topographical character of the place, and the sanitary conditions where it is prevalent; to describe the habits of the afflicted, their diet, occupations, and any conditions or circumstances of life that would seem to aggravate the disease; to suggest if they considered that it was hereditary, related to yaws, syphilis, or any other disease, and if they knew of instances where it had been communicated by contagion or by sexual intercourse; to note whether the afflicted were permitted to mix with others in the colony; to indicate what public provision was made for the reception and treatment of those with the disease who were poor; to estimate how long it had been in the colony, and if they had observed any results from hygienic, dietetic, and medical treatment, or if they had any cures to report; and, finally, to estimate the proportion of leprosy patients in the overall population of the colony. As a counterpoint to the replies from the colonies, the definitive work of the Norwegian authorities Danielssen and Boeck (1847) is quoted as arguing that leprosy seems “to pass over one generation, and to reappear in the next” (ibid, p. lxviii). This time, the seed and soil metaphor serves to express the latent tendencies of the disease — the 18 LEPROSY AND THE ELUSIVE longer the disease lies dormant, the greater its intensity when it re- emerges: “If it has spared the first generation, it as a general rule appears in all the individuals of the second, who transmit the germ of the disease to succeeding generations. Tolerably often, it seemed to pass over the second or third generations, and to reappear in the fourth generation, and then to spread in all directions, so to speak, with a new energy” (p. lxviii). vol. 10 (supplement 1):13-40, 2003 Cross-pollination: the Royal College of Physicians, Danielssen and Boeck, Vandyke Carter, and Hansen The opposing view promises to “reconcile the pre-existing and non-concordant opinions” in which “the implication of the system … is a secondary consequence of the primary local implantation of the malady.” Here Carter is heralding Hansen’s opinion, which he is careful to distinguish from “earlier and more crude hypotheses 19 JO ROBERTSON of the extraneous origin of leprosy.” At the same time, he calls for “renewed investigation, to be pursued in accordance with modern means and attainments” (ibid, p. 72). The shift to observations and conclusions based on experimental evidence accompanies Carter’s introduction of Hansen’s work. Hansen’s 1869 paper, excerpted and translated as an appendix in Vandyke Carter’s (1874), would appear to demonstrate Carter’s ideal researcher. Hansen’s observations identify structural elements which he suggests are characteristic of leprous productions. In this description, minute observation, aided by the microscope, combines with a descriptive repertoire that is still within the trajectories of morbid anatomy and attention to structural detail that I have been tracing. In describing the commencement of the softening of the tubercles, he notes “round, oblong, and spindle-shaped cells” containing nuclei and, in addition, “one or more large and small, round, yellow, granular masses” (Hansen, cited in Carter, 1874, Appendix A, p. i). The nucleus and clear part of the cell can be colored with carmine; the yellow masses remain unchanged. These masses lie in the cavity of the cell. When a cell contains a single mass, it looks like a signet ring, with the tinted mass in the middle. In some cells, these masses seem quite distinct from the rest of the cell, but in other cells they seem to be amalgamated with the rest of the cell. There are also other cells, similar in form, containing protoplasm that is partly clear and partly finely granular, and which is mostly filled with fat-granules. As the softening of the tubercle progresses, these cells change. Their color becomes a more intense yellowish brown. Amidst these changes in the cells, rounded masses or corpuscles are found which vary in size from a quarter of the size of white blood cells to six or eight times as large. These seem to have been set free from the cells. Large collections of these masses can be detected. These may be found adhering to each other and, more often, they can be found enclosed in a colorless envelope. História, Ciências, Saúde — Manguinhos, Rio de Janeiro LEPROSY AND THE ELUSIVE simplest shape, appearing as a homogenous mass, indented at the middle like a dumb-bell, or further indented as a trefoil. They are tough and elastic. As seen either in motion or at rest, the outlines of the projecting hemispheres on their under surface are clearly visible. These same elements were, Hansen comments, described, but less correctly, in Danielssen’s work in 1862 as pigmented clusters of fatty granules and were identified by him as characteristic results of the leprous products. This conclusion was also confirmed by Virchow (cited in Carter, 1874, p. ii). Hansen (ibid, p. iv) comments that during his earliest examination of the morbid products in leprosy, he “almost constantly” and to his astonishment “met with these peculiar structural elements.” He locates these rounded masses of leprous character in the lymph glands, liver, and spleen, and in a section taken from the ulnar nerve. simplest shape, appearing as a homogenous mass, indented at the middle like a dumb-bell, or further indented as a trefoil. They are tough and elastic. As seen either in motion or at rest, the outlines of the projecting hemispheres on their under surface are clearly visible. These same elements were, Hansen comments, described, but less correctly, in Danielssen’s work in 1862 as pigmented clusters of fatty granules and were identified by him as characteristic results of the leprous products. This conclusion was also confirmed by Virchow (cited in Carter, 1874, p. ii). Hansen (ibid, p. iv) comments that during his earliest examination of the morbid products in leprosy, he “almost constantly” and to his astonishment “met with these peculiar structural elements.” He locates these rounded masses of leprous character in the lymph glands, liver, and spleen, and in a section taken from the ulnar nerve. Hansen (p. iv) is confident that what he is observing is the result of a “necrobiotic process,” and that there is “some peculiar property in them, which is connected with such process.” But he adds: “I am not acquainted with any either described, or conceivable, structures or properties, which quite correspond with the forms in question, and with their constant peculiar tinting.” The anatomical changes that he has noted are completely different to anything he or anyone else has observed “in preparations taken from cases of necrosis and chronic ulcerations.” Therefore, the changes that he observes are, he concludes, evidence of a specific leprous affection. Hansen (p. vol. 10 (supplement 1):13-40, 2003 Cross-pollination: the Royal College of Physicians, Danielssen and Boeck, Vandyke Carter, and Hansen Like the signet-ring-like cells, he identifies large orange masses surrounded by an extremely thin enveloping material raised on one side. The centers have a space like a vacuole with clear contents. This space is sometimes so large that there is no other space around it, except for a very narrow brownish ring. There is always a sharp boundary. The interior is minutely granular and deep-tinted and more or less translucent. He observes that across the most clouded and dark-tinted specimens, “other underlying elements may be clearly enough perceived; and this is most apparent in the more monstrous forms” (Hansen, ibid). He has not come to any conclusion about the history and mode of origin of these bodies. He does not know if they have been produced by the enlargement of the smaller elements that he has just described or by amalgamation of them. He describes them as large and, in their 20 LEPROSY AND THE ELUSIVE LEPROSY AND THE ELUSIVE iv) writes: If one next turns to the affections of the liver and spleen, one cannot but suppose — even without paying regard to the appearance of the brown elements — from the fact of their connection with the small round cells, from the many-nucleated cells with their degeneration into fat granule masses, and from the conjoined state of the lymphatic glands, that these affections are of leprous character. When, besides all these, one finds certain peculiar looking structures — brown-tinted masses — the conclusion seems inevitable, that here are altogether specific leprous products. In Leprosy: in its clinical and pathological aspects, Hansen is more certain of what he is looking at. Examination by microscope of a section of fresh nodules reveals little else but cells, with distinct nuclei, usually the size of white corpuscles, or larger. With an even higher power, “one sees in the fluid of the preparation small straight rods, which are not destroyed by the addition of potash. These are the lepra bacilli, and thus were they first discovered in the year 1871” (Hansen et al., 1895, p. 31). After preparation, the rods are colored faint brown, and they can be found lying mostly in the cells. In a fresh preparation, they can be seen moving actively. Building on his previous descriptions of the softening nodule, with its central part, with its distinct brown color, which when examined 21 JO ROBERTSON JO ROBERTSON under a microscope reveals larger or smaller clumps of a brownish color that are very granular and that lie in the cells and can be located in all the other organs affected with leprosy, he writes that they may very well serve as diagnostic indication for leprous affections because, in his experience, they can always present, except in very young nodules. Subsequent investigations have shown that these brown clumps are “nothing else but collections of lepra bacilli broken down into granules.” Neisse has called them “globi” (ibid, p. 40). He is certain that the bacilli therefore occur in the cells. Additional observations lead him to conclude that the bacilli must increase very, very slowly and that they probably produce a toxin in very small quantities which “causes no particular injury to the organism, since patients, in spite of numerous nodules with millions or milliards of bacilli, may remain in pretty good health for years” (ibid). This toxin only acts immediately around the bacilli. História, Ciências, Saúde — Manguinhos, Rio de Janeiro História, Ciências, Saúde — Manguinhos, Rio de Janeiro LEPROSY AND THE ELUSIVE have bearing on an ultimate solution” (Hansen, 1976, p. 92). As a result, another world of observation is opened up — in finer detail. At the same time, the familiar metaphors still have their uses. In considering the clinical variations in leprosy, Hansen (1976, pp. 79, 80) asks if these depend upon the virulence of the bacilli. He concludes that the virulence of the bacilli depend “not so much on any constant character of their own, as in the soil in which they live.” In this case, the botanical metaphor serves to express climatic variations. In Hansen’s experience, maculo-anaesthetic cases are more numerous where the climate is dry, and nodular cases can be found where the climate is moist. He reasons that the exposure of the skin to the influence of the weather may affect the disease. Against this hypothesis, he also argues that “it is also possible that the bacilli always possess the same virulence, and that it is solely dependant on the soil in which they live, whether they multiply freely or no.” Here the ‘soil’ may be responsible for modifying the virulence of the bacillus or it may actually be more or less receptive to an unvarying bacillus. vol. 10 (supplement 1):13-40, 2003 LEPROSY AND THE ELUSIVE As a result, the blood vessels are dilated and white corpuscles migrate to the site. The bacilli multiply in the cells. In some cells the bacilli lie in separate collections; in others they fill the whole cell body but never penetrate into the nucleus. He also identifies bacilli that break down into granules as ones that have degenerated. In his numerous attempts to cultivate the bacilli, he has managed only to attain granules. As the bacilli at first multiply in the cells, and the breaking down appears most definitely and freely when the cells are crammed full of bacilli, it is equally possible that it is the result of diminished nutrition, and as they break down more rapidly in the internal organs, it is also possible, indeed probable, that the higher temperature in these organs favors disintegration. As we have unfortunately not been able to cultivate the bacilli, it is at present impossible to form a conclusion. At all events, we regard the transformation into granules as a degeneration, and believe that the bacilli this altered are dead (Hansen et al., 1895, pp. 42-3). He also remarks that: “As we do not know the manner and method of the primary infection of the organ, we must devote our attention to the search for discoveries like those described above, and to the localization of the bacilli in general, in order to form an idea of the method of action of the bacilli” (ibid, p. 39). What sort of conceptual break do these observations indicate? Perhaps they simply indicate an intensification of the practices of observation. Hansen looked at the structure of the morbid changes in the same way as did Wilson (1867). Perhaps Hansen simply had a stronger microscope. Perhaps Hansen did not allow his looking to be so strongly influenced by the conceptual lens of earlier disease models, even though his looking is still informed by those models. He says that he was influenced by Darwin’s scientific research and reasoning “to set aside every preconceived opinion and to diagnose from every approach that might 22 LEPROSY AND THE ELUSIVE A virulent metaphor While Hansen identifies the bacillus and Carter emphasizes a modern scientific approach to research based on observation, the soil/seed metaphor serves to express an intensification of concern about the disease that was not only medical but also circulated in heated public debates. This represented a revivification of the metaphor so that it carried both new understandings of the disease, gestured towards the still unknown aspects of leprosy, and conveyed underlying fears and anxieties. The final section of the Report on Leprosy by the Royal College of Physicians includes an article by Erasmus Wilson (1867), ‘Observations on the true leprosy or elephantiasis, with cases.’ Erasmus Wilson’s case studies create a powerful impression of the dangers of transplantation in the colonies. The vivid images that they present may have fuelled the debates that followed. For Wilson, the predisposing cause of leprosy is “long residence in countries in which the disease is endemic’ or, alternatively, “birth in an infected country takes the place of long residence.” The period of latency may be months and even years. His case studies describe eighteen Europeans (and one native of Hindustan) who had all lived in either India, Ceylon, Mauritius, or the West Indies. Each emerges as a narrative of diseased bodies charted over time and categorized according to race, gender, age, pursuit, and predecessors. These cases (along with a number of others) were circulated and recirculated in the debates about the disease which took place after the release of the report and up until the turn of the century. All are Europeans, except one, and all from the colonies: a sixteen-year-old 23 JO ROBERTSON JO ROBERTSON boy, born in Ceylon and vaccinated, for smallpox, with attenuated bacteria from a native child; his older brother; a young man of seventeen years, born in Bombay; a 21-year-old male, born in Jamaica; a young woman who had been living in Mauritius; a 43- year-old captain in the Indian army; a sixty-year-old man “in the judicial service of India,” who had lived in the East for nearly twenty years; a captain in the Indian army; a sixty-year-old colonel who had lived in the West Indies; a 26-year-old wife of an officer in the Indian army; a 19-year-old Hindustani woman; a young medical officer in the Indian army who had originally contracted syphilis; “one of the chiefs of the Bengal medical establishment” (Wilson, 1867, p. A virulent metaphor 242), who had lived in India for forty years; and a merchant in Mauritius for 29 years. y The collective effect of the descriptions of the symptoms of these cases is of a metamorphosis in disposition as well as physical appearance — a degenerative descent in which they are poised on the boundary between what constitutes a human being and something else. A mother notes alterations in “the appearance of [a child’s] countenance” and a change in behavior: “He shunned amusements; was fond of sitting alone and secluding himself” (Wilson, 1867, pp. 235, 237, 238); the features of another developed changes which “gave an occasional gleam of savageness to his countenance;” another’s vital functions seem to slow down to the extent that she experienced “coldness of extremities … and a certain listlessness, heaviness, sleepiness, and indisposition for exertion of every kind.” Another was “dejected, listless, and melancholy,” unable to sleep at night and sitting for hours during the day “without occupation and without attempting to make any exertion.” Their faces and their skin were altered. The features were “spread out,” “enlarged and flattened.” The skin became covered in spots that changed from “beautiful pink” to “purple” and finally “dirty brown.” It thickened around the eyebrows, nose, lips, chin, and ears, giving the face “a frowning and dejected expression” (Wilson, 1867, p. 237). Facial hair fell out. In one case, the skin was “yellowish brown with a purplish almost livid blush” and “the brow was heavy and frowning, the eye sunken, anemic, and glistening, and the general expression of features listless and melancholic.” Another looked like a Satyr: His features were large and of a deep red-brown or copper color; the forehead was deeply wrinkled and studded with tubercles; two of the tubercles at the upper angles of the forehead resembling young horns; the brow was thickened, heavy, frowning and deprived of hair; the eyes suffused with redness. The voice was hoarse and sonorous (p. 239). Another begins to look like “a native”: “in his infancy he was somewhat darker in complexion than his brother and sister … but 24 História, Ciências, Saúde — Manguinhos, Rio de Janeiro LEPROSY AND THE ELUSIVE LEPROSY AND THE ELUSIVE during the last few years, and especially the last twelve months, has become swarthy, and at present is darker than a native of India” (p. 240). The ability to speak deteriorated. A virulent metaphor The hands and feet altered, the bones retracting so that the shape was lost. For example: “[He] had lost a phalanx from the little finger of one of his hands, the rest of the fingers were bent in different directions and the hands distorted. He was unable to use his hands and was incapable of walking” (p. 244). Evidently all Europeans, particularly those with a constitutional predisposition, at the outskirts of Empire were potentially in peril. Children, young men, young women, and old and distinguished men were not safe from becoming animal, becoming native, becoming afflicted with leprosy — and such threats of atavistic reversion and racial degeneration were embodied in the figure of the leper. By implication, the price to be paid for venturing away from ‘home’ was loss of the defining characteristics that gave one entry into society and established one’s social, racial, and imperial identity. The marks of leprosy heralded a lingering process of dying in which one’s vitality was lessened in agonizingly incremental degrees. Leprosy’s representation, as bringing about a metamorphosis in the bodies of colonizers, dramatically externalized anxieties about living in tropical climates and mixing with peoples of other races. Previously healthy families or those with a constitutional predisposition, upon exposure to the soil of the colonies, ran the risk of developing leprosy. Transplanted seed could ‘grow’ in unpredictable ways. vol. 10 (supplement 1):13-40, 2003 Dissemination In 1887, a case study by W. T. Gairdner, Professor of Medicine in the University of Glasgow, produced a sensation in medical circles, and seemed to contribute an irrefutable instance of the contagion of leprosy by inoculation (specifically vaccination) in a chain of infection which originated with “a native child” (Gairdner, 1887b, p. 1269). The story concerns a child brought to England by his parents with a referral from a doctor who had been one of Gairdner’s pupils. In consultation with another expert, it was concluded that the child had leprosy, and Gairdner was surprised that the referring doctor had not recognized it, knowing that the child had come from a region where the disease was endemic. Gairdner let the referring doctor know the diagnosis, and eventually received a reply from him indicating that he had already known that it was leprosy but had deliberately chosen not to tell the parents or Gairdner. Out of a reluctance to “have the credit of having discovered for the first time what a gentleman so much more familiar with the disease might 25 JO ROBERTSON JO ROBERTSON have been supposed to have overlooked,” Gairdner informed the parents that the referring doctor had known it to be leprosy all the time. After a number of years, Gairdner was called back to the now rapidly deteriorating child, where he learnt from the parents, who had had further contact with the referring doctor, the reason for his unaccountable reluctance to disclose the disease as leprosy. The referring doctor had made a terrible blunder: He had vaccinated his own boy with virus derived from a native child in a leprous family, and as I understood (though perhaps not definitely so stated) that leprosy had declared itself in the native child after the vaccination; and, further, that (using his own child as a vaccinifer) he had vaccinated our patient directly from him (Gairdner, 1887b, p. 1269). He had vaccinated his own boy with virus derived from a native child in a leprous family, and as I understood (though perhaps not definitely so stated) that leprosy had declared itself in the native child after the vaccination; and, further, that (using his own child as a vaccinifer) he had vaccinated our patient directly from him (Gairdner, 1887b, p. 1269). História, Ciências, Saúde — Manguinhos, Rio de Janeiro História, Ciências, Saúde — Manguinhos, Rio de Janeiro Dissemination He had “known,” not only that the child had leprosy but where it had come from — from his own child; that three children (the native child, the referring doctor’s child, and the child who had been presented to Gairdner) had the disease; and that two of them had contracted it at the hand of the colonial doctor. The referring doctor was now dead, but his child, now an orphan, was attending school in Britain.6 This presented Gairdner with ‘a difficult dilemma’ — should he do anything about this child, as a possible source of infection, and what would be the consequences for the child, in a foreign land? He consulted other expert medical practitioners, who reassured him that the child did not present a danger to other children, but knowing one of the medical officers at the school, he let him know of “the extraordinary circumstances” (ibid., p. 1270). As a result, the child was “sent for and privately examined” and “beyond all doubt, considered to be a case of leprosy.” The medical officers then decided not to “sound the alarm” so as to avoid disturbing the boy’s education. But some time later, Gairdner was called to the school by the school authorities and, because of an outbreak of contagious eczema and a deterioration in the general health of the child, “it was no longer expedient that he should remain at the school” (ibid). The child’s guardian was informed, and although the child was suffering from “a mild type” of the disease, and there were no “breaches” on the surface of the skin and no discharge; and although Dr. Anderson, who supplied the other opinion, was certain that he did not represent a danger to the other children at the school, Gairdner “did not feel able to give an unqualified assent” (ibid.) to that opinion, and the child’s education at that school came to an end. The responses to this ‘confession’, as they appeared in the British Medical Journal from June to November of 1887, were instantaneous, sustained, and conflicting. Dissemination The Acting Surgeon General from Trinidad wanted to know more details about the case and was rather skeptical: were the parents of the child, who was first vaccinated, European?; if they were not, then did they have any taint of leprosy?; was blood inoculated or only lymph?; was it possible to inoculate a person 26 LEPROSY AND THE ELUSIVE with leprosy from the lymph? (Pasley, 1887, p. 270). Gairdner (1887a, p. 799) responded to Pasley’s request for information with a further profession of reluctance and dutiful responsiveness, stating that he had simply reported what he had seen. But the result was that John Hillis (1887, p. 1022), the Late Medical Superintendent of the British Guiana Leper Asylums, wrote, calling for the College to reconsider their 1867 report on the basis that “much light has been thrown on the pathology of the disease.” One reader commented that the whole medical profession owed a “deep debt of gratitude” to Gairdner “for his simple and clear statements concerning the communicability of leprosy by inoculation” (Jelly, 1887, p. 176). In contrast, the cautious Beaven Rake (1887a, p. 646), the Medical Superintendent of the Trinidad Leper Asylum, assembled a summary of the case for and against communicability and hereditary transmission, arguing that “no one knows what bacteriology may do for us in the future,” but the matter was far from “set at rest.” This story galvanized the medical profession in Britain and in the colonies because it seemed to present evidence of transmission by inoculation, specifically by vaccination against smallpox. In addition, its power was contained in the image of double penetration that vaccination with the bacillus presented — unwitting contamination with an invading micro-organism by Western lancet. Did it encapsulate something of the compromised position that the colonizer found himself in? His penetration, by vaccination, of the black skin was responsible for the eventual transfer of an invading bacillus into the young body of his own son and heir: the sins of the fathers visited on the next generation, the very process of degeneration expressed by Nordau (1968), and, at the same time, a concrete embodiment of the supposed effects of miscegenation. The seed/soil metaphor resonated. vol. 10 (supplement 1):13-40, 2003 Colonial soil and Imperial alarm Discussions about leprosy and its contagiousness focused implicitly and explicitly upon its potential to stage a ‘return’ commensurate with its activity in Europe in the past. These medical and popular debates sharpened in focus until they became debates about how to contain the contaminating agents at their point of origin: segregation in the colonies became the issue. Editorials in the British Medical Journal in November 1887 expressed what must have been a growing concern about the threat that the disease was coming to pose. The editorials opposed, on the one hand, the views of those who argued that any proof of infection, however isolated, was sufficient cause for alarm, to those who, in a leading article in the Times newspaper, supported the Report on Leprosy by the Royal College of Physicians that the disease was no more contagious than syphilis, and compulsory detention was unnecessary.7 27 vol. 10 (supplement 1):13-40, 2003 JO ROBERTSON JO ROBERTSON The evidence of bacterial activity was drawn on to support what was considered to be justifiable concern: the discovery of the bacillus; the proven connection between the bacillus and the disease; the proof that, whatever part of the world the diseased body is discovered in, the bacillus is present; and the presence of the bacillus in dead bodies were all sufficient evidence to conclude that “if it is the human body which, living or dead, harbors the parasite which causes leprosy, it ought to be accepted as a matter of common prudence that healthy persons should avoid as far as possible contact with lepers living or dead” (Brit. Med. Journ., 1887, p. 1056). Questions of the “liberty of the subject” were considered subordinate to the importance of protecting the healthy and possibly bringing an end to the disease.8 The editorial is thus positioned between the concern of the alarmists and the optimism of the Times by suggesting that the number of people with leprosy who had entered England had been underestimated. It concluded that “without sounding a note of alarm, or considering that there is any occasion at present for compulsory measures in England, we are yet unable to consider the presence of lepers … as being absolutely free from danger” (Brit. Med. Journ., 1887, p. 1056). História, Ciências, Saúde — Manguinhos, Rio de Janeiro História, Ciências, Saúde — Manguinhos, Rio de Janeiro Colonial soil and Imperial alarm Another editorial in the same month noted that the question of the contagiousness of leprosy was a question uppermost in the thoughts of those in the medical establishment and in the Government. It reassured its readers that the prevalence of the disease amongst populations that are “under the care of the British Government” was being noted. g In 1889, H. P. Wright, who had already written of his concerns in the Times, published Leprosy an imperial danger, intensifying the attack on the 1867 Report on Leprosy. Wright personified it, demonized it, expressed its trajectory through metaphors of invasion, and, most significantly of all, equated the individual suffering from the disease with the disease itself — the leper as a breeding ground for leprosy became the disease: “In leper lands, that which produces leprosy is not the soil, as in malaria; nor water, as with so many infectious maladies; nor decaying food; nor destitution, as in lathyrism, pellagra, &c. It is the leper” (Wright, 1889, pp. 15, 37, 31, 12, 99, 86, 116, 122, 16). He suggested that lepers might “fertilize” the soil with “their bacilli and spores,” contaminating a district “for a period more or less lengthy.” Consequently, if a person lived where lepers lived, even if they did not come into “close proximity,” there was always the possibility that “you may be attacked by the disease, and that in a very short period.” Eventually, in Wright’s rhetoric, an attack from the disease leprosy becomes a “leper attack.” The disease was also given demonical dimensions. It “manifests itself;” it is an evil that spreads with terrifying rapidity to be stamped out; it is a foul disease; “a frightful scourge ever threatening, and slowly advancing;” and it “threatens to become the scourge of the whole earth.” Its progress throughout 28 LEPROSY AND THE ELUSIVE LEPROSY AND THE ELUSIVE history was figured as the “rapid propagation of a scourge,” albeit an arbitrary one: sometimes moving slowly, sometimes with “a fearful rapidity,” other times with a “primitive intensity.” It invaded, attacked, abounded, prevailed, and ravaged. Most frighteningly, it was immortal: “It is ever alive, ever reviving, threatening without cessation all who approach its haunts.” pp It was communicated between “races,” and was a threat to the “white races.” Any country “which allows itself to be freely visited by a race infected with the malady” will itself be affected (Wright, 1889, pp. Colonial soil and Imperial alarm 10 (supplement 1):13-40, 2003 JO ROBERTSON JO ROBERTSON JO ROBERTSON In their attempts to reassure, the editors of the British Medical Journal constantly reiterated that “leprosy is rarely seen in this country;” “cases of leprosy in this country are very uncommon;” “there is no evidence that the disease spreads by contagion in England;” “we are satisfied that there is no cause for alarm;” “we are satisfied that on the part of the general public there is no reason for fear or anxiety” (ibid, p. 722). In support of this editorial, the statistics of cases presented to the Dermatological Society in the United Kingdom were published in the same issue of the Journal (p. 734). These efforts must not have defused public concern because a further editorial in June suggested that “the leprosy question is becoming one of the questions of the day” (Brit. Med. Journ., 1889a, p. 1364). It welcomed public discussion in the hope that attention to “this great pest” would result in convincing governments that the disease was contagious and so lead to “enforcing compulsory segregation.” Subsequent concern about the disease became increasingly focused on leprosy in the colonies. It began to be monitored with increasing attention and an eye to the possibility of its “coming home.” Lepers in India were reported as uncontrolled and uncontrollably spreading germs by sitting on iron railings outside a school attended by European children, selling fruit, and contaminating the wells of the city. They were depicted as interchangeable with the bacteria: “The Principal of St Xavier’s College stated that the lepers rubbed their sores against the iron railings surrounding the Elphinstone High School, and that the boys afterwards sat upon them” (Brit. Med. Journ., 1889b, p. 1261). There was a call for additional powers so that the Health Department could “deal effectively with the evil” (ibid.) and a suggestion made that police powers could also be increased. A letter to the Journal in June summarized the spirit of the times: the 1867 Report was “dangerous and full of false conclusions” and as a result “we are now threatened with it at home,” but “timely preventative measures in our Indian and Colonial possessions” will take care of the problem. “If we legislate in India and in the colonies, enough will be done; we shall check the disorder at the spring head” (Simms, 1889, p. 1491). Colonial soil and Imperial alarm 5, 13, 14, 37, 39-40, 55, 92, 93); some were more ready to receive it than others: the yellow and black races were more susceptible than the white; although some races presented an “aptitude for maturing the leprous agent,” none “can claim absolute immunity;” it was caught from colored men and slaves who had been given responsibility for caring for one’s children. He argued that it spread “wherever an infected race” was “brought into contact under favorable conditions with a non-infected one.” The invasion by leprosy and an invasion by another “race” become indistinguishable, particularly where the Chinese are concerned: “The invasion of a country by leprosy has always coincided with the introduction of lepers into that country; and races which have avoided intercourse with leprous people have remained intact.” Most importantly, Wright was explicit about the potential threat that leprosy posed to England. The disease, he predicted, “will ruthlessly invade our colonies” and again become a “common scourge throughout Europe.” This concern was exacerbated in 1889 and 1890 by the unfortunate conjunction of a series of events: the death of the well-known Catholic priest, Father Damien, in the leper colony at Molokai, Hawaii; the discovery of leprosy in an Irishman who had never been out of the country (Hawtrey Benson, 1889, p. 860); an experiment upon a condemned criminal, Keanu, by Dr. Arning, in the Sandwich Isles;9 and British and American alarm at the discovery of a leprous Swedish immigrant who had crossed the Atlantic. g A flurry of attention was concentrated on the potential for an outbreak in Great Britain. An editorial in the British Medical Journal (1889) at the end of March, entitled ‘Leprosy in the United Kingdom,’ seeking to allay alarm, conceded with some justification that the subject had come to preoccupy both medical discussion and “the public mind.” It explained how the medical mind had been impressed with the discovery of the leprosy bacillus, with Arning’s experiment with Keanu, and how the popular imagination had been riveted by the death of Damien: “For these and other reasons the subject of leprosy has recently cropped up from time to time in magazines and newspapers, in addition to being a subject of discussion in medical journals” (Brit. Med. Journ., 1889c, p. 721). 29 vol. História, Ciências, Saúde — Manguinhos, Rio de Janeiro Attempted cultivation in India: the 1891 Leprosy Commission In 1891, the Leprosy Commission in India set about investigating the disease, and for a brief moment of triumph thought that they had isolated the bacterium outside the body.10 The Leprosy Commission had grown out of the National Leprosy Fund instituted on the death of Father Damien. Its first meeting was held on June 17, 1889, and the Prince of Wales was president. Its second meeting was held as a subscription dinner at the Hotel Metropole, London (Tebb, 1893, p. 295). The Prince of Wales’ speech was recorded in the Times the next day. He described the “wide prevalence of leprosy in the Indian Empire as an undoubted fact” and also expressed the general impression that “the disease is increasing in India, as well as in many of our colonies” (Times, Jan. 14, 1890, p. 7). The Prince of Wales’ address was followed by one from Sir Andrew Clarke, the president of the Royal College of Physicians. He described the increase in the disease in frightening terms: “The evidence was conclusive that not only did leprosy exist in larger measures than in recent years, but that new germ centers were springing up in various quarters and the old centers were widening, and before England and the civilized world there was looming a condition of affairs which might, by growth, threaten civilization” (Times, Jan. 14, 1890, p. 7). Colonial India was imagined as the soil in which the germ was multiplying, and as such an appropriate site for its investigation and attempted cultivation. The Fund appointed a Commission of three (from the Royal College of Physicians, the Royal College of Surgeons, and the General Committee of the National Leprosy Fund) with two representatives from the India Auxiliary Committee to investigate the disease in India.11 They left England on October 23, 1890, finished their research in late 1891, and prepared their report. They had been sent to do what the 1867 Report had failed to do, but their efforts were no less free from censure and controversy. According to Tebb (1893, p. 298), the publication of their report was held up on the excuse that the statistics on leprosy in India had not yet been completed, but, in reality, because their conclusions were “strongly objected to.”12 One facet of this extremely comprehensive report involved research on the bacillus. Colonial soil and Imperial alarm One study presented sixteen cases which it used to develop an argument for “a system of precaution, of segregation, … regulations influenced and dictated by a spirit of Christian charity,” and a “duty imperative upon England” to stamp out the disease (Donnet, 1889, pp. 301-5). The push for legislation intensified, and South Africa and New South Wales enacted laws to detain those diagnosed with the disease. The Journal (1890a, p. 1047) was full of praise for the measures enacted in these colonies: “the public of England would be making a very great mistake if they supposed, because they heard of isolated cases of leprosy in distant parts of the colony, that the matter was 30 LEPROSY AND THE ELUSIVE LEPROSY AND THE ELUSIVE not being dealt with by the Government of the colony.” In fact, the article maintained that in no part of the world were such responsible measures being taken. Prompted by the discovery of several Europeans with the disease, a Leprosy Bill was passed in New South Wales with “promptitude and uncompromising thoroughness” on November 20, 1890 (Brit. Med. Journ., 1891, p. 779). vol. 10 (supplement 1):13-40, 2003 Attempted cultivation in India: the 1891 Leprosy Commission The members of the Commission carried out a series of investigations in two separate teams. Rake, Buckmaster, and Thomson 31 JO ROBERTSON JO ROBERTSON conducted bacteriological investigations at Almora, while Barclay and Kanthack did the same at Sabathu. They converged on the laboratory at Simla. By June 6, the Journal reported the “Apparently successful cultivation of the bacillus leprae” by A.A. Kanthack and Surgeon-Major Barclay (members of the Leprosy Commission), a preliminary communication in which they triumphantly announced: “We have succeeded in isolating and cultivating from leprous tissues, removed under all aseptic precautions from patients intra vitam, a bacillus which may fairly claim to be the true bacillus of leprosy” (Kanthack et al., 1891b, p. 1222). In this article, they describe obtaining free bacilli which they claimed were morphologically identical with the bacilli of leprosy and which could be stained by the Koch-Ehrlich method. Some qualification about the characteristics of the bacillus had to be made. It differed from the leprosy bacillus found in the tissues because it absorbed the aqueous methyl blue dye more rapidly and did not retain the fuchsine staining as tenaciously, but they were optimistic that they would be able to produce bacillus that would be equally resistant as that obtained from tissue samples, and they sent their result to several continental laboratories for criticism (ibid, p. 1223). Then, on June 20, the Lancet (1891, p. 1397) also announced that Rake and Buckmaster had succeeded in cultivating the leprosy bacillus in serum. In the meantime, Kanthack and Barclay (1891a, p. 331) were looking forward to a successful animal experiment in order to substantiate their claims. Two of the appendices of the report detail the laboratory investigations. The investigators examined the distribution of the bacillus within bodily fluids (including blood, blisters, what they termed juice from tubercles, ulcers, and nerves); secretions (such as saliva); and excreta. They looked for the bacillus in the soil, water, fish, and flies. They conducted vaccination experiments, attempts at cultivation, and experimented with animals to investigate transmission. In order to show that their cultivated bacillus was indeed M. leprae, they needed to be able to demonstrate its effect, and their best chance of doing this was using animal experimentation. Frustratingly, this was not achieved. Their ultimate conclusions in the report were dramatically modified from those that had been reported in the medical journals. História, Ciências, Saúde — Manguinhos, Rio de Janeiro Twentieth century attempts at cultivation Seventy-two years later, in ‘A review of leprosy research’ given by Dr. R.J.W. Rees in March, 1963, to the Medical Research Council on the contribution made by the Council (either at the National Institute for Medical Research or under the auspices of the Leprosy Committee), he states that: The continuing failure to culture the human leprosy bacillus in vitro or to transmit the infection to experimental animals as a routine procedure has seriously restricted the scope of both fundamental and applied research in leprosy. Even the simplest, though basically essential, laboratory techniques for studying an infectious disease cannot be used in leprosy. For example, it is impossible to prepare a specific vaccine against leprosy or to test in vitro for chemotherapeutically active drugs. As a result progress of research in leprosy has been limited.13 In some ways nothing much had changed. The bacillus had still not been amenable to cultivation, nor had it been transmitted to experimental animals. Fascinatingly though, the animal model for leprosy research was just beginning to take off: experiment with M. lepraemurium in 1958, the measured growth of bacilli in the mouse footpad in 1960, and harvesting of M. leprae from the nine-banded armadillo from 1971. It might be argued that while M. leprae continued to be as elusive as ever, analogies with the functioning of the bacillus in a few extremely specific animals enabled scientists to outwit it. How, in this era of investigation that draws on animal models, the analogies between natural growth and the mysteries of leprosy were reinvented or discarded remains to be examined. Attempted cultivation in India: the 1891 Leprosy Commission Attempts to determine the pathogenic character of the bacilli they had cultivated were unsuccessful and so “it was impossible to affirm with certainty that the cultivation of the bacillus had been accomplished.” So they were forced to confess: From the numerous recorded experiments of observers in various parts of the world, and from our own attempts at inoculation, we consider it extremely doubtful whether a true leprosy, such as we recognize clinically, can be produced in animals. In absence of this step, Koch’s postulates remain unfulfilled, and it is impossible to say whether the cultures we obtained from leprous tissues and fluids are growths of leprosy bacilli or not (Leprosy in India, 1893, p. 439). 32 História, Ciências, Saúde — Manguinhos, Rio de Janeiro LEPROSY AND THE ELUSIVE Ironically, the colonial soil of India had not been conducive to the cultivation of the bacillus. vol. 10 (supplement 1):13-40, 2003 Mapping the genome of an uncultivated bacillus A search of the genome can be conducted by gene name, region in the genome, gene function, by DNA or protein patterns, or by a search of the DNA sequence or protein sequence. Results are presented as a list or a drawing, and the DNA or protein sequence of a single gene can be viewed or downloaded (Jones, 2001, pp. 470-7). The whole sequence of the chromosome is available. Its density, length, number of genes, pseudogenes, and other respective genetic components are enumerated. In the era of the genome, it would seem that the botanical metaphor has given way to one in which information is revealed through processes of translation and decoding. Firstly, it is possible to describe something of the evolutionary changes that have taken place. M. leprae has undergone a loss of genes and a subsequent loss of ability to respond to different environments. It is described as a decaying genome: one that has undergone considerable downsizing during its evolution. It has a trajectory and an ancestry — a former self that was more complex. Less than half of the genome contains functional genes. Its “immediate ancestor may have already undergone reductive evolution and … a single clone then expanded and … disseminated globally” (Eiglmeier et al., 2001a, p. 390). As well as a history, it is imagined as having a greater degree of agency. Adaptation has been selective and self- interested. Chromosomal rearrangements and gene deletions and duplications have had a profound effect on the biology of M. leprae and in turn on leprosy itself (Cole et al., 2001, p. 459). Yet this decay is evidence of a reduction in redundant functions so that the major repair pathways are still intact (Dawes et al., 2001, p. 411). Mapping makes it possible to answer some questions. M. leprae strains from different origins exhibit no obvious, important genome diversity (Eiglmeier et al., 2001b, p. 465). The genome still retains its full complement of heat shock proteins, explaining why, given that its optimal growth temperature is 32oC, it multiplies in the extremities of the body. It is possible to determine the uniqueness of the metabolic pathways of the genome, as well as compare them to that of M. tuberculosis, indicating that the survival of M. leprae is dependent on a specialized niche. Mapping the genome of an uncultivated bacillus The mapping of the genome of M. leprae has produced another dimension for understanding M. leprae14 so that the logic of the botanical metaphor may no longer be necessary in the face of the powerful rhetorical repertoire commanded by DNA. Mapping and decoding DNA enables access to the “language of life,” and genomes are variously described as “the book of life,” “the code of codes,” and as “blueprints,” “information,” “lexicons,” and “encyclopedias” (Bacsik, 2002, p. 2). DNA is understood as “a biochemical language that we are learning to read by learning to write” (ibid.). Its mysteries can be disentangled by identification and matching. The schematic language of the gene is written in layers and has only to be read. M. leprae can now be conceptualized as a series of genes that can be grouped and described, 33 JO ROBERTSON JO ROBERTSON often by analogy with those identified in another genome, such as M. tuberculosis. Technologies other than the microscope are available to view it. Instead of a sample of tissue in serum under a microscope, it now also exists as a genome database that can be explored via Leproma, a genome browser located on the web at http://genolist.pasteur.fr/ leproma. A search of the genome can be conducted by gene name, region in the genome, gene function, by DNA or protein patterns, or by a search of the DNA sequence or protein sequence. Results are presented as a list or a drawing, and the DNA or protein sequence of a single gene can be viewed or downloaded (Jones, 2001, pp. 470-7). The whole sequence of the chromosome is available. Its density, length, number of genes, pseudogenes, and other respective genetic components are enumerated. In the era of the genome, it would seem that the botanical metaphor has given way to one in which information is revealed through processes of translation and decoding. often by analogy with those identified in another genome, such as M. tuberculosis. Technologies other than the microscope are available to view it. Instead of a sample of tissue in serum under a microscope, it now also exists as a genome database that can be explored via Leproma, a genome browser located on the web at http://genolist.pasteur.fr/ leproma. História, Ciências, Saúde — Manguinhos, Rio de Janeiro História, Ciências, Saúde — Manguinhos, Rio de Janeiro Mapping the genome of an uncultivated bacillus It has extremely reduced genes for dealing with respiration and an oxygen-rich environment, and hence exists successfully in an intracellular environment, which has relatively constant conditions. The pathogenicity of the mycobacterium depends on its ability to survive in the macrophage or in the Schwann cell (Eiglmeier et al., 2001a, p. 395). As such it is characterized as “extremely specialized” and it is “irreversibly committed … to a lifestyle characterized by slow growth 34 LEPROSY AND THE ELUSIVE LEPROSY AND THE ELUSIVE and necessarily slow central metabolism” (Wheeler, 2001, pp. 402-3). The basis for resistance to dapsone and rifampicin is able to be understood (Grosset et al., 2001, pp. 429, 431). In addition, the susceptibility of M. leprae to other drugs can be determined. Yet some questions are not answered. In considering the metabolic pathways that are retained by the mycobacterium, Wheeler (2001, pp. 405, 406) comments that “there is something unusual about this whole area of purine and pyrimidine metabolism in M. leprae. Why should the biosynthetic pathways have been retained in such a host- dependent pathogen?” He speculates that “maybe it is part of the mechanism that allows the leprosy bacillus to survive and grow within the rather metabolically inert Schwann cells.” Wheeler also speculates about the inability to grow the bacillus in vitro: Why cannot M. leprae be grown axenically; do the lesions in energy metabolism only allow interrupted growth when conditions are just right in the host? Are media too toxic, at least in aerobic conditions? With a massive loss of regulatory functions have those that would allow M. leprae to adapt to axenic culture been lost? One of the most troubling questions associated with M. leprae in the nineteenth and twentieth centuries, arising out of the newly developed sciences of microbiology and bacteriology, has given rise to many other questions in the twenty-first century. The question of in vitro cultivation is now a question about the metabolism of energy, appropriate media, and evolutionary adaptation. Interestingly, the rhetoric of the genome, that of writing and reading, of decoding, translation, and making all clear, is being used to explain the bacillus as it was understood and continues to be understood in botanical terms. The ‘new’ questions are still as much about the right ‘soil’ or appropriate conditions to facilitate growth. NOTES 1 Hansen published his findings ‘Causes of leprosy’ as part of his annual report for 1873 to the Norwegian Medical Society. He says that he identified the bacillus in 1871 (Hansen et al., 1895, p. 31). 1 Hansen published his findings ‘Causes of leprosy’ as part of his annual report for 1873 to the Norwegian Medical Socie He says that he identified the bacillus in 1871 (Hansen et al., 1895, p. 31). 2 “The various clinical manifestations in leprosy are the results of the variations in the tissue response of the host to the presence of leprosy bacilli in the body. In other words, they are determined by the host-parasite relationship” (Dharmendra, cited in Hastings, 1985, p. 88). 3 A chapter in Hastings (1985) is devoted to shifts in classification, highlighting a struggle between the need for both clinical and histological classifications. The extent of this ongoing reclassification process can only be appreciated by tracing some of its mutations. 3 A chapter in Hastings (1985) is devoted to shifts in classification, highlighting a struggle between the need for both clinical and histological classifications. The extent of this ongoing reclassification process can only be appreciated by tracing some of its mutations. 4 In leprosy, both the reference points for measuring the incubation period — the time of infection and the time of onset of disease — are difficult to define; the former because of the lack of adequate immunological tools and the latter because of the insidious nature of the onset of leprosy (WHO web pages http://www.who.int/lep/disease/disease.htm). 5 A history of attempts to cultivate the bacillus is outlined in Ryan and McDougall (1988). 6 The copy of the Brit. Med. Journ. from which I am summarizing this story has the name of the public school that the child was attending pencilled in the margin. 7 The editorial indicated that M. Besnier, the physician of the Hopital Saint-Louis, had delivered an address at the Academie de Medecine on October 11 on the ‘Nature, origin, and propagation of leprosy,’ and Archdeacon Wright had addressed a letter to the Times on November 8 entitled ‘The spread of leprosy.’ Both expressed concern and alarm that the disease was contagious. 8 A number of studies of leprosy in the Middle Ages in Europe and Great Britain were produced that supported this argument. Mapping the genome of an uncultivated bacillus Ironically, while leprosy research has for such a long time drawn its logic from a view of the world in which a seed and soil metaphor expressed many different aspects of the activity of the disease, in spite of the newly available technology of elucidation, the bacillus itself continues to be unreceptive to cultivation. 35 35 vol. 10 (supplement 1):13-40, 2003 JO ROBERTSON 13 Public Record Office: FD7/1190. Medical Research Council: Tropical Medicine Research Board: special subject ‘nvitation to Dr. R.J.W. Rees to address noon session 8th March 1963.’ Rees then goes on to summarize the leprosy program at the institute. One facet of this was the use of murine leprosy as a model for human leprosy. This work is conducted on the basis of an extended analogy between murine leprosy and human leprosy. Both were chronic infections caused by acid-fast bacilli existing predominantly as intra-cellular parasites and causing surprisingly little damage to the host cell. Projects included attempts to measure the viability of murine leprosy bacilli, its rate of multiplication in tissue culture, and the application of the results of studies on murine leprosy to human leprosy in collaboration with the Research Unit (MRC/Malayan Government), Sungei Buloh leprosarium, Malaya. 11 Beaven Rake, George Buckmaster, and Alfred Kanthack were appointed from the College, the General Committee, and the Surgeons, respectively. The appointees from India were Surgeon-General Barclay and Deputy Sanitary Commissioner Surgeon-General S. J. Thompson. História, Ciências, Saúde — Manguinhos, Rio de Janeiro NOTES The waning of the disease in the Middle Ages was attributed to the natural horror with which the general population responded to the disease, so that the afflicted were inevitably shunned: for example, James Y. Simpson (1842, 1841); in 1895, as part of a collection of Prize Winning Essays published by the Sydenham Society, George Newman wrote On the history of the decline and final extinction of leprosy as an endemic disease in the British Isles; and, demonstrating the longstanding concern with the disease, Charles A. Mercier wrote Leper houses and medieval hospitals, in 1915. 9 Dr. Arning presented a paper at the First Dermatological Congress in Prague, June 10-12, 1889, describing how he had obtained permission to suggest to a condemned criminal, Keanu, a choice between death by execution or becoming a subject in a medical experiment. Keanu cooperated with the latter option in September 1884 and was injected with leprous tissue. In December 1887, he showed unmistakable symptoms of the disease. In September 1888, he was diagnosed with fully developed leprosy. Arning concluded that the disease could be conveyed by inoculation, specifically vaccination (‘The inoculation of leprosy,’ in Brit. Med. Journ., pp. 90-1, Jan. 11, 1890). Subsequent medical reports in the Brit. Med. Journ. on April 19, 1890, p. 909 and pp. 917-8, revealed that members of his immediate family had already been exposed to the disease, and the case sank into oblivion. Most of the literature of the time that argued the case for contagion referred to this ethically suspect experiment for support. 10 Articles in the Brit. Med. Journ. trace the journey taken by the Commission. The Journal recorded this in the following articles: ‘The Leprosy Commission in India,’ Feb. 7, 1891, p. 296; ‘The Leprosy Commission in India,’ Feb. 28, 1891, p. 475; ‘The Leprosy Commission in India,’ May 9, 1891, p. 1031; ‘Leprosy Commission,’ May 23, 1891, p. 1137; ‘Apparently successful cultivation of the Bacillus Leprae,’ by A.A. Kanthack and Surgeon-Major Barclay (Members of the Leprosy Commission), Jun. 6, 1891, p. 1222; ‘Pure cultivation of the leprosy bacillus,’ Jun. 20, 1891, p. 1330; and ‘Cultivation of the leprosy bacillus in serum,’ Jun. 27, 1891, p. 1395. p The Lancet also traced the journey and experiments of the Commission in the following: ‘The Leprosy Commission,’ Feb. 7, 1891, p. 324; ‘The Leprosy Commission,’ Feb. 28, 1891, p. 500; ‘The bacillus of leprosy,’ Jun. LEPROSY AND THE ELUSIVE 14 The analysis of the first complete M. leprae cosmid sequence was conducted in 1993. The genome sequencing project was coordinated by Stewart Cole and Bart Barrell, and supported by the Association Française Raoul Follereau, ILEP, the Heiser Program for Research in Leprosy and Tuberculosis of The New York Community Trust, the World Health Organization, and the Institut Pasteur. Additional funding was provided by the Wellcome Trust: The complete genome sequence of the TN strain of Mycobacterium leprae comprises 3,268,203 bp, with a G+C content of 57.79%. The start of the sequence is the first base of the dnaA gene, close to the origin of replication. The TN strain was initially isolated from a patient in Tamil Nadu, India, then subsequently passaged in a nine-banded armadillo at the National Institute for Medical Research, at Mill Hill in London. DNA was prepared from bacteria isolated from the liver and used either to construct a cosmid library in Lorist6 or a whole-genome shotgun library in pUC18. For details of the cosmid library see K. Eiglmeier; N. Honoré; S.A.,Woods; B. Caudron; S.T., Cole. ‘Use of an ordered cosmid library to deduce the genomic organization of Mycobacterium leprae,’ Molecular Microbiology, 7:2, 1993, pp. 197-206. Information about and data from previously sequenced cosmids is still available (http://www sanger ac uk/Projects/ 14 The analysis of the first complete M. leprae cosmid sequence was conducted in 1993. The genome sequencing project was coordinated by Stewart Cole and Bart Barrell, and supported by the Association Française Raoul Follereau, ILEP, the Heiser Program for Research in Leprosy and Tuberculosis of The New York Community Trust, the World Health Organization, and the Institut Pasteur. Additional funding was provided by the Wellcome Trust: The complete genome sequence of the TN strain of Mycobacterium leprae comprises 3,268,203 bp, with a G+C content of 57.79%. The start of the sequence is the first base of the dnaA gene, close to the origin of replication. The TN strain was initially isolated from a patient in Tamil Nadu, India, then subsequently passaged in a nine-banded armadillo at the National Institute for Medical Research, at Mill Hill in London. DNA was prepared from bacteria isolated from the liver and used either to construct a cosmid library in Lorist6 or a whole-genome shotgun library in pUC18. For details of the cosmid library see K. Eiglmeier; N. Honoré; S.A.,Woods; B. Caudron; S.T., Cole. ‘Use of an ordered cosmid library to deduce the genomic organization of Mycobacterium leprae,’ Molecular Microbiology, 7:2, 1993, pp. 197-206. Information about, and data from, previously sequenced cosmids is still available (http://www.sanger.ac.uk/Projects/ NOTES 20, 1891, p. 1397; ‘The Leprosy Commission,’ Jun. 27, 1891, p. 1440; ‘The Leprosy Commission in India,’ Aug. 8, 1891, p. 303; ‘The Leprosy Commission,’ Aug. 29, 1891, p. 498; ‘The Leprosy Commission,’ Oct. 10, 1891, p. 827. The Lancet also traced the journey and experiments of the Commission in the following: ‘The Leprosy Commission,’ Feb. 7, 1891, p. 324; ‘The Leprosy Commission,’ Feb. 28, 1891, p. 500; ‘The bacillus of leprosy,’ Jun. 20, 1891, p. 1397; ‘The Leprosy Commission,’ Jun. 27, 1891, p. 1440; ‘The Leprosy Commission in India,’ Aug. 8, 1891, p. 303; ‘The Leprosy Commission,’ Aug. 29, 1891, p. 498; ‘The Leprosy Commission,’ Oct. 10, 1891, p. 827. 11 Beaven Rake, George Buckmaster, and Alfred Kanthack were appointed from the College, the General Committee, and the Surgeons, respectively. The appointees from India were Surgeon-General Barclay and Deputy Sanitary Commissioner Surgeon-General S. J. Thompson. 12 The reasons that the recommendations of the Leprosy Investigation Commission were objected to by the executive committee of the National Leprosy Fund are explained in Tebb, but also in Buckinghams (2002, pp. 174-8). 36 História, Ciências, Saúde — Manguinhos, Rio de Janeiro y p , gy, , , pp Information about, and data from, previously sequenced cosmids is still available (http://www.sanger.ac.uk/Projects/ M_leprae/cosmids.shtml and http://genolist.pasteur.fr/Leproma/help/project.html). História, Ciências, Saúde — Manguinhos, Rio de Janeiro BIBLIOGRAPHIC REFERENCES An account of the lazaretto in the Island of Madeira: with an inquiry into the various diseases called leprosy. London, Smith and Son. ‘Technologies of inscription: archival iterability and the semiotics of genomic language,’ unpublished doctorate, University of Florida. 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K. Lewis. Nordau, Max Degeneration. Lincoln/London, University of Nebraska Press. 1968 Newman, George On the history of the decline and final extinction of leprosy as an ende 1895 disease in the British Isles. London, Royal Historical Society. Pasley, C. Burgoyne ‘Communicability of leprosy.’ Brit. Med. Journ., pp. 270-1. July 30, 1887 Rake, Beaven. ‘The question of communicability and heredity of leprosy.’ Sept. 17, 1887a Brit. Med. Journ., pp. 646-7. Rake, Beaven. ‘Experimental investigations on leprosy.’ Brit. Med. Journ., pp. 275-6. Feb. 5, 1887b R T J d E l b O f d di l t d t Hawtrey Benson, J. ‘Leprosy in the United Kingdom.’ Brit. Med. Journ., p. 860. Apr. 13, 1889 Hillis, John ‘The spread of leprosy,’ Brit. Med. Journ., pp. 1022-3, 5 v. 5. 1887 Jelly, William ‘Communicability of leprosy.’ Brit. Med. Journ., p. 176. July 23, 1887 Jones, L; Moszer, I. and ‘Leproma: a Mycobacterium leprae genome browser.’ Cole, S. T. Leprosy Review, 72, pp. 470-7. 2001. Kanthack, A. A. and ‘Pure cultivation of the leprosy bacillus.’ Surgeon-Major Brit. Med. Journ., pp. 1330-1. Barclay, A. Jun. 20, 1891a Kanthack, A. A. and ‘Apparently successful cultivation of the Bacillus leprae.’ Surgeon-Major Brit. Med. Journ., pp. 1222-3. Barclay, A. Jun. História, Ciências, Saúde — Manguinhos, Rio de Janeiro LEPROSY AND THE ELUSIVE 6, 1891b Lambert, Agnes ‘Leprosy: present and past.’ 1884 Nineteenth Century, 90, pp. 210-27; 91, pp. 467-89. Lancet ‘The bacillus of leprosy,’ p. 1397. 1891 Leprosy in India: report Calcutta: Superintendent of Government Printing. of the Leprosy Commission in India 1890-91 1893 Mackenzie, Morrell ‘The dreadful revival of leprosy.’ 1890 Wood’s medical and surgical monographs, 5:3, pp. 603-27. Maxwell, James Observations on yaws and its influence in originating leprosy. 1839 Edinburgh, Maclachlan, Stewart. Mercier, Charles A. Leper houses and medieval hospitals. 1915 London, H. K. Lewis. Nordau, Max Degeneration. Lincoln/London, University of Nebraska Press. 1968 Newman, George On the history of the decline and final extinction of leprosy as an endemic 1895 disease in the British Isles. London, Royal Historical Society. Pasley, C. Burgoyne ‘Communicability of leprosy.’ Brit. Med. Journ., pp. 270-1. July 30, 1887 Rake, Beaven. ‘The question of communicability and heredity of leprosy.’ Sept. 17, 1887a Brit. Med. Journ., pp. 646-7. Rake, Beaven. ‘Experimental investigations on leprosy.’ Brit. Med. Journ., pp. 275-6. Feb. 5, 1887b Ryan Terence, J. and Essays on leprosy by Oxford medical students. Hawtrey Benson, J. ‘Leprosy in the United Kingdom.’ Brit. Med. Journ., p. 860. Apr. 13, 1889 Hillis, John ‘The spread of leprosy,’ Brit. Med. Journ., pp. 1022-3, 5 v. 5. 1887 Nordau, Max Degeneration. Lincoln/London, University of Nebraska Press. 1968 Newman, George On the history of the decline and final extinction of leprosy as an endemic 1895 disease in the British Isles. London, Royal Historical Society. Pasley, C. Burgoyne ‘Communicability of leprosy.’ Brit. Med. Journ., pp. 270-1. July 30, 1887 Rake, Beaven. ‘The question of communicability and heredity of leprosy.’ Sept. 17, 1887a Brit. Med. Journ., pp. 646-7. Rake, Beaven. ‘Experimental investigations on leprosy.’ Brit. Med. Journ., pp. 275-6. Feb. 5, 1887b ‘Experimental investigations on leprosy.’ Brit. Med. Journ., pp. 275-6. Rake, Beaven. Feb. 5, 1887b Rake, Beaven. Feb. 5, 1887b Ryan Terence, J. and Essays on leprosy by Oxford medical students. McDougall, A.C. (eds.) Oxford, Slade Hospital Department of Dermatology. 1988 McDougall, A.C. (eds.) Oxford, Slade Hospital Department of Dermatology. 1988 Simms, Frederick ‘Etiology of leprosy.’ Brit. Med. Journ., p. 1491. Jun. 29, 1889 39 vol. 10 (supplement 1):13-40, 2003 JO ROBERTSON Simpson, James Y. 1842 Tebb, William 1893 Times Jan. 7, 1890 Wheeler, Paul, R. 2001 Wilson, Erasmus 1867 Worboys, Michael 2000 Wright, H. P. 1889 Simpson, James Y. Submitted on February 2003. Approved on June 2003. LEPROSY AND THE ELUSIVE ‘Antiquarian notices of leprosy and leper hospitals in Scotland and England: 1842 parts I-III.’ The Edinburgh Med. and Surg. Journ., part I 56, pp. 301-30, 1841; part II 57, pp. 121-56, 1842; part III 57, pp. 394-429. Tebb, William The recrudescence of leprosy and its causation: a popular treatise. London, 1893 Swan Sonnenschein. Times ‘The National Leprosy Fund,’ p. 7. Jan. 7, 1890 Wheeler, Paul, R. ‘The microbial physiologist’s guide to the leprosy genome.’ 2001 Leprosy Review, 72, pp. 399-407. Wilson, Erasmus ‘Observations on the true leprosy or elephantiasis, with cases.’ In George 1867 Edward Eyre and William Spottiswood, Report on leprosy by the Royal College of Physicians prepared for and published by her Majesty’s Secretary of State for the Colonies, with an appendix. London, pp. 231-44. Worboys, Michael Spreading germs: disease theories and medical practice in Britain, 1865-1900. 2000 United Kingdom, Cambridge University Press. Wright, H. P. Leprosy an imperial danger. 1889 London, Churchill. ‘Antiquarian notices of leprosy and leper hospitals in Scotland and England: parts I-III.’ The Edinburgh Med. and Surg. Journ., part I 56, pp. 301-30, 1841; part II 57, pp. 121-56, 1842; part III 57, pp. 394-429. The recrudescence of leprosy and its causation: a popular treatise. London, Swan Sonnenschein. Tebb, William 1893 The recrudescence of leprosy and its causation: a popular treatise. London, Swan Sonnenschein. Times Jan. 7, 1890 ‘The microbial physiologist’s guide to the leprosy genome.’ Leprosy Review, 72, pp. 399-407. ‘Observations on the true leprosy or elephantiasis, with cases.’ In George Edward Eyre and William Spottiswood, Report on leprosy by the Royal College of Physicians prepared for and published by her Majesty’s Secretary of State for the Colonies, with an appendix. London, pp. 231-44. Spreading germs: disease theories and medical practice in Britain, 1865-1900. United Kingdom, Cambridge University Press. Leprosy an imperial danger. London, Churchill. Wheeler, Paul, R. 2001 Wilson, Erasmus 1867 Submitted on February 2003. Approved on June 2003. 40
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Alternative Fuels for Internal Combustion Engines
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Received: 20 May 2020; Accepted: 31 July 2020; Published: 6 August 2020 Abstract: The recent transport electrification trend is pushing governments to limit the future use of Internal Combustion Engines (ICEs). However, the rationale for this strong limitation is frequently not sufficiently addressed or justified. The problem does not seem to lie within the engines nor with the combustion by themselves but seemingly, rather with the rise in greenhouse gases (GHG), namely CO2, rejected to the atmosphere. However, it is frequent that the distinction between fossil CO2 and renewable CO2 production is not made, or even between CO2 emissions and pollutant emissions. The present revision paper discusses and introduces different alternative fuels that can be burned in IC Engines and would eliminate, or substantially reduce the emission of fossil CO2 into the atmosphere. These may be non-carbon fuels such as hydrogen or ammonia, or biofuels such as alcohols, ethers or esters, including synthetic fuels. There are also other types of fuels that may be used, such as those based on turpentine or even glycerin which could maintain ICEs as a valuable option for transportation. Keywords: biofuels; fuels; synthetic fuels; internal combustion engine; alternative fuels Review Alternative Fuels for Internal Combustion Engines Jorge Martins * and F. P. Brito MEtRICs, Universidade do Minho, 4710-057 Braga, Portugal; francisco@dem.uminho.pt * Correspondence: jmartins@dem.uminho.pt Jorge Martins * and F. P. Brito MEtRICs, Universidade do Minho, 4710-057 Braga, Portugal; francisco@dem.uminho.pt * Correspondence: jmartins@dem.uminho.pt www.mdpi.com/journal/energies   Received: 20 May 2020; Accepted: 31 July 2020; Published: 6 August 2020 Received: 20 May 2020; Accepted: 31 July 2020; Published: 6 August 2020 1. Introduction This is surprising, as it is accompanied by a growing acceptance of Plug-in Hybrid Electric Vehicles (PHEVs) that are able to run for several tens of kilometres without burning fossil fuel and therefore without producing pollution locally (in city centres, for example) [11]. But these vehicles rely on ICEs for some of their operation, namely in long trips, once the battery has been depleted. Under these conditions, the ICE usually runs on fossil fuel, therefore producing a non-negligible amount of pollutants and CO2 during operation [12]. Therefore, what policy-makers likely aim with the strong limitations to conventional vehicles is the reduction of the emission of fossil CO2 and the elimination of pollutant emissions within the city limits, which can be jointly achieved by the use of electrified (PHEV) vehicles [3] This seems to be the direction most OEMs (original equipment manufacturers) are taking. Volvo, for example, had vowed to stop developing “conventional” (non-electrified) vehicles from 2019, only hybrid and battery electric ones [13]. As of 2020 this has been mostly fulfilled although some of them are only “mild-hybrids” which have a bigger starter generator that displays some braking energy recovery and limited engine assist [14]. Similar commitments can be seen in other OEMs. Nevertheless, the announcements and proposed timeframes towards electrification or even ICE development abandonment are not always completely fulfilled [15,16]. However, GHG (greenhouse gases) reduction in the transport sector can hugely benefit from the use of ICE using CO2 neutral fuels [17]. This is particularly important in sectors, such as heavy-duty and aviation, where energy density plays an important role. A recent paper about future trends in transport [4] calculated that, with the present battery technology, electric passenger airplanes would require between 14 and 31 times its maximum take-offweight in batteries to store the energy that they usually carry as jet fuel. Also, the time for battery charging, using 80 Tesla superchargers would take over one day to fully recharge the battery equivalent of an Airbus 320 fuel tank. In terms of large vessels, the 170 GWh of energy that some these types of container ships carry in their tanks to power the engines would require batteries over five times their dead weight and would take years to recharge [4]. 1. Introduction Since the beginning of the industrial age, the burning of fossil fuels has been releasing to the atmosphere the carbon that was slowly sequestered more than 50 million years ago and stored as coal, oil, natural gas and other types of fossil fuels sources such as shale gas and shale oil. The combustion of these fuels produces, besides pollutants, carbon dioxide (CO2) which has a major effect of trapping the solar heat within the atmosphere (greenhouse effect), is expected to aim to the warming up of the Earth and the severity of the climate [1]. The general public and most policy makers perceive electric vehicles as a good alternative to fossil fuel-based transportation [1–3]. However, regarding emissions linked to vehicle use, electric vehicles are as green as the electricity they consume [4]. In a country such as Poland [5] or Australia, where most of the electricity is produced from coal, the running of electric vehicles will increase the contribution to the greenhouse effect comparing to the same car running in Norway or Brazil, where most of the electricity is produced from renewable energy sources [6]. Additionally, the evaluation of the sustainability of EVs must take into account the whole life cycle of the vehicle, including sensible issues such as the mining of the materials used in batteries and electric motors, as well as battery end-of-life [1,4,7,8]. Nonetheless, although it is a hot topic with a lot of promising developments [1], the detailed sustainability comparison between conventional and electric mobility is out of the scope of the present study and is only treated to provide some background. Now, a large number of countries, regions and cities are proposing the ban on so-called “conventional” vehicles within the next decades [9,10] Normally, what policy makers refer to when using this term are cars, buses or lorries propelled by an Internal Combustion Engine (ICE) that burns a fossil fuel. So, if one of these specifications is eliminated from a vehicle, it will no longer be “conventional”. This leaves three types of vehicles: electric, hybrid-electric and alternative-fuel burning. Energies 2020, 13, 4086; doi:10.3390/en13164086 www.mdpi.com/journal/energies 2 of 33 Energies 2020, 13, 4086 Nevertheless, some references to the future ruling out of so-called “polluting” vehicles are specified in terms of a ban on Internal Combustion (IC) Engines altogether, or in terms of a ban on vehicles that burn specific fuels such as diesel or gasoline [4]. 1. Introduction These estimations seem to be too pessimistic as they do not take into account the difference in efficiencies between electric motors and IC engines, which would cut in half the energy needed, but are high enough to illustrate the impracticality of electrification for large carriers to travel over very long distances unless highly disruptive changes take place in battery technology. But renewable fuels and/or biofuels may be good propositions for these types of transport. 2. Hybrid Vehicles Of course, the real-world emissions of vehicles are different from the reported emissions of new CO2 emissions may become negligible for cases where the long trips are less than 25% of the total mileage [12]. This was reported for the case where a compact and efficient range extender with two different operating conditions (one for efficiency another one for extra power) was implemented. Moreover, this configuration would allow low fuel consumption, fairly low complexity (comparatively to parallel hybrids) and low system cost [12]. E e gie , , O EE E IE o with two different operating conditions (one for efficiency another one for extra power) was implemented. Moreover, this configuration would allow low fuel consumption, fairly low complexity (comparatively to parallel hybrids) and low system cost [12]. Of course, the real-world emissions of vehicles are different from the reported emissions of new Of course, the real-world emissions of vehicles are different from the reported emissions of new vehicles. Real driving emissions tests tried to address this issue and are now part of the emissions certification process. They are done under charge depleting (CD—mostly electric mode) and charge sustaining (CS—driving with a stable low state of charge using mostly the engine) modes. The official fuel consumption and emissions is a weighted average between the CD and CS modes, with the weighting factor being the so-called “utility factor” (UF) [22]. The UF used in Europe is based on the driving statistics described by the SAE J2841 standard [23]. But these tests are made to new model cars, not to cars being currently driven in roads. There is a loophole in this certification because some users will not use plug-in capability of the vehicle as often as desirable, relying excessively on the charge sustaining mode. Of course, to attain a more realistic measure of the emissions of fleet vehicles, the data mining of this information would be required in these vehicles, but this is still not the case. However, as long as there is an economic incentive in terms of energy cost in order to use electricity, it seems that a hybrid architecture would be well suited for a gradual transition towards full BEV mobility once their challenges have been overcome. Now, the sustainability of hybrid vehicles could be further improved by the use of fuels with lower GHG and pollutant emissions, as discussed in continuation. vehicles. 3. Fuels 3. Fuels They have a very high energy density in terms of mass and volume, enabling the vehicles to have an enormous range. Gaseous fuels require y If a fuel contains oxygen or nitrogen, as these elements do not burn, its HV is lower than others composed just by carbon and hydrogen. In fact, when an alcohol (composed of C, H and O) burns, the oxygen atoms present in the burned gases are in the form of CO2 or H2O, mostly the latter [23,24] Liquid fuels are more appropriate for vehicle propulsion. They have a very high energy density in terms of mass and volume enabling the vehicles to have an enormous range Gaseous fuels require If a fuel contains oxygen or nitrogen, as these elements do not burn, its HV is lower than others composed just by carbon and hydrogen. In fact, when an alcohol (composed of C, H and O) burns, the oxygen atoms present in the burned gases are in the form of CO2 or H2O, mostly the latter [23,24]. Liquid fuels are more appropriate for vehicle propulsion. They have a very high energy density in terms of mass and volume, enabling the vehicles to have an enormous range. Gaseous fuels require pressurized tanks and have a much lower energy density, resulting in much larger and heavier tanks for the same amount of stored energy (Figure 1, [25]). y If a fuel contains oxygen or nitrogen, as these elements do not burn, its HV is lower than others composed just by carbon and hydrogen. In fact, when an alcohol (composed of C, H and O) burns, the oxygen atoms present in the burned gases are in the form of CO2 or H2O, mostly the latter [23,24] Liquid fuels are more appropriate for vehicle propulsion. They have a very high energy density in terms of mass and volume, enabling the vehicles to have an enormous range. Gaseous fuels require pressurized tanks and have a much lower energy density, resulting in much larger and heavier tanks for the same amount of stored energy (Figure 1, [25]). Liquid fuels are more appropriate for vehicle propulsion. They have a very high energy density in terms of mass and volume, enabling the vehicles to have an enormous range. 2. Hybrid Vehicles Real driving emissions tests tried to address this issue and are now part of the emissions certification process. They are done under charge depleting (CD—mostly electric mode) and charge sustaining (CS—driving with a stable low state of charge using mostly the engine) modes. The official fuel consumption and emissions is a weighted average between the CD and CS modes, with the weighting factor being the so-called “utility factor” (UF) [22]. The UF used in Europe is based on the driving statistics described by the SAE J2841 standard [23]. But these tests are made to new model cars, not to cars being currently driven in roads. There is a loophole in this certification because some users will not use plug-in capability of the vehicle as often as desirable, relying excessively on the charge sustaining mode. Of course, to attain a more realistic measure of the emissions of fleet vehicles, the data mining of this information would be required in these vehicles, but this is still not the case. However, as long as there is an economic incentive in terms of energy cost in order to use electricity, it seems that a hybrid architecture would be well suited for a gradual transition towards full BEV mobility once their challenges have been overcome. Now, the sustainability of hybrid vehicles could be further improved by the use of fuels with lower GHG and pollutant emissions, as discussed in continuation. 3. Fuels 3. Fuels Fuels for IC engines (and fuel cells) are usually a combination of hydrogen (H) and carbon (C) atoms, but occasionally the fuel may also have other elements such as oxygen (O) or nitrogen (N). Fossil fuels (such as petrol or diesel) are, usually, a mixture of different components (hydrocarbons) composed of H and C. Each component has its own physical properties, such as density, boiling temperature and heating value (HV). One of the problems of IC engines is the very high standard for exhaust emissions which requires expensive and bulky after-treatment of the exhaust gases, which also reduces the fuel efficiency of the vehicle. Fuels for IC engines (and fuel cells) are usually a combination of hydrogen (H) and carbon (C) atoms, but occasionally the fuel may also have other elements such as oxygen (O) or nitrogen (N). Fossil fuels (such as petrol or diesel) are, usually, a mixture of different components (hydrocarbons) composed of H and C. Each component has its own physical properties, such as density, boiling temperature and heating value (HV). One of the problems of IC engines is the very high standard for exhaust emissions which requires expensive and bulky after-treatment of the exhaust gases, which also reduces the fuel efficiency of the vehicle. y If a fuel contains oxygen or nitrogen, as these elements do not burn, its HV is lower than others composed just by carbon and hydrogen. In fact, when an alcohol (composed of C, H and O) burns, the oxygen atoms present in the burned gases are in the form of CO2 or H2O, mostly the latter [23,24]. y If a fuel contains oxygen or nitrogen, as these elements do not burn, its HV is lower than others composed just by carbon and hydrogen. In fact, when an alcohol (composed of C, H and O) burns, the oxygen atoms present in the burned gases are in the form of CO2 or H2O, mostly the latter [23,24] If a fuel contains oxygen or nitrogen, as these elements do not burn, its HV is lower than others composed just by carbon and hydrogen. In fact, when an alcohol (composed of C, H and O) burns, the oxygen atoms present in the burned gases are in the form of CO2 or H2O, mostly the latter [23,24]. Liquid fuels are more appropriate for vehicle propulsion. 2. Hybrid Vehicles Electrified vehicles need not specifically be battery electric vehicles (BEV), but there are various levels of hybrid vehicles, from the standard plug-in (parallel) hybrids, in which the engine provides mechanical traction power, to the extended-range (series) hybrids, in which traction is solely made by electric motors and the engine merely generates electricity, to fuel-cell hybrids. All these types of hybrid cars use some type of fuel that is burned in an ICE, with the exception of the fuel-cell hybrids. These latter vehicles use hydrogen (or another hydrogen-rich fuel such as alcohols or ammonia) which does not burn, but goes through a different process normally involving a catalysis through a Proton Exchange Membrane (PEM) producing electricity, water or water and CO2, when the molecule of the fuel also comprises carbon atoms [18,19]. While hybrid systems tend to duplicate systems, which might be a disadvantage in terms of cost and maintenance needs, it might provide a positive trade-offin the short to midterm, as it allows to minimize the main current limitations of electric mobility: energy storage cost, density, reliability and charging time [8,20]. While some of these limitations are no longer critical for small urban vehicles, which do not need large storage, they are still critical for driving patterns needing frequent long trips, which would require huge, expensive energy storage systems needing long recharging times or very high power fast charging stations, which also have their own challenges [21]. For instance, the authors reported that the Plug-in hybrid well-to-wheel 3 of 33 Energies 2020, 13, 4086 CO2 emissions may become negligible for cases where the long trips are less than 25% of the total mileage [12]. This was reported for the case where a compact and efficient range extender with two different operating conditions (one for efficiency another one for extra power) was implemented. Moreover, this configuration would allow low fuel consumption, fairly low complexity (comparatively to parallel hybrids) and low system cost [12]. Energies 2020, 13, x FOR PEER REVIEW 3 of 40 with two different operating conditions (one for efficiency another one for extra power) was implemented. Moreover, this configuration would allow low fuel consumption, fairly low complexity (comparatively to parallel hybrids) and low system cost [12]. 3. Fuels 3. Fuels Gaseous fuels require pressurized tanks and have a much lower energy density, resulting in much larger and heavier tanks for the same amount of stored energy (Figure 1, [25]). yg p g y [ ] Liquid fuels are more appropriate for vehicle propulsion. They have a very high energy density in terms of mass and volume, enabling the vehicles to have an enormous range. Gaseous fuels require pressurized tanks and have a much lower energy density, resulting in much larger and heavier tanks for the same amount of stored energy (Figure 1, [25]). Figure 1. Percent increase of volume and mass of fuel tanks in relation to petrol (55 L tank), [25]. Battery electric vehicles, for example, require huge volumes for their batteries (Table 1), adding mass and cost [8]. Nevertheless, the advances in battery technology have been slow but steady, with emerging technologies gaining traction. Examples are the use of high capacity cathode (e.g., metal id ) d d t i l l t l t ith hi h id ti t ti l d t l i b tt i hi h Figure 1. Percent increase of volume and mass of fuel tanks in relation to petrol (55 L tank), [25]. Battery electric vehicles, for example, require huge volumes for their batteries (Table 1), adding mass and cost [8]. Nevertheless, the advances in battery technology have been slow but Figure 1. Percent increase of volume and mass of fuel tanks in relation to petrol (55 L tank), [25]. Figure 1. Percent increase of volume and mass of fuel tanks in relation to petrol (55 L tank), [25]. Figure 1. Percent increase of volume and mass of fuel tanks in relation to petrol (55 L tank), [25]. Figure 1. Percent increase of volume and mass of fuel tanks in relation to petrol (55 L tank), [25]. Battery electric vehicles, for example, require huge volumes for their batteries (Table 1), adding mass and cost [8]. Nevertheless, the advances in battery technology have been slow but steady, with e e i tech olo ies ai i t actio E a ples a e the use of hi h capacity cathode (e etal Battery electric vehicles, for example, require huge volumes for their batteries (Table 1), adding mass and cost [8]. 3. Fuels 3. Fuels Nevertheless, the advances in battery technology have been slow but 4 of 33 Energies 2020, 13, 4086 steady, with emerging technologies gaining traction. Examples are the use of high capacity cathode (e.g., metal oxide) and anode materials, electrolytes with high oxidation potential and metal-air batteries which replace the positive electrode with an air electrode [26]. Another advantage of liquid fuels is their straightforward and fast refuelling. Gaseous fuels are more complicated and take longer to refuel, while electric vehicles require complex, expensive and time-consuming procedures. Table 1. Storage energy in volume for fuels and batteries [25]. Fuel Stored Energy (MJ/L) diesel 36 petrol 33 biodiesel 33 LPG 25 LNG (@ −162 ◦C) 22 ethanol 21 methanol 16 CNG (@300 bar) 12 H2 (liq. @ −253 ◦C) 8.5 H2 (comp. @ 250 bar) 2.5 battery Li-ion 0.9–1.35 battery Pb-acid 0.3 Table 1. Storage energy in volume for fuels and batteries [25]. Fuel Stored Energy (MJ/L) diesel 36 petrol 33 biodiesel 33 LPG 25 LNG (@ −162 ◦C) 22 ethanol 21 methanol 16 CNG (@300 bar) 12 H2 (liq. @ −253 ◦C) 8.5 H2 (comp. @ 250 bar) 2.5 battery Li-ion 0.9–1.35 battery Pb-acid 0.3 Table 1. Storage energy in volume for fuels and batteries [25]. In general, in terms of CO2 production of fossil fuels, the more carbon the molecule has, the higher is the production of this gas. Table 2 shows the potential for CO2 production of various fuels (in terms of LHV) compared to petrol (100). Please note that hydrogen is not a natural fuel, so it has to be produced from other sources, that may be fossil, therefore generating CO2, not in its burning, but during its production. Table 2. Potential for CO2 production of various fuels (adapted from [27]). Fuel CO2 Emissions Petrol 100 Diesel 102 LPG 87 Natural gas 75 Hydrogen 0 Table 2. Potential for CO2 production of various fuels (adapted from [27]). 4. Biofuels It is important to know whether a fuel is based on renewable energy, such as crops, as when it burns it does not increase the level of fossil CO2 in the atmosphere but marginally (if process and process emissions are taken into account [28], so there is an important division between different fuels is if they are generated from fossil or from renewable sources (biofuels) (Figure 2). However, as it will be seen later, sometimes it is difficult to assess whether the fuel is “bio” (from renewable energy) or not. For example, biodiesel is seen as a biofuel, but the 10% of methanol required for the transesterification process is usually produced from natural gas, a fossil fuel (Figure 2). Some fuels (such as hydrogen or ammonia) can be produced from fossil sources (hydrogen from natural gas or oil) but they can also be produced entirely from renewable sources. This is the case for the hydrogen produced from hydrolysis of water using renewable electricity such as solar or wind, as well as photochemical cells. Other so-called solar fuels can also incorporate CO2 using the same methods [29]. Also, biomass can be converted to other more convenient fuel forms through the incorporation of solar energy, which aids the pyrolysis process [30]. 5 of 33 ogas is Energies 2020, 13, 4086 methane present in considered biofuel Figure 2. Types of fossil and biofuels that can be used in Internal Combustion Engines [25]. Figure 2. Types of fossil and biofuels that can be used in Internal Combustion Engines [25]. Figure 2. Types of fossil and biofuels that can be used in Internal Combustion Engines [25]. Figure 2. Types of fossil and biofuels that can be used in Internal Combustion Engines [25]. Important Properties A list of the most common properties for different fuels gathered from several sources [27–38] can be seen in Table 3. Fuel density and the heating value are important for the determination of the quantity of energy available in the fuel, in terms of volume or mass. The heating value can be specified in terms of higher heating value (HHV) or lower heating value (LHV). The difference between HHV and LHV is the heat related to the condensation of the produced water. Obviously, for carbon, HHV has the same value than LHV, as there is no water produced by its combustion. 4. Biofuels Usually the energy content of liquid and solid fuels is characterized by their LHV, as normally the combustion gases are exhausted at temperatures high enough as not to enable condensation. The extremes, in terms of LHV, are carbon (33 MJ/kg, although a common value for coal is 27 MJ/kg) and hydrogen (120 MJ/kg), and in most hydrocarbons their LHV is a function of the H/C ratio. Gaseous fuels such as methane have a high LHV as its H/C ratio is one of the highest The energy density of This brings another discussion about the fuels, because some of them (such as hydrogen) can be seen as “energy carriers” rather than “energy sources”. Electricity is an energy carrier, as it may be produced from different energies sources (renewables such as solar and wind or fossil such as carbon or nuclear) in a specific location, but then it is transported to the place where the energy is needed, such as houses. The electricity may be transported over large distances by the electrical network or it may be transported by the vehicles within chemical-electric batteries. In this respect, hydrogen, ammonia and other synthetic fuels (such as Fischer-Tropsch petrol or diesel) can also be considered “energy carriers”. If these fuels are produced entirely from renewable sources, then they are considered biofuels. So, these referred fuels (hydrogen, ammonia, synthetic hydrocarbons) may be considered fossil fuels, if they are produced from fossil origins, or they can be partially or entirely considered biofuels. Therefore, the SAME FUEL can be considered a fossil fuel or a biofuel. The methane present in the natural gas is fossil, whereas the same methane contained in the biogas is considered biofuel. the fuel on a mass ba Important Properties Energies 2020, 13, x; doi: FOR PEER REVIEW www.mdpi.com/journal/energies but not so relevant for stationary applications. A list of the most common properties for different fuels gathered from several sources [27–38] can be seen in Table 3. Fuel density and the heating value are important for the determination of the quantity of energy available in the fuel, in terms of volume or mass. The heating value can be specified in terms of higher heating value (HHV) or lower heating value (LHV). The difference between HHV and LHV is the heat related to the condensation of the produced water. Obviously, for carbon, HHV has the same value than LHV, as there is no water produced by its combustion. 6 of 33 Energies 2020, 13, 4086 Table 3. Properties for some fuels (adapted from [27–38]). the fuel on a mass ba Important Properties Fuel Chemical Formula ρLiquid (kg/m3) TBoiling (◦C) Latent Heat of Vaporization (kJ/kg) Tignition (◦C) Tadiabatic (◦C) LHV (MJ/kg) LHVLiquid (MJ/L) HHVMixture (kJ/L) A/Fstoich RON CN Viscosity (@40 ◦C) (cSt) Flash Point (◦C) Reid Vapour Pressure (@38 ◦C) (kPa) Flammability Limits (% vol.) O2 (%) Acetylene C2H2 621 −84 614 305 2334 48.5 4.0 13.2 40 2.5–75 Ammonia NH3 682 −33 1370 650 1803 18.6 2.8 6.1 110 0.3 - 15–28 Av gas 715 25–170 440 44 32 ~14.5 115 −40 30–90 1.5–7.6 Biodiesel C17H32O2 850-885 250–350 220 2000 37 33 ~13 - 45–65 3.5–5.5 62 ~11 Biogas CH4 - −162 510 580 1954 24 3.1 17 120 Butane C4H10 580 −0.6 386 405 1975 46.5 26 4.2 15.6 102 1.8–8.4 Carbon C 700 33 11.5 Carbon monoxide CO 609 2120 12 2.4 12.5–74 Coal - 450 27 11.5 - DEE C2H5 O C2H5 714 34 160 1980 34 34 11.1 >125 0.23 110 1.9–36 Diesel CnH1.8n 820–870 180–360 ~270 ~210 43 36 3.9 ~14 - 45–55 2.0–3.5 60–80 <1.5 0.6–8 DMC C3H6O3 1079 90 418 458 15.8 17 4.64 109 16.7 10.8 4.2–12.9 53.3 DME CH3 O CH3 667 −25 375 320 2020 28.8 19 3.4 9.0 - 55–60 0.18 −41 800 3.4–19 35 DMF CH3 C4H2 O CH3 895 93 340 286 33.7 3.4 10.7 119 67 3.4–18.6 DMM CH3O CH2O CH3 865 319 237 22.4 30 42.1 ETBE C6H14O 770 307 12.2 −25 1.4–10 Ethanol C2H5 OH 790 78 900 365 1965 26.8 21 9.0 110 8 1.5 12 16 4.3–19 35 FAGE 962 34.2 11.2 70 11.9 Fuel oil (“thick”) ~960 >180 ~230 ~260 1995 41 36 ~14 - 0.7–5 F-T diesel CnH2n+2 785 175–355 315 43.9 15.0 79 3.5 Glycerine C3H5(OH)3 1260 290 670 390 19 4.2 5 170 3–19 52.2 HVO 776 43.9 15.0 82 2.65 Hydrogen H2 70 −253 455 500 2510 120 8.5 3.0 34.1 106 4–75 Isopropanol C3H7 OH 790 82 740 30.3 24 10.3 106 12 7 of 33 Energies 2020, 13, 4086 Table 3. Cont. the fuel on a mass ba Important Properties Fuel Chemical Formula ρLiquid (kg/m3) T Boiling (◦C) Latent Heat of Vaporization (kJ/kg) Tignition (◦C) Tadiabatic (◦C) LHV (MJ/kg) LHV Liquid (MJ/L) HHV Mixture (kJ/L) A/Fstoich RON CN Viscosity (@40 ◦C) (cSt) Flash Point (◦C) Reid Vapour Pressure (@38 ◦C) (kPa) Flammability Limits (% vol.) O2 (%) LPG (95% propane) 540 −43 425 455 1980 46 25 3.3 15.6 110 2.1–9.5 MeFo C2H4O2 957 31.5 464 450 15.8 4.64 115 −19 >100 5–20 53.3 Methane CH4 500 −162 510 580 1963 50 22 3.1 17 120 −188 5–15 Methanol CH3 OH 800 65 1100 470 1950 19.7 16 3.5 6.4 115 5 0.75 11 32 7–36 50 MTBE CH3 O C4H9 740 55 340 1990 35.2 26 11.7 117 −11 2.4–8 18 Natural Gas (CNG, LNG) 500 −162 419 580 1954 47 21 3.1 17 120 5–15 Nitromethane CH3NO2 1130 100 600 500 2272 11 12 8.7 1.7 52.5 PL 918 40.5 17.6 2.5 Petrol RON98 CnH1.87n 720–780 25–210 ~350 ~260 1995 44 33 3.8 ~14.5 98 13–17 0.7 −40 55–100 1.3–8 PODE3-4 or OME3-4 CH3O(CH2O) nCH3 1019 18 19.1 71 Propane C3H8 520 −43 437 455 1977 46.1 24 3.3 15.7 112 −104 1170 2.1–9.5 TBA C4H9 OH 790 83 570 32.5 26 11.2 113 11 2.4–8 22 Turpentine C10H16 860–900 150–180 285 370 44.4 14.2 20–25 2.5 38 <1 0.7 8 of 33 Energies 2020, 13, 4086 Usually the energy content of liquid and solid fuels is characterized by their LHV, as normally the combustion gases are exhausted at temperatures high enough as not to enable condensation. The extremes, in terms of LHV, are carbon (33 MJ/kg, although a common value for coal is 27 MJ/kg) and hydrogen (120 MJ/kg), and in most hydrocarbons their LHV is a function of the H/C ratio. Gaseous fuels such as methane have a high LHV, as its H/C ratio is one of the highest. The energy density of the fuel on a mass base would be an important parameter for mobile applications (namely, aerospace) but not so relevant for stationary applications. Energies 2020, 13, x FOR PEER REVIEW 6 of 4 Another important property is the heating value of a stoichiometric air-fuel mixture in terms o p y pp Another important property is the heating value of a stoichiometric air-fuel mixture in terms of volume at atmospheric pressure. the fuel on a mass ba Important Properties Th l f th t i hi t i i f l ti (A/F) i i di ti f th H/C ti f th The value for the stoichiometric air-fuel ratio (A/F) is an indication of the H/C ratio of the hydrocarbon and/or the amount of oxygen (or nitrogen) in its molecule. The value for the stoichiometric air-fuel ratio (A/F) is an indication of the H/C ratio of th hydrocarbon and/or the amount of oxygen (or nitrogen) in its molecule. The RON (Research Octane Number) and CN (Cetane Number) used to classify commercia petrol and diesel fuels, respectively, are related to the way the fuel self-ignites. High RON number a e ood fo S a k I itio (SI) e i e he ea hi h CN u be a e ood fo die el e i e I The RON (Research Octane Number) and CN (Cetane Number) used to classify commercial petrol and diesel fuels, respectively, are related to the way the fuel self-ignites. High RON numbers are good for Spark Ignition (SI) engines, whereas high CN numbers are good for diesel engines. In fact, these two numbers are opposed [39,40] (Figure 4). are good for Spark Ignition (SI) engines, whereas high CN numbers are good for diesel engines. In fact, these two numbers are opposed [39,40] (Figure 4). High values for RON indicate a very difficult auto-ignition behaviour, whereas high values fo CN specify fuels that auto ignite easily Observing Figure 4 it may be seen that there is a clear relation High values for RON indicate a very difficult auto-ignition behaviour, whereas high values for CN specify fuels that auto-ignite easily. Observing Figure 4 it may be seen that there is a clear relation between these numbers. The curve fit yields the following formulae: y g g y curve fit yields the following formulae: CN = 56 − 0.39 RON (1) CN = 56 −0.39 RON (1) RON = 105 −0.145 CN −0.333 CN2 (2) (1) (1) (2) CN = 56 − 0.39 RON RON = 105 −0.145 CN −0.333 CN2 RON = 105 − 0.145 CN − 0.333 CN2 (2) Other physical properties presented in Table 3 are viscosity, flash point and Reid vapour pressure. The viscosity values show the potential for the fuel to be injected as a fine spray, very important for diesel engines. the fuel on a mass ba Important Properties This shows the amount of energy that can be introduced into an IC Engine per cycle, which affects torque and power [25]. The importance of this parameter can be illustrated with Hydrogen: although it has the highest HV in terms of mass, its value in terms of volume (of its mixture with air) is one of the lowest (Table 3; Figure 3). Naturally, this parameter affects storage space, which is also critical for mobile applications. Another important property is the heating value of a stoichiometric air fuel mixture in terms o volume at atmospheric pressure. This shows the amount of energy that can be introduced into an IC Engine per cycle, which affects torque and power [25] The importance of this parameter can b illustrated with Hydrogen: although it has the highest HV in terms of mass, its value in terms o volume (of its mixture with air) is one of the lowest (Table 3; Figure 3). Naturally, this paramete affects storage space, which is also critical for mobile applications. Figure 3. Heating values for the fuel (per mass) and for the stoichiometric air-fuel mixture (per unit 0 20 40 60 80 100 120 140 6.47 14.5 14.6 34.3 Lower heating value (MJ/kg) FUEL methanol diesel petrol hydrogen 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 6.47 14.5 14.6 34.3 Lower heating value (MJ/m3) FUEL-AIR MIXTURE methanol diesel petrol hydrogen Figure 3. Heating values for the fuel (per mass) and for the stoichiometric air-fuel mixture (per unit volume—[25]). Figure 3. Heating values for the fuel (per mass) and for the stoichiometric air-fuel mixture (per unit Figure 3. Heating values for the fuel (per mass) and for the stoichiometric air-fuel mixture (per unit volume—[25]). volume—[25]). The latent heat of vaporization is responsible for the cooling effect on the mixture when the fu is vaporized Alcohols have a high value so their mixture with air enters the engine at low The latent heat of vaporization is responsible for the cooling effect on the mixture when the fuel is vaporized. Alcohols have a high value, so their mixture with air enters the engine at low temperatures, even when supercharging is used [25]. is vaporized. Alcohols have a high value, so their mixture with air enters the engine at low temperatures, even when supercharging is used [25]. the fuel on a mass ba Important Properties The Reid vapour pressure is a measure of the volatility of a fuel and is very important for fuels The Reid vapour pressure is a measure of the volatility of a fuel and is very important for fuels used in SI engines, mainly when they were carburetted fuelled. The flammability limits show the proportions (% in volume) where a spark may ignite the fuel-air mixture. Hydrogen is the fuel with the widest limits for flammability, which is very important to burn very lean mixtures. The burning of very lean mixtures in ICEs has various advantages such as high engine efficiency and low pollutant emissions [25]. p p y y p used in SI engines, mainly when they were carburetted fuelled. The flammability limits show the proportions (% in volume) where a spark may ignite the fuel-air mixture. Hydrogen is the fuel with the widest limits for flammability, which is very important to burn very lean mixtures. The burning of very lean mixtures in ICEs has various advantages such as high engine efficiency and low pollutant emissions [25]. Oxygenated fuels, as the name refers, have oxygen in their molecule, so their energy density is reduced by this fact. Half the mass of methanol is oxygen and its LHV is less than half of that of petrol, but as its stoichiometric A/F is also almost half of the petrol, the HV of the air-methanol mixture is, although lower, at about the same level as for petrol. But, as the high latent heat of Oxygenated fuels, as the name refers, have oxygen in their molecule, so their energy density is reduced by this fact. Half the mass of methanol is oxygen and its LHV is less than half of that of petrol, but as its stoichiometric A/F is also almost half of the petrol, the HV of the air-methanol mixture is, although lower, at about the same level as for petrol. But, as the high latent heat of methanol greatly reduces the air temperature entering the engine, its density is increased by this fact and much more air enters the engine, effectively increasing engine power output (by around 6%, [42]). The fuel with the maximum potential for power boost is nitromethane. the fuel on a mass ba Important Properties For example, diesel fuel has lower viscosity than biodiesel, so the biodiesel spray is 9 of 33 Energies 2020, 13, 4086 coarser than the conventional diesel spray. On the other hand, ethanol and mainly DME and DEE exhibit much less viscosity, so their injection can be made in fine droplet sprays [41]. Energies 2020, 13, x FOR PEER REVIEW 1 of 40 Figure 4. Relation between CN and RON for various oxygenate and hydrocarbon fuels (adapted from [39,40]). Figure 4. Relation between CN and RON for various oxygenate and hydrocarbon fuels (adapted from [39,40]). Figure 4. Relation between CN and RON for various oxygenate and hydrocarbon fuels (adapted from [39,40]). Figure 4. Relation between CN and RON for various oxygenate and hydrocarbon fuels (adapted from [39,40]). Other physical properties presented in Table 3 are viscosity, flash point and Reid vapour pressure. The viscosity values show the potential for the fuel to be injected as a fine spray, very important for diesel engines. For example, diesel fuel has lower viscosity than biodiesel, so the biodiesel spray is coarser than the conventional diesel spray. On the other hand, ethanol and mainly DME and DEE exhibit much less viscosity, so their injection can be made in fine droplet sprays [41]. Flash point is the temperature at which the liquid releases enough vapour to produce a stoichiometric mixture with air, therefore sustaining a flame, so it is a property related to safety. If the flash point of a fuel (such as diesel) is well above room temperature, a leak of it will not enable combustion. On the other hand, if the fuel has its flash point below 38 ◦C (100 ◦F) it is considered flammable [23]. y, j p p y [ ] Flash point is the temperature at which the liquid releases enough vapour to produce a stoichiometric mixture with air, therefore sustaining a flame, so it is a property related to safety. If the flash point of a fuel (such as diesel) is well above room temperature, a leak of it will not enable combustion. On the other hand, if the fuel has its flash point below 38 °C (100 °F) it is considered flammable [23]. 5. Hydrogen Hydrogen is one of the simplest molecules, with just two atoms joint together, each one with just one proton and an electron. Normally it is in gas form and, unless at very high pressure and/or at very low temperature, its energy density (in terms of volume, or MJ/L–Figure 3) is extremely low (0.11 MJ/L at atmospheric conditions). And this is one of the disadvantages of this fuel: even at very high pressures (750 bar) or very low temperatures (liquefied at 20 K) its energy density is much lower than that of most other liquid fuels (4.7 and 8.6 MJ/L, respectively [43]) And it takes a significant amount of energy to pressurize the hydrogen or to liquefy it, a value that is a significant proportion of its own HV. When compared to other liquid hydrocarbons, one litre of liquid hydrogen actually has less hydrogen (atoms) than a litre of a conventional fuel (and the conventional fuel also has, in addition, carbon atoms). Some recent developments promise the use of materials to store hydrogen at much lower pressures, such as Liquid Organic Hydrogen Carrier (LOHC) systems [44–46]. However, these methods are complicated, need pressure and/or temperature control and require time for the storage (hydrogenation) and for the recovery (dehydrogenation), often requiring catalysts when liquids are used [47,48]. Yet, these technologies might become viable in the future for specific applications, namely in large-scale stationary cases where the economy of scale eventually compensates for the added complexity. One other important disadvantage of hydrogen is that its tiny molecule can escape through materials that usually are not permeable to other gases. This requires the use of specific materials for piping and storage, including particular specifications for welding [49]. Although hydrogen can be used in ICEs, its major advantage is to be used, as energy carrier, in fuel cells, where it produces nothing but electricity and water. Hydrogen production from electricity is usually done by water electrolysis in a process that may have an efficiency between 52% and 67%, so 60% seems a good average value. The hydrogen is then used to produce electricity in fuel cells with efficiencies ranging from 50 to 60% (we will use 55%) [18]. Furthermore, it is necessary to store the hydrogen as compressed gas at 350 to 700 bar or as liquid at 20K. the fuel on a mass ba Important Properties Although it has a low HV of 12 MJ/kg, the 1.7 stoichiometric A/F enables a huge amount of fuel to be injected at each cycle, increasing the engine power output to more than 2.3 times the value for petrol [42]. Energies 2020, 13, 4086 10 of 33 10 of 33 5. Hydrogen This requires 15 MJ/kg for the compression up to 700 bar [50] and 50 MJ/kg for the liquefaction of H2 [50]. This leads to overall efficiencies of electricity-to-electricity of 29% (compressed H2) and 19.5% (liquid H2) when hydrogen is used as an energy carrier. A novel electrolysis process called high temperature electrolysis or steam electrolysis (at 700 to 1000 ◦C, much higher than the water critical temperature and at high pressures), shows a potential for much higher efficiencies [51]. As said before, petrol and diesel also display more hydrogen content on a volume basis than liquid hydrogen, which makes their synthetic versions also good energy carriers, probably better than hydrogen. But hydrogen has some important properties to be used in ICEs as an additive. It’s very high combustion speed (much higher than petrol) improves the combustion of other fuels even at low fractions (less than 5%). This is beneficial for fuels which burn slowly, such as ammonia [52]. When the percentage of hydrogen is high or when it is burned on its own (pure), the higher adiabatic flame temperature generates a high quantity of NOx, so it is common to inject water as a means to reduce the maximum temperature, especially when supercharging is used. One of the problems of hydrogen is its potential for auto-ignition, as the activation energy (of a spark) required for ignition is very low (0.01 mJ). This fact interferes with the measurement of its knock behaviour and the value for its octane number. The measurement of RON requires an intake temperature of 149 ◦C, too high for the use of hydrogen. Usually the RON for hydrogen is reported as higher than 100 (Table 3), but some researchers [31] report values as low as 60. Others report RON of over 130 when lean mixtures are used. When the intake is at atmospheric temperature hydrogen shows a very high RON, enabling compression ratios (CR) higher than 14.5:1 without knock, probably helped by its very high combustion velocities [53]. But, as discussed earlier, the major problem with hydrogen is its energy density. 50 L of petrol can be stored in a 72 L tank (weighing 84 kg, including the fuel), whereas the same amount of energy in 11 of 33 Energies 2020, 13, 4086 hydrogen (19 kg) requires a cylindrical tank with 272 L and 129 kg ([54]; see Figure 1). 5. Hydrogen So, the main interests of hydrogen seem to be its potential for use in PEM fuel cells, the absence of CO2 emissions and its use as an energy carrier and energy storage in stationary applications (although at very high pressures or volumes). If other types of high performance fuel cells capable of consuming liquid fuels are developed, other biofuels (with the potential for not producing fossil CO2) are used and other synthetic fuels may be used as energy carriers, what will be the benefit of hydrogen? It is our opinion that, if that is the case, the justification for the “hydrogen economy” will lose a lot of its appeal and other high energy density (liquid) synthetic fuels and/or biofuels will likely replace it. So, it seems that the major advantage of hydrogen in transportation is its higher energy density compared to batteries, which makes fuel cell hybrid electric cars a better proposition than full electric vehicles, with higher range and much lower refueling times. 6. Alcohols The most common alcohol used in propulsion is ethanol, with vast amounts being deployed in Brazil and USA in the so called “flex-fuel” engines. These SI engines can burn petrol, straight ethanol, or any mixture of these two fuels. Although the stoichiometry of both fuels is very different (AFR of 14.5 for petrol and 9.0 for ethanol—see Table 3), the injection system uses the lambda sensor in the exhaust to assess the richness of the mixture and to adjust it. If, for example, the engine is running on straight petrol and the driver fills the tank with ethanol, when the new fuel reaches the injectors, they produce a lean mixture (less fuel than required) but, within one or two seconds the lambda sensor reads the strength of the mixture, sends the information to the ECU (electronic control unit) and the right amount of fuel is then injected to the cylinders and from that point onwards the ECU (electronic control unit) of the engine assumes that fuel. Therefore, only during this very short period the driver may feel some swift glitch in the engine, but then it works seamlessly afterwards. Methanol is another alcohol occasionally used, mainly in the USA and mainly for racing engines. Ethanol and mostly methanol are exceptional racing fuels for various reasons. They have a high value for RON and a high latent heat of vaporization (see Table 3) leading to cold and dense mixtures entering the engine (more mass) and allowing the use of high compression ratio (CR), which brings higher efficiencies and power [25]. Ethanol is mainly produced from the enzymatic breakdown of starch (grains) leading to sugar and then to ethanol. In the USA the base is corn but in Brazil, where the sugar cane is used, the first transformation is avoided, therefore greatly enhancing the overall efficiency of production. g y g y p Methanol is mainly produced from natural gas from the steam reforming equation: CH4 + H2O→CO + 3H2 (3) (3) followed by a catalytic reaction between CO and hydrogen: ed by a catalytic reaction between CO and hydrogen: followed by a catalytic reaction between CO and hydrogen: CO + 2H2→CH3OH (4) (4) One of the less known advantages of alcohol combustion is the so-called “alcohol bonus”. which in terms of volumes is: 2.5 V (reactants)→2 V (products) (8) 2.5 V (reactants)2 V (products) (8) 2.5 V (reactants)→2 V (products) (8) 2.5 V (reactants)2 V (products) (8) (8) (8) 2.5 V (reactants)→2 V (products) 2.5 V (reactants)2 V (products) Thus, for the same volume of reactants (2.5) only two volumes are produced when petrol is used, but three are produced when methanol is used. This means there is a substantial higher volumetric expansion when alcohol is used. This can be seen on the indicated diagram, where the methanol shows higher pressure during expansion (Figure 5). This is considering that both petrol and methanol are entirely vaporized when entering the engine, although it is much more difficult to vaporize methanol than petrol, as the latent heat of vaporization of the former (1100 kJ/kg) is much higher than that of the latter (350 kJ/kg), so the relation is even higher. Thus, for the same volume of reactants (2.5) only two volumes are produced when petrol is used, but three are produced when methanol is used. This means there is a substantial higher volumetric expansion when alcohol is used. This can be seen on the indicated diagram, where the methanol shows higher pressure during expansion (Figure 5). This is considering that both petrol and methanol are entirely vaporized when entering the engine, although it is much more difficult to vaporize methanol than petrol, as the latent heat of vaporization of the former (1100 kJ/kg) is much higher than that of the latter (350 kJ/kg), so the relation is even higher. gasolina metanol Volume PMS PMI patm Pressão TD Volume BDC Figure 5. Indicated diagram for petrol and methanol. petrol methanol Pressure Figure 5. Indicated diagram for petrol and methanol. gasolina metanol Volume PMS PMI patm Pressão TD Volume BDC Figure 5. Indicated diagram for petrol and methanol. petrol methanol Pressure Figure 5. Indicated diagram for petrol and methanol. gasolina petrol Figure 5. Indicated diagram for petrol and methanol. Figure 5. Indicated diagram for petrol and methanol. As methanol (and ethanol) have much higher latent heat of vaporization than petrol and, for the same power, the amount of injected mass is also considerable higher, the total heat required for the total vaporization of the fuel is much higher when alcohols are used. This creates a cooling effect on the intake mixture, even when supercharging is used. which in terms of volumes is: This is beneficial for motorsports, as the thermal loads of engine internals are very high. With the use of alcohols, a supercharged engine can work without intercooling and face no thermal problems or knock. As methanol (and ethanol) have much higher latent heat of vaporization than petrol and, for the same power, the amount of injected mass is also considerable higher, the total heat required for the total vaporization of the fuel is much higher when alcohols are used. This creates a cooling effect on the intake mixture, even when supercharging is used. This is beneficial for motorsports, as the thermal loads of engine internals are very high. With the use of alcohols, a supercharged engine can work without intercooling and face no thermal problems or knock. p As the RON values for alcohols are higher than those for petrol (see Table 3), the compression ratio of the engines can be increased without knock, therefore improving power and efficiency. As the adiabatic flame temperature of alcohols is lower than that of petrol, the thermal losses to the combustion chamber are reduced, further improving the overall efficiency. As the RON values for alcohols are higher than those for petrol (see Table 3), the compression ratio of the engines can be increased without knock, therefore improving power and efficiency. As the adiabatic flame temperature of alcohols is lower than that of petrol, the thermal losses to the combustion chamber are reduced, further improving the overall efficiency. p g y Other advantages of the alcohols are the fact that, unlike petrol, they mix very well with water, enabling firefighting by the use of water. Throwing water into a petrol fire usually aggravates the problem, as the water is denser than petrol, so the fuel floats over it and spreads easily. Other advantages of the alcohols are the fact that, unlike petrol, they mix very well with water, enabling firefighting by the use of water. Throwing water into a petrol fire usually aggravates the problem, as the water is denser than petrol, so the fuel floats over it and spreads easily. But alcohols also have some problems. The methanol flame has no colour, so it is very difficult to assess whether a fire is taking place. 6. Alcohols When methanol, is burned, the equation is: CH3OH + 1.5 O2→CO2 + 2 H2O (5) (5) which in terms of moles (which translate into volume) gives: which in terms of moles (which translate into volume) gives: 2.5 V (reactants)→3 V (products) (6) 2.5 V (reactants)→3 V (products) (6) When petrol is used (considering CH2): CH2 + 1.5 O2→CO2 + H2O (7) CH2 + 1.5 O2→CO2 + H2O (7) Energies 2020, 13, 4086 12 of 33 (7) 7.2. DEE 7.2. DEE Diethyl ether (DEE) is a volatile ether conventionally produced as a by-product of ethylene hydration during the production of ethanol, but it can also be produced from the reaction of sulphuric acid with ethanol or by catalytic dehydration of ethanol. As its cetane number is very high (ignition temperature of 160 °C, one of the lowest, Table 3), DEE is used as an IC Engine starting agent, both in SI and diesel engines Diethyl ether (DEE) is a volatile ether conventionally produced as a by-product of ethylene hydration during the production of ethanol, but it can also be produced from the reaction of sulphuric acid with ethanol or by catalytic dehydration of ethanol. As its cetane number is very high (ignition temperature of 160 ◦C, one of the lowest, Table 3), DEE is used as an IC Engine starting agent, both in SI and diesel engines. in SI and diesel engines. With such a high cetane number (up to 158 [41]) and being able to be stored as a liquid at atmospheric conditions (Tboiling = 34 °C, Table 3), this fuel seems to be a good candidate for use in Compression Ignition (CI) engines. It can be added to diesel and, during WWII in Japan, it was used as an additive (up to 5%) in aeroplane engines [41]. Its use as a straight CI engine fuel may have problems as it does not have lubricity and it has tendency to oxidation, producing peroxides. Some organic peroxides are dangerously reactive because they combine both fuel (carbon) and oxygen in the same compound. As it has oxygen in its molecule and has no C-C bonds, its burning does not produce smoke. With such a high cetane number (up to 158 [41]) and being able to be stored as a liquid at atmospheric conditions (Tboiling = 34 ◦C, Table 3), this fuel seems to be a good candidate for use in Compression Ignition (CI) engines. It can be added to diesel and, during WWII in Japan, it was used as an additive (up to 5%) in aeroplane engines [41]. Its use as a straight CI engine fuel may have problems as it does not have lubricity and it has tendency to oxidation, producing peroxides. Some organic peroxides are dangerously reactive because they combine both fuel (carbon) and oxygen in the same compound. 7.2. DEE 7.2. DEE As it has oxygen in its molecule and has no C-C bonds, its burning does not produce smoke. which in terms of volumes is: Also, as the heat required for full vaporization of the alcohols is high, mixture preparation may be a problem [55], and a large proportion of liquid may enter the cylinders and “wash” away the oil from the cylinder surfaces, enabling piston-cylinder contact. Also, in cold countries (even in south Brazil) a small tank of petrol is required to start the engine, as ethanol or methanol does not have enough vapour pressure to produce an ignitable mixture. Also, ethanol and especially methanol, tend to induce heavy corrosion on various metals and also other materials such as rubber. But alcohols also have some problems. The methanol flame has no colour, so it is very difficult to assess whether a fire is taking place. Also, as the heat required for full vaporization of the alcohols is high, mixture preparation may be a problem [55], and a large proportion of liquid may enter the cylinders and “wash” away the oil from the cylinder surfaces, enabling piston-cylinder contact. Also, in cold countries (even in south Brazil) a small tank of petrol is required to start the engine, as ethanol or methanol does not have enough vapour pressure to produce an ignitable mixture. Also, ethanol and especially methanol, tend to induce heavy corrosion on various metals and also other materials such as rubber. As the flammability limits of alcohols are much wider than those of petrol, the engines may work with much leaner mixtures, improving the efficiency of the engine and reducing all the pollutants. Energies 2020, 13, 4086 As the flamma 13 of 33 y work Despite being a clean and efficient fuel, the major contribution from methanol to transportation is its use to the biodiesel production in the transesterification process [56]. p g y g g p Despite being a clean and efficient fuel, the major contribution from methanol to transportation is its use to the biodiesel production in the transesterification process [56]. Figure 6. Formation of dimethyl ether from methanol. Figure 6. Formation of dimethyl ether from methanol. Figure 6. Formation of dimethyl ether from methanol. Figure 6. Formation of dimethyl ether from methanol. The compressibility of DME is much higher than diesel, which increases the required energy for fuel compression, although it does not require the huge injection pressures required for fine diesel spray formation. However, it cannot be used directly on diesel injection systems, as it has poor lubrication properties, but a small amount of biodiesel may be added to enable the lubricity. Also, its low density and low heating value require higher injection mass flowrates than diesel but in overall it has the potential for producing more power from the same engine using diesel [56]. The compressibility of DME is much higher than diesel, which increases the required energy for fuel compression, although it does not require the huge injection pressures required for fine diesel spray formation. However, it cannot be used directly on diesel injection systems, as it has poor lubrication properties, but a small amount of biodiesel may be added to enable the lubricity. Also, its low density and low heating value require higher injection mass flowrates than diesel but in overall it has the potential for producing more power from the same engine using diesel [56]. 7. Ethers 7. Ethers Ethers are molecules with an atom of oxygen connecting two radicals that usually are similar. For example, dimethyl ether (DME) is composed by two identical methyl radicals connected by the oxygen atom. They are highly flammable liquids or gases which, therefore, may be used in IC engines. 7 1 DME Ethers are molecules with an atom of oxygen connecting two radicals that usually are similar. For example, dimethyl ether (DME) is composed by two identical methyl radicals connected by the oxygen atom. They are highly flammable liquids or gases which, therefore, may be used in IC engines. 7.1. DME 7.1. DME Dimethyl ether (DME) is the simplest ether and is a gas at atmospheric pressure, but it is easily condensed by applying pressure (<10 bar), so it may be stored in similar containers as propane. Its cetane number (CN > 55) is higher than that of diesel (see Table 3), has a low ignition temperature (320 ◦C), its viscosity is very low and, as it is composed of 35% oxygen and it has no C-C bonds, its combustion is smoke free [57]. As it is highly volatile, its mixture preparation with air is much easier than diesel, which makes it a perfect compression ignition fuel. Also, it burns fast and without knock (silent combustion—[41]), it has a potential for higher efficiency but it produces more NOx than diesel [56]. Dimethyl ether (DME) is the simplest ether and is a gas at atmospheric pressure, but it is easily condensed by applying pressure (<10 bar), so it may be stored in similar containers as propane. Its cetane number (CN > 55) is higher than that of diesel (see Table 3), has a low ignition temperature (320 °C), its viscosity is very low and, as it is composed of 35% oxygen and it has no C-C bonds, its combustion is smoke free [57]. As it is highly volatile, its mixture preparation with air is much easier than diesel, which makes it a perfect compression ignition fuel. Also, it burns fast and without knock (silent combustion—[41]), it has a potential for higher efficiency but it produces more NOx than diesel [56]. DME (and other ethers) can be produced by the dehydration of two alcohol molecules, also producing water (Figure 6). It can also be produced from the “black liquor”, a by-product of pulp and paper production, or from lignite-cellulose biomass, which makes it a second-generation biofuel. [56]. DME (and other ethers) can be produced by the dehydration of two alcohol molecules, also producing water (Figure 6). It can also be produced from the “black liquor”, a by-product of pulp and paper production, or from lignite-cellulose biomass, which makes it a second-generation biofuel. 8. Esters (Biodiesel) Common esters are better known as biodiesel and they are good substitutes for fossil diesel fuel in CI engines. They are produced from vegetable oils (and other fats) in esterification or, more commonly, Energies 2020, 13, 4086 Common est 14 of 33 iesel fuel transesterification processes (Figure 7). In the latter process a triglyceride reacts with an alcohol, in the presence of a catalyst, producing the ester and glycerol. Methods for glycerol usage will be discussed in a following chapter. commonly, transesterification processes (Figure 7). In the latter process a triglyceride reacts with an alcohol, in the presence of a catalyst, producing the ester and glycerol. Methods for glycerol usage will be discussed in a following chapter. transesterification processes (Figure 7). In the latter process a triglyceride reacts with an alcohol, in the presence of a catalyst, producing the ester and glycerol. Methods for glycerol usage will be discussed in a following chapter. commonly, transesterification processes (Figure 7). In the latter process a triglyceride reacts with an alcohol, in the presence of a catalyst, producing the ester and glycerol. Methods for glycerol usage will be discussed in a following chapter. Figure 7 T a e te ifi atio o e Figure 7. Transesterification process. Figure 7 Transesterification process Figure 7. Transesterification process. Fi 7 T ifi i Figure 7. Transesterification process. igu e . a ses e i ica io p ocess The esters of different vegetable oils (rape seed, soy, peanut, sunflower, etc.) are known as biodiesel or FAME (fatty acid methyl ester), if they are produced from methanol. They are usually produced using methanol, but it is possible to produce biodiesel using ethanol. In this case the process is slower and has lower efficiency, but the final product may be considered 100% biofuel, if bioethanol is used and if the vegetable oil is 100% bioproduced. While methanol can also be produced from renewable sources, usually it is derived from natural gas. One of the advantageous properties of biodiesel is its lubricity. When sulphur was removed from diesel, its lubricity was reduced The esters of different vegetable oils (rape seed, soy, peanut, sunflower, etc.) are known as biodiesel or FAME (fatty acid methyl ester), if they are produced from methanol. They are usually produced using methanol, but it is possible to produce biodiesel using ethanol. 8. Esters (Biodiesel) In this case the process is slower and has lower efficiency, but the final product may be considered 100% biofuel, if bioethanol is used and if the vegetable oil is 100% bioproduced. While methanol can also be produced from renewable sources, usually it is derived from natural gas. One of the advantageous properties of biodiesel is its lubricity. When sulphur was removed from diesel, its lubricity was reduced drastically, and the solution was the addition of 2% biodiesel to restore it. drastically, and the solution was the addition of 2% biodiesel to restore it. Biodiesel, although with slightly different properties according to the original oil it was made from, is a fuel with higher cetane number than diesel, has no sulphur, the CO and HC emissions are lower [58] and is a biodegradable liquid. In terms of disadvantages, it has higher viscosity than diesel, produces higher values of NOx and is not stable (oxidises) during prolonged storage. Also, the production process is inefficient (is intensive in energy), its heating value is lower than diesel and it Biodiesel, although with slightly different properties according to the original oil it was made from, is a fuel with higher cetane number than diesel, has no sulphur, the CO and HC emissions are lower [58] and is a biodegradable liquid. In terms of disadvantages, it has higher viscosity than diesel, produces higher values of NOx and is not stable (oxidises) during prolonged storage. Also, the production process is inefficient (is intensive in energy), its heating value is lower than diesel and it may attack elastomers. p p gy g may attack elastomers. For the same amount of injected fuel as diesel, the power reduction using biodiesel should be about 10%, but engine data shows only a reduction of 5% [37], showing a higher efficiency. For the same vehicle energy consumption, the author [58] measured an increase of only 3.5% (in volume) and a reduction of 6% in terms of energy (in fuel) used, when compared to fossil fuel in a long (12,350 km) trip in South America. This also shows a better engine efficiency on the use of biodiesel when For the same amount of injected fuel as diesel, the power reduction using biodiesel should be about 10%, but engine data shows only a reduction of 5% [37], showing a higher efficiency. 8. Esters (Biodiesel) For the same vehicle energy consumption, the author [58] measured an increase of only 3.5% (in volume) and a reduction of 6% in terms of energy (in fuel) used, when compared to fossil fuel in a long (12,350 km) trip in South America. This also shows a better engine efficiency on the use of biodiesel when compared to diesel [58]. p g y compared to diesel [58]. It is important to state that the referred comparative tests were performed in common-rail engines. When using traditional pump-pipe-injector systems the lower compressibility and higher cetane number of the biodiesel generates an earlier and faster combustion which further improves efficiency (and increases NOx) if the engine is developed to minimize NOx emissions. But the lower compressibility of the biodiesel does not interfere with the injection in common-rail systems, so the higher efficiency in these type of engines is only explained by the better combustion potential of the It is important to state that the referred comparative tests were performed in common-rail engines. When using traditional pump-pipe-injector systems the lower compressibility and higher cetane number of the biodiesel generates an earlier and faster combustion which further improves efficiency (and increases NOx) if the engine is developed to minimize NOx emissions. But the lower compressibility of the biodiesel does not interfere with the injection in common-rail systems, so the higher efficiency in these type of engines is only explained by the better combustion potential of the biodiesel [58]. biodiesel [58]. Biodiesel has some disadvantages in relation to diesel. It solidifies at higher temperature (~0 °C) which may be problematic in cold countries. Also, the cold weather additives for diesel do not work for biodiesel, so other additives have to be developed. Biodiesel produced from animal fats (a Biodiesel has some disadvantages in relation to diesel. It solidifies at higher temperature (~0 ◦C) which may be problematic in cold countries. Also, the cold weather additives for diesel do not work for biodiesel, so other additives have to be developed. Biodiesel produced from animal fats (a significant proportion of Brazil biodiesel, 25%) has a much higher solidification temperature (~15 ◦C) [59]. significant proportion of Brazil biodiesel, 25%) has a much higher solidification temperature (~15 °C) [59]. In prolonged storage it may oxidize and, as it is a biofuel, it may be a source of bacteriologic contamination [59]. Iodine Value (IV) Iodine Value (IV) The hydrogenation of oil occurs when the double bonds of the unsaturated oil are transformed into single bonds and hydrogen atoms are included where the iodine atoms were placed, as seen in Figure 8 right The hydrogenation of oil occurs when the double bonds of the unsaturated oil are transformed into single bonds and hydrogen atoms are included where the iodine atoms were placed, as seen in Figure 8, right. Figure 8, right. A fuel with a higher degree of unsaturation has a higher IV, usually produces higher values of NOx [62–64] and has lower stability to oxidation. With that in mind, in Europe the IV of the biodiesel is limited to 120, restricting biodiesel produced from unsaturated oils such as sunflower or soy [64] and allowing rapeseed oil based biodiesel. This restriction imposes limitations to the biodiesel production of southern European countries and imports from Brazil and the USA (usually from soy) a d the e i a la e debate about it Biodie el e ifi atio i the USA B a il a d Au t alia do ot A fuel with a higher degree of unsaturation has a higher IV, usually produces higher values of NOx [62–64] and has lower stability to oxidation. With that in mind, in Europe the IV of the biodiesel is limited to 120, restricting biodiesel produced from unsaturated oils such as sunflower or soy [64] and allowing rapeseed oil based biodiesel. This restriction imposes limitations to the biodiesel production of southern European countries and imports from Brazil and the USA (usually from soy) and there is a large debate about it. Biodiesel specifications in the USA, Brazil and Australia do not restrict IV. and there is a large debate about it. Biodiesel specifications in the USA, Brazil and Australia do not restrict IV. Biodiesel produced from animal fats, a highly saturated fat, induces a reduction in NOx [63] and its IV is also low [65]. Brazil uses a mixture of biodiesel produced from soy oil (75%) and tallow fat (25%), enabling the reduction of the high IV from the soy oil biodiesel [65]. Iodine Value (IV) Iodine Value (IV) The iodine value (IV) is a measure of the level of unsaturation of the biodiesel or oils. This is an easy test to perform, basically it consists on measuring the amount of iodine that can be added to saturate 100 g of the fuel. The degree of unsaturation relates to the number of double bonds (Figure 8a) between carbon atoms and shows its stability to oxidation and/or to polymerization. A biodiesel from an unsaturated oil has various double bonds. When iodine is added, two atoms are connected to the carbon atoms that were previously connected by the double bond (Figure 8b). The higher the iodine value of the biodiesel (oil or fat), the lower the melting temperature. So, biodiesels produced from animal fat (saturated) have melting points usually above 15 ◦C [59]. The iodine value (IV) is a measure of the level of unsaturation of the biodiesel or oils. This is an easy test to perform, basically it consists on measuring the amount of iodine that can be added to saturate 100 g of the fuel. The degree of unsaturation relates to the number of double bonds (Figure 8a) between carbon atoms and shows its stability to oxidation and/or to polymerization. A biodiesel from an unsaturated oil has various double bonds. When iodine is added, two atoms are connected to the carbon atoms that were previously connected by the double bond (Figure 8b). The higher the iodine value of the biodiesel (oil or fat), the lower the melting temperature. So, biodiesels produced from animal fat (saturated) have melting points usually above 15 °C [59]. (a) (b) Figure 8. Partial carbon-hydrogen chain showing a double bond (a) and the inclusion of two iodine ato (b) Figure 8. Partial carbon-hydrogen chain showing a double bond (a) and the inclusion of two iodine atoms (b). (b) (a) (a) (b) Figure 8. Partial carbon-hydrogen chain showing a double bond (a) and the inclusion of two iodine atoms (b) Figure 8. Partial carbon-hydrogen chain showing a double bond (a) and the inclusion of two iodine atoms (b). Iodine Value (IV) Iodine Value (IV) However, biodiesel produced from anchovies, an unsaturation fat, has an IV of 185 and tends to reduce the emission of NOx (in 11%) when compared to diesel fuel for similar conditions [66] proving that at least in some Biodiesel produced from animal fats, a highly saturated fat, induces a reduction in NOx [63] and its IV is also low [65]. Brazil uses a mixture of biodiesel produced from soy oil (75%) and tallow fat (25%), enabling the reduction of the high IV from the soy oil biodiesel [65]. However, biodiesel produced from anchovies, an unsaturation fat, has an IV of 185 and tends to reduce the emission of NOx (in 11%), when compared to diesel fuel for similar conditions [66], proving that, at least in some cases, there is no direct link between IV and NOx emissions. 8. Esters (Biodiesel) Acrolein, which is a toxic substance, is seen as a problem for the biodiesel burning. However, acrolein is a by-product of glycerol burning and biodiesel should have almost no glycerol. In fact, Energies 2020, 13, 4086 contamination [59] Acrolein, whi 15 of 33 owever, a study [60] showed that biodiesel exhaust emissions may present a lower risk to human health than diesel emissions in IC engines. study [60] showed that biodiesel exhaust emissions may present a lower risk to human health than diesel emissions in IC engines. There are other processes for the production of biodiesel other than esterification and transesterification. One of the routes involves a mixture of biomass and water (to keep it moisten) undergoing a high temperature (300–350 ◦C) and high pressure (120–180 bar) process (hydro thermal upgrading—HTU) to remove part (85%) of its oxygen [61]. The resulting oil can be physically or chemically refined into biodiesel. This is a second-generation process. There are other processes for the production of biodiesel other than esterification and transesterification. One of the routes involves a mixture of biomass and water (to keep it moisten) undergoing a high temperature (300–350 °C) and high pressure (120–180 bar) process (hydro thermal upgrading — HTU) to remove part (85%) of its oxygen [61]. The resulting oil can be physically or chemically refined into biodiesel. This is a second-generation process. cases, there is no d 9. Vegetable Oils Energies 2020, 13, x; doi: FOR PEER REVIEW www.mdpi.com/journal/energies 9. Vegetable Oils The first diesel engines developed by Rudolf Diesel were fuelled by vegetable oils and only later was mineral diesel oil used. One of the problems of using raw vegetable oils is their very high viscosity. It is possible to reduce the viscosity by increasing the temperature of the vegetable oil to The first diesel engines developed by Rudolf Diesel were fuelled by vegetable oils and only later was mineral diesel oil used. One of the problems of using raw vegetable oils is their very high viscosity. It is possible to reduce the viscosity by increasing the temperature of the vegetable oil to values similar to those of diesel prior to injection. But oils and fats have various levels of saturation (double bonds) indicated by their iodine value (IV). The higher the IV, the higher is the probability of the oil or fat to polymerize at high temperature, leading to the formation of heavy and sticky deposits (gums) at the injector tips and piston rings, resulting in a damaged engine. Energies 2020, 13, 4086 The previous f are called oxygenat 16 of 33 ch they nd they 16 of 33 ch they nd they 10. Other Oxygenate Fuels that there is a strong correla reduction Furthermore thes The previous fuels (alcohols, ethers and esters) have oxygen in their molecule, for which they are called oxygenate fuels. These oxygen atoms significantly improve the fuel combustion and they reduce the potential for particulate matter (PM) production. A work by Harlt [67] showed (Figure 9) that there is a strong correlation between oxygen mass content of the fuel and relative soot (PM) reduction. Furthermore, these researchers proved that oxygenate fuels with higher hydrogen content (higher H/C ratio) tend to further reduce soot formation. Therefore, light oxygenate fuels such as DME or DMM (dimethoxy methane) are better suited as far as soot emissions are concerned. While DME has a high cetane number (~60), DMM, has a relatively low CN of 30 (see Table 3), which reduces the CN number of the mixture diesel-DMM, increasing its ignition delay [68]. But its addition to diesel fuel greatly reduces PM production. DMM has low lubricity so it cannot be used as straight fuel on a diesel engine, without additives for lubricity and cetane number enhancement [67]. reduction. Furthermore, these researchers proved that oxygenate fuels with higher hydrogen content (higher H/C ratio) tend to further reduce soot formation. Therefore, light oxygenate fuels such as DME or DMM (dimethoxy methane) are better suited as far as soot emissions are concerned. While DME has a high cetane number (~60), DMM, has a relatively low CN of 30 (see Table 3), which reduces the CN number of the mixture diesel-DMM, increasing its ignition delay [68]. But its addition to diesel fuel greatly reduces PM production. DMM has low lubricity so it cannot be used as straight fuel on a diesel engine, without additives for lubricity and cetane number enhancement [67]. Diesel engines and recent Spark-Ignition direct injection engines suffer from PM production, as the time for fuel preparation is scarce. In these types of engines, the fuel injection takes place very late in the cycle, reducing the time required for proper fuel-air mixture, leading to PM production. Oxygenate fuels, such as alcohols and ethers, are known for PM production reduction as they lack C- C bonds [69]. Figure 9. Soot (PM) reduction as a function of fuel oxygen content [67]. Petrol Figure 9. Soot (PM) reduction as a function of fuel oxygen content [67]. Figure 9. Soot (PM) reduction as a function of fuel oxygen content [67]. Figure 9. 10. Other Oxygenate Fuels that there is a strong correla reduction Furthermore thes Soot (PM) reduction as a function of fuel oxygen content [67]. Dimethyl carbonate (DMC) and methyl formate (MeFo) are knock resistant (with high RON) esters which are suitable for direct-injection (DI) high-compression Spark-Ignition (SI) engines [70,71]. These fuels also offer the potential for significantly PM production reduction of SI-DI engines, which is a beneficial surplus. They are both used in the chemical industry as solvents and other applications. Both DMC (C3H6O3) and MeFo (C2H4O2) have equal elemental ratios, so they have the Diesel engines and recent Spark-Ignition direct injection engines suffer from PM production, as the time for fuel preparation is scarce. In these types of engines, the fuel injection takes place very late in the cycle, reducing the time required for proper fuel-air mixture, leading to PM production. Oxygenate fuels, such as alcohols and ethers, are known for PM production reduction as they lack C-C bonds [69]. applications. Both DMC (C3H6O3) and MeFo (C2H4O2) have equal elemental ratios, so they have the same heating value (Table 3) and similar knocking resistance. Tests comparing to petrol showed a higher fuel consumption for these oxygenates, but the engine power output was significantly increased (by 13%) as a result of a higher mixture heating value when compared to a petrol-air Dimethyl carbonate (DMC) and methyl formate (MeFo) are knock resistant (with high RON) esters which are suitable for direct-injection (DI) high-compression Spark-Ignition (SI) engines [70,71]. These fuels also offer the potential for significantly PM production reduction of SI-DI engines, which is a beneficial surplus. They are both used in the chemical industry as solvents and other applications. Both DMC (C3H6O3) and MeFo (C2H4O2) have equal elemental ratios, so they have the same heating value (Table 3) and similar knocking resistance. Tests comparing to petrol showed a higher fuel consumption for these oxygenates, but the engine power output was significantly increased (by 13%) as a result of a higher mixture heating value when compared to a petrol-air mixture. But the better improvement was in terms of PM reduction, where the particulate number (PN) was reduced by one (DMC) and two (MeFo) orders of magnitude [69] compared to diesel. In terms of novel oxygenate fuels for compression ignition engines, the oxymethylene ethers OMEs (CH3O(CH2O)nCH3) seem very promising [72]. These fuels are also known by the name 17 of 33 Energies 2020, 13, 4086 polyoxymethylene dimethyl ethers (PODEn). 10. Other Oxygenate Fuels that there is a strong correla reduction Furthermore thes This is a class of different fuels, with n varying from 1 (dimethoxy methane or DMM) to more than 5. However, for n = 1 we have the DMM which is very volatile (almost like the DME, which is n = 0) and for n = 2 the fuel has a low flash point [73] so the more usable fuels have the n ranging from 3 to 4 (OME3-4 or PODE3-4). Higher values of n have very high melting points and may precipitate when mixed with diesel fuel. Diesel engine tests of this fuel (PODE3-4 mixed with diesel) showed the potential for a faster combustion, lower PM production and slightly higher NOx emissions. The engine efficiency improved for all conditions, when compared to straight diesel fuel and the CO and HC emissions were lowered [73]. These fuels have the added benefit of being able to be produced from biomass feedstock [74] and do not have C-C bonds, therefore burning easily and cleanly. 11. Synthetic Fuels—Fischer-Tropsch Process It is possible to produce synthetic liquid fuels from more traditional fuels such as coal, natural gas or hydrogen. The processes are initiated by the production of syngas (a mixture of hydrogen and carbon monoxide) which then passes through catalytic reactions, such as the Fischer-Tropsch (F-T) process, leading to the production of liquid hydrocarbons [75]. The ratio between H2 and CO on the syngas and the type of catalyst determines the types of hydrocarbons produced, which can be similar to petrol, diesel or lubricating oil. The relevant equations are the following: nCO + (2n + 1) H2 →CnH2n+2 + nH2O (paraffins) (9) nCO + 2nH2 →CnH2n + nH2O (olefins) (10) (9) (10) nCO + 2nH2 →CnH2n + nH2O (olefins) Diesel F-T has a higher compressibility than fossil diesel (which is not an issue for common-rail engines), has a higher cetane number (Table 3) and the potential for NOx and PM (particulate matter) production is lower [36]. As these synthetic fuels are sulphur free, their combustion is very clean with low PM emitting potential. However, it seems to be sensitive to EGR (exhaust gas recirculation) levels, producing high levels of smoke above a certain value of EGR. But diesel F-T may have different formulations with different distillation curves, which changes some of its properties [76]. These synthetic fuels are hydrocarbons, which do not have oxygen in their molecule, so the reduction of PM production cannot be attributed to that element, as is for oxygenate fuels. These fuels are seldom known as GTL (gas to liquid). If the base fuel to produce the syngas is biomass, the name changes to BTL (biomass to liquid) and are considered second generation biofuels. During WWII the axis countries had huge shortages of oil, so most of the required fuels and lubricants were produced with these techniques (synthetic fuels) from coal (called CTL—coal to liquid, [77]), like some decades later did South Africa to overcome the oil embargo they were subjected to [78]. and also, with limited air: The major differences between ICL and DCL are: - DCL uses just one step and is more energy efficient - ICL is easier to control (“design”) the type of produ - DCL uses just one step and is more energy efficient; The production of huge quantities of CO2 (almost twi - DCL uses just one step and is more energy efficient; The production of huge quantities of CO2 (almost twi - ICL is easier to control (“design”) the type of produced fuel. p g q ( WTW basis, [79]) is one of the major drawback of these process The production of huge quantities of CO2 (almost twice the overall emission of fossil fuels, in a WTW basis, [79]) is one of the major drawback of these processes. Other problems are the large amounts of necessary thermal energy and water. The required water consumption is in the region of 1m3 (1 ton) per each barrel of fuel production [77] and the coal (bituminous) consumption is between 0.73 to 1.04 ton per barrel, according to Sasol experience [77]. Suitable catalysts are essential for each process. 11 2 BTL amounts of necessary thermal energy and water. The required water consumption is in the region of 1m3 (1 ton) per each barrel of fuel production [77] and the coal (bituminous) consumption is between 0.73 to 1.04 ton per barrel, according to Sasol experience [77]. Suitable catalysts are essential for each process. 11.2. BTL 11.1. CTL CTL (coal to liquid) fuels burn cleaner than fossil petrol or fossil diesel, as they are specifically produced to be burned in a particular type of engine. There are two methods to produce them. The indirect coal liquefaction (ICL) requires the crushing of the coal, which then is exposed to high temperature and high pressure together with water (steam) and oxygen to produce the syngas: C + H2O →CO + H2 (11) (11) and also, with limited air: and also, with limited air: C + 1 2 O2 →CO (12) (12) Energies 2020, 13, 4086 Then, the syng 18 of 33 quired Then, the syngas is transformed into liquid fuels by the F-T process. According to the required fuel (petrol, kerosene, diesel or lubricant), the F-T process has to be fed with the right proportions of H2 and CO, so it is possible to change these proportions, for example by: H2 and CO, so it is possible to change these proportions, for example by: CO + H2O +  H2 + CO2 (13) CO + H2O + →H2 + CO2 (13) DCL) the pulverized coal is exposed to hydrogen (hydrogenation) sures (pyrolysis), resulting in a syncrude liquid, which is then (13) tion) then In the direct coal liquefaction (DCL) the pulverized coal is exposed to hydrogen (hydrogenation) also at high temperatures and pressures (pyrolysis), resulting in a syncrude liquid, which is then refined. The Belgius process involves the mixing of coal with heavy oil recovered from the process and hydrogen at high pressures and temperatures, resulting in a liquid hydrocarbon: refined. The Belgius process involves the mixing of coal with heavy oil recovered from the process and hydrogen at high pressures and temperatures, resulting in a liquid hydrocarbon: nC + (n+1) H2  CnH2n+2 (14) nC + (n+1) H2 →CnH2n+2 (14) ICL and DCL are: more energy efficient; (14) The major differences between ICL and DCL are: - DCL uses just one step and is more energy efficient - ICL is easier to control (“design”) the type of produ 11.5. Gasification Fuels—VGO Gasification fuels can be obtained from biomass using forest residues (small branches and leaves) and black liquor (a paper and pulp production by-product), which produce syngas at high temperature and pressure, followed by the F-T process. Different plastics (namely those non-recyclable) can undergo a gasification process where, after decontamination, the gases are condensed in a high-quality oil (VGO—vacuum gas oil) that may be distilled into petrol and diesel. Such a processing plant could be a floating platform used to eliminate and treat the enormous amounts of plastic that litter vast parts of the oceans. The process to produce VGO is already used for the recovery of fossil heavy oils, where the heating at low pressure allows the heavy oil to boil at much lower temperatures than at atmospheric pressure. This prevents the production of cokes and therefore increase the liquid fuel production. 11.4. HVO Vegetable oils can undergo processes of cracking and/or hydrogenation similar to those in oil refineries, leading to oxygen free linear paraffinic hydrocarbons usually called HVO (hydrogenated vegetable oils) and propane. HVO are not biodiesels, as they have no oxygen in their molecule and have properties similar to fossil diesel. The hydrogen brakes the links between the glycerol and the fatty acids and deoxidize the hydroxyl and carboxyl groups, leading to a hydrocarbon without oxygen [83]. Part of the carbon is used to “brake” the glycerol and generate propane. So, this process consumes fat and hydrogen and produces long chain hydrocarbons (diesel fuel) using catalysts and high temperature (300 ◦C) and pressure (50 to 180 bar). CO and CO2 are also formed as by-products of the process. The removal of the oxygen alters some of the properties. It reduces the lubricity and its PM (smoke) production and heating value are between those of biodiesel and diesel. The cetane number of HVO is very high (CN = 82: Table 3) which will require a remapping of the engines, namely of the injection advance. Although its heating value is somehow higher than diesel, its lower density results in a lower heating value per unit volume. 11.2. BTL Some Some of the processes for transforming solid biomass (plants, wood, crops, straw –lignocellulosic) into liquid fuels (Figure 10) are similar to the above referred processes. The solid biomass is burned under a low oxygen environment (gasification) or is reacted with steam (high pressure and temperature, although lower than CTL) in the presence of adequate catalysts to produce syngas, which is then transformed into liquid fuels using the F-T process. Or, the biomass goes through a pyrolysis process (Figure 10), producing a pyrolysis oil, that is processed and distilled into the required liquid fuels. However, these processes are very biomass intensive (6 ton of biomass to produce 1 ton of BTL, [80]). lignocellulosic) into liquid fuels (Figure 10) are similar to the above referred processes. The solid biomass is burned under a low oxygen environment (gasification) or is reacted with steam (high pressure and temperature, although lower than CTL) in the presence of adequate catalysts to produce syngas, which is then transformed into liquid fuels using the F-T process. Or, the biomass goes through a pyrolysis process (Figure 10), producing a pyrolysis oil, that is processed and distilled into the required liquid fuels. However, these processes are very biomass intensive (6 ton of biomass to produce 1 ton of BTL, [80]). Figure 10. Various processes for the conversion of biomass into engine fuels (BTL). Figure 10. Various processes for the conversion of biomass into engine fuels (BTL). Figure 10. Various processes for the conversion of biomass into engine fuels (BTL). Figure 10. Various processes for the conversion of biomass into engine fuels (BTL). There are other processes for the transformation of lignocellulosic biomass into fuels such as the second-generation hydrolysis process (Figure 10) followed by the fermentation of the sugar into ethanol Energies 2020, 13, 4086 19 of 33 and the old process of anaerobic digestion. However, this process requires novel ways of increasing the bioconversion efficiency, such as performing the pre-treatment leading to cell wall degradation [81]. 11.3. GTL and the old process of anaerobic digestion. However, this process requires novel ways of increasing the bioconversion efficiency, such as performing the pre-treatment leading to cell wall degradation [81]. 11.3. GTL 11.3. GTL The easier way of using the F-T process is using a mixture of natural gas (methane) and steam in a catalyst bed, where it produces the syngas: CH4 + H2O + →CO + 3H2 (15) CO + H2O + →H2 + CO2 (16) (15) (16) These are called gas-to-liquid or GTL. One way to produce BTL fuels is using GTL plants and “hybridizing” them to accept syngas produced from biomass, what is sometimes called hybrid BGTL plants. These plants have a maximum efficiency of around 22% of production fuel derived from biomass [82]. 11.7. Electrofuels 11.7. Electrofuels ( p ) p [ ] Electro-fuels are very expensive to produce as the energy efficiency of its production is very low, requiring much more energy in the production process than the available in the fuel. Bannon [87] refers overall efficiency values of 73%, 22% and 13% for vehicles running on batteries, on hydrogen fuel-cell and on IC engines burning electrofuel, respectively (Figure 11). So, it is more logical to use the electricity directly stored in batteries in electric vehicles. However, this is not possible in aviation, where the heavy batteries prevent its use. Therefore, airplanes need liquid fuels to fly, so electro-fuels seem to have a future in the huge market for the renewable energy use in the air transport [51,87]. And bear in mind the proposed 50% of EU aviation renewable fuel (electrofuel) by 2050 [85]. Figure 11. Vehicle overall (WTW) efficiency for electric, fuel cell and electrofuel burning in a IC engine (based on [87]). Figure 11. Vehicle overall (WTW) efficiency for electric, fuel cell and electrofuel burning in a IC engine (based on [87]). Figure 11. Vehicle overall (WTW) efficiency for electric, fuel cell and electrofuel burning in a IC engine (based on [87]). Figure 11. Vehicle overall (WTW) efficiency for electric, fuel cell and electrofuel burning in a IC engine (based on [87]). When comparing to biofuels, electrofuels from zero-carbon renewable sources (solar, wind, etc.) have much less sustainability risks and use one order of magnitude less land [51] Additionally, the requirement for water is far inferior and there is no risk for groundwater pollution (through chemical fertilization, such as nitrogen). However, if the electricity production is based on any amount of carbon intensity, its inherent low WTW efficiency causes it to produce high levels of CO2. For example, electrofuels produced from the European energy mix average grid [6] production would have a CO2 intensity three times higher than current fossil fuels [51]. In terms of cost, the values are extremely high, more than five times the cost of fossil fuels and, therefore, much more than the price for biofuels [88]. 11.7. Electrofuels 11.7. Electrofuels The denomination electrofuels has been gaining importance in the last decade [28]. It does mean renewable energy-based fuels which are of non-biologic origin, so they are not based on crops. The EC [85] introduced the term Indirect Land Use Change (ILUC) to account for the consequences (sustainability) of the production of biofuels on the land use. As a sustainability measure, the biofuels produced through ILUC will not be included in terms of renewable targets after 2030. The electro-fuels, such as hydrogen and its derivatives, are basically produced from the (renewable) electricity and water by electrolysis or other physical/chemical processes. Then the hydrogen is combined with the carbon in the CO2 (through carbon monoxide) to produce the syngas required for the synthesis (Fischer-Tropsch) process [86]. The denomination electrofuels has been gaining importance in the last decade [28]. It does mean renewable energy-based fuels which are of non-biologic origin, so they are not based on crops. The EC [85] introduced the term Indirect Land Use Change (ILUC) to account for the consequences (sustainability) of the production of biofuels on the land use. As a sustainability measure, the biofuels produced through ILUC will not be included in terms of renewable targets after 2030. The electro- fuels, such as hydrogen and its derivatives, are basically produced from the (renewable) electricity and water by electrolysis or other physical/chemical processes. Then the hydrogen is combined with the carbon in the CO2 (through carbon monoxide) to produce the syngas required for the synthesis (Fischer-Tropsch) process [86]. Electro-fuels are very expensive to produce as the energy efficiency of its production is very low, requiring much more energy in the production process than the available in the fuel. Bannon [87] refers overall efficiency values of 73%, 22% and 13% for vehicles running on batteries, on hydrogen fuel-cell and on IC engines burning electrofuel, respectively (Figure 11). So, it is more logical to use the electricity directly stored in batteries in electric vehicles. However, this is not possible in aviation, where the heavy batteries prevent its use. Therefore, airplanes need liquid fuels to fly, so electro-fuels seem to have a future in the huge market for the renewable energy use in the air transport [51,87]. And bear in mind the proposed 50% of EU aviation renewable fuel (electrofuel) by 2050 [85]. 11.6. Pyrolysis Fuels (PL) Pyrolysis is a high temperature reaction without air contact. It produces gases, liquids and solids, being the liquid fractions (PL) the important ones for IC engines. Various substances, such as plastics, biomass and used tires can be used as raw material. Using the latter material (tires), the resulting liquid has a high heating value (over 40 MJ/kg [84]) and some other properties are similar to fossil Energies 2020, 13, 4086 Energies 2020, 13, x FOR 20 of 33 12 of 40 diesel, but the cetane number is very low (17.6—Table 3), which allows it to be used mixed with fossil diesel (or biodiesel) only in small percentages. liquid has a high heating value (over 40 MJ/kg [84]) and some other properties are similar to fossil diesel, but the cetane number is very low (17.6—Table 3), which allows it to be used mixed with fossil diesel (or biodiesel) only in small percentages. 11.8. Solar Fuels Solar fuels are new concepts for renewable liquid fuel production. These fuels are produced from solar energy through direct or indirect techniques. They can be electrofuels, where the required electricity is generated from photovoltaic sources, or fuels generated from processes involving photochemical, thermochemical or biochemical (photosynthesis) involving solar energy [29]. The solar energy in these processes are used for breaking the water and/or the CO2 producing the H2 and CO (syngas) required for the subsequent Fischer-Tropsch process. So, some of the earlier mentioned synthetic fuels produced using electricity or thermal energy can also be created using solar energy, through photovoltaic and/or thermal concentration. Enzymatic conversion of CO2 can also be accomplished with the use of solar energy, leading to chemicals such as methane and CO [93]. One process involves the reaction of CO2 and H2O at high temperatures (~1400 ◦C) in the presence of a cerium oxide catalyst, followed by hydrolysis at 800 ◦C, producing H2 and CO [94]. This process of producing liquid hydrocarbons through the processing of the syngas is already being explored commercially, although requires further optimisation involving the use of metal oxides in powerful solar concentrators [29]. Photochemical and photoelectrochemical systems have the active light-absorbing materials directly integrated into the cathode and/or anode electrodes which are placed in contact with the electrolyte. The photosensitized electrodes convert light into an electric current that is then used to split the water into hydrogen and oxygen. The combination of photovoltaic and electrochemical processes is also a promising technology as it allows the separate optimization of both processes [95]. Thermochemical processes have a lot of potential when using very high values of solar concentration [29]. However, the integration of the process into the solar reactor originates significant thermal losses and various other difficulties for upscaling installations have hindered the viability of this approach, with efficiencies lower than 10% [29], prior to considering its use in IC engines, where the efficiency barely reaches the 40% mark or in fuel cells (~60% efficiency). 11.7. Electrofuels 11.7. Electrofuels One of the important factor on the production of electrofuels is the production of h d b l l h h ll h l ff h h When comparing to biofuels, electrofuels from zero-carbon renewable sources (solar, wind, etc.) have much less sustainability risks and use one order of magnitude less land [51] Additionally, the requirement for water is far inferior and there is no risk for groundwater pollution (through chemical fertilization, such as nitrogen). However, if the electricity production is based on any amount of carbon intensity, its inherent low WTW efficiency causes it to produce high levels of CO2. For example, electrofuels produced from the European energy mix average grid [6] production would have a CO2 intensity three times higher than current fossil fuels [51]. In terms of cost, the values are extremely high, more than five times the cost of fossil fuels and, therefore, much more than the price for biofuels [88]. One of the important factor on the production of electrofuels is the production of 21 of 33 Energies 2020, 13, 4086 hydrogen by electrolysis, which conventionally has a low efficiency [51]. However, high temperature electrolysis, or steam electrolysis, is a novel process where the hydrogen and oxygen are generated at temperatures between 700 and 1000 ◦C [89] with much higher efficiencies. The higher efficiencies are, in part, a result of the high temperature of the steam and because the heat required for these high temperatures comes from the subsequent F-T process itself [51] Another possibility of producing electrofuels is through the high-temperature co-electrolysis of CO2 and H2O [90] using solid oxide electrolysis cells (SOECs) [91]. These promising advanced electrochemical energy storage and conversion devices have high conversion efficiencies and convert directly CO2 and water into syngas, leading directly to the production of F-T fuels, but are still in an early stage of development [92]. 13. Nitromethane Nitromethane (CH3NO2—Table 3) is a fuel with oxygen and nitrogen beyond the usual carbon and hydrogen, known for its explosive behaviour and by the huge power improvement it can offer to powerful engines. As it has a high proportion of O and N (52.5% of oxygen and 75.4% of N + O), its heating value is low but, as it has a very low stoichiometric A/F (1.7, Table 3), its mixture with air, in a volume base, carries much more energy than any other fuel, a massive 2.3 times the mixture of air-petrol (see Table 3). The very low A/F requires large amounts of injected fuel (8.5 times more mass, compared to petrol) which, allied to the high latent heat of vaporization (almost twice of the petrol, see Table 3), requires huge amounts of heat to vaporize. Nitromethane is used mostly on the “top fuel” category of drag racing, where the consumption can be 25 L for a race of 300 m ran in 3.6 s and where the finishing speeds are in excess of 530 km/h. Strangely these 10,000 cv plus engines have no cooling other than the latent heat of the fuel. Some other applications are its use as an additive (~5%), usually of methanol-based fuels, such as those for aero models. 12. Dimethylfuran (DMF) Dimethylfuran (DMF—CH3-C4H2-O-CH3—Table 3) is a biofuel with the potential to substitute petrol, as it has properties between petrol and ethanol. Its RON is even higher than for ethanol, but its knocking behaviour is slightly lower than ethanol, probably because its latent heat of vaporization is much lower (similar to gasoline), preventing the effective mixture cooling of the ethanol [96]. Also, its laminar flame speed is lower than for ethanol, being even slightly lower than for petrol [97]. Its boiling temperature is high (93 ◦C, Table 3), which makes it a less volatile fuel and more practical for transport and storage, although it may be difficult to start a cold engine. Unlike ethanol and methanol, it is insoluble in water, reducing some of the alcohol’s storage problems. DMF can be obtained from fructose, so it may be a biofuel produced through a chemical or biochemical route using a direct process using catalysts and its production consumes about one third of the energy required for the production of ethanol [98], where its low latent heat of vaporization helps. Energies 2020, 13, 4086 22 of 33 14. Acetylene This fuel (C2H2) was occasionally used in IC engines, mostly during the world wars, as there were no oil-based fuels for the general public. It is produced from the reaction of calcium carbide and water: Ca C2 + 2H2O →C2H2 + Ca (OH)2 (17) (17) Ca C2 + 2H2O →C2H2 + Ca (OH)2 This process was commonly used in the pit gasometers and on the front lamps of old cars and was occasionally produced in-board (in the trunk of the car) and supplied to the engine. This process was commonly used in the pit gasometers and on the front lamps of old cars and was occasionally produced in-board (in the trunk of the car) and supplied to the engine. Acetylene RON is 40 (Table 3), so it is unsuitable for today’s high compression ratio (CR) engines, but it has a fast flame propagation speed, which may reduce knock occurrence. One of the best properties of acetylene is its adiabatic temperature, one of the highest, that can exceed the 3000 ◦C when burned with straight oxygen, but this is not an advantage for an IC engine. 15. Ammonia Figure 12 Torque and spark timing required for the mixing of ammonia to petrol (adapted from Figure 12. Torque and spark timing required for the mixing of ammonia to petrol (adapted from [100]). Figure 12 Torque and spark timing required for the mixing of ammonia to petrol (adapted from Figure 12. Torque and spark timing required for the mixing of ammonia to petrol (adapted from [100]). [100]). It can be burned in CI engines when mixed with diesel, but it would be advantageous to be mixed with biodiesel or DME as these fuels have higher cetane numbers (CN) [104]. Ammonia causes irritation in small amounts and may be lethal in higher concentrations. However, its distinct and strong smell and the fact that is lighter than air, reduces its risks. Ammonia is largely used in the word, having specific production, storage and delivery installations and procedures, so it has been extensively tested throughout the world. Also, unlike petrol it is not carcinogenic, its combustion does not produce smoke and it is much less prone to explosions [104] Thus, the combustion of straight ammonia in a SI engine is not easy but it is possible if measures are taken to ensure that the referred combustion deficiencies (very high ignition energy, flammability limits and slow combustion) are overcome, for example using multiple spark location and very high energy ignition and compact combustion chambers [101]. Supercharging seems to be a good option [102] and some researchers used plasma ignition with good results [103]. As one of the major problems is its slow combustion, engine conditions of low charge and high speed can only be achieved using a combustion “promotor” such as hydrogen [53], petrol or even diesel fuel, enabling a better ignibility and increased combustion velocity. E i 2020 13 d i FOR PEER REVIEW d i /j l/ i does not produce smoke and it is much less prone to explosions [104]. At the moment, ammonia is produced from natural gas (70%) and from coal (30%) by the Haber- Bosch process [105] where hydrogen and nitrogen react (3H2 + N2  2NH3) in an iron oxide catalyst at temperatures ranging from 380 to 500 °C. Ammonia was produced from renewable energy (hydro) by water hydrolyses in the 40s, but high production costs and the aging of the facilities originate production to stop on the 80s [106]. 15. Ammonia Ammonia (NH3) is an important substance used as a fertilizer throughout the world with a yearly production of over 150 million tons. More than half the world population rely on the enhanced crop production boosted by the nitrogen in the ammonia, but its production is very high energy intensive (uses about 2% of the total energy consumed in the world) and it produces approximately 1% of the CO2 emissions worldwide. In general, the production of one molecule of NH3 results in the emission of a molecule of CO2. In nature, there is the production of ammonia by the decomposition (rotting) of vegetable and animal wastes by bacteria. It can also be produced during the pyrolysis of coal, as a by-product of coke and coal gas production. In these cases, the ammonia appears as ammonium hydroxide, a liquid normally used as a cleaning agent usually known as “ammonia”. Ammonia is also used in absorption cycles refrigeration systems. Anhydrous ammonia (without added water) may be a substitute for petrol in SI engines or even diesel engines and the main interest is to use it as an “energy carrier”, to substitute the electricity (or the hydrogen) as a means to transport energy from the place where is produced (wind farm or nuclear power station) to the location where will be used, such as to power vehicles. As there is no carbon in its molecule, it does not produce any CO2, CO nor HC. Comparing it to hydrogen (another energy carrier), one litre of liquid ammonia (@10 bar and 25 ◦C) has 30% more hydrogen than 1 L of liquid hydrogen (@-253 ◦C). Therefore, it makes much more sense to use ammonia as an energy carrier than hydrogen. In terms of NOx production, the burning of ammonia will produce some thermally (Zeldovich mechanism) but its combustion also produces NOx because its molecule has nitrogen atoms. However, ammonia has a relatively low adiabatic temperature, lower than usual hydrocarbons and much lower than hydrogen (Table 3) reducing the potential for Zeldovich NOx production. 23 of 33 C, Table he very Energies 2020, 13, 4086 Other crucial p 3) a very high ene In terms of heating value, ammonia has a low value (18.6 MJ/kg, Table 3), less than half of petrol and the comparison gets worse when done in volume, where it has slightly more than 1/3 of the energy of petrol. 15. Ammonia Also, as ammonia is a gas at atmospheric conditions, it requires a pressure tank, cylindrical or toroidal, similar to those for LPG, where only 80% of the volume can be filled, which reduces the vehicle range. g p g p p difficulties on its use as an IC engine fuel. Other problems are the narrow limits for ignition (flammability, see Table 3) and the reduced speed for flame propagation, five times slower than petrol [99] and 30 times slower than hydrogen [52]. Adding 4% of ammonia to petrol reduced its burning speed in 15% [99]. As a result, the spark timing required for the burning of the petrol-ammonia mixture increases with the percentage of ammonia (Figure 12, [100]). g Other crucial properties of ammonia are the very high auto-ignition temperature (651 ◦C, Table 3), a very high energy required for ignition (much higher than the required for petrol) and the very high latent heat of vaporization (2450 kJ/kg, compared to 380 for petrol), which introduces further difficulties on its use as an IC engine fuel. Other problems are the narrow limits for ignition (flammability, see Table 3) and the reduced speed for flame propagation, five times slower than petrol [99] and 30 times slower than hydrogen [52]. Adding 4% of ammonia to petrol reduced its burning speed in 15% [99]. As a result, the spark timing required for the burning of the petrol-ammonia mixture increases with the percentage of ammonia (Figure 12, [100]). Thus, the combustion of straight ammonia in a SI engine is not easy but it is possible if measures are taken to ensure that the referred combustion deficiencies (very high ignition energy, flammability limits and slow combustion) are overcome, for example using multiple spark location and very high energy ignition and compact combustion chambers [101]. Supercharging seems to be a good option [102] and some researchers used plasma ignition with good results [103]. As one of the major problems is its slow combustion, engine conditions of low charge and high speed can only be achieved using a combustion “promotor” such as hydrogen [53], petrol or even diesel fuel, enabling a better ignibility and increased combustion velocity. Figure 12. Torque and spark timing required for the mixing of ammonia to petrol (adapted from Figure 12. Torque and spark timing required for the mixing of ammonia to petrol (adapted from [100]). 15. Ammonia It can be burned in CI engines when mixed with diesel, but it would be advantageous to be mixed with biodiesel or DME as these fuels have higher cetane numbers (CN) [104]. Ammonia causes irritation in small amounts and may be lethal in higher concentrations. However, its distinct and strong smell and the fact that is lighter than air, reduces its risks. Ammonia is largely used in the word, having specific production, storage and delivery installations and procedures, so it has been extensively tested throughout the world. Also, unlike petrol it is not carcinogenic, its combustion does not produce smoke and it is much less prone to explosions [104]. Energies 2020, 13, x; doi: FOR PEER REVIEW www.mdpi.com/journal/energies At the moment, ammonia is produced from natural gas (70%) and from coal (30%) by the Haber-Bosch process [105] where hydrogen and nitrogen react (3H2 + N2 →2NH3) in an iron oxide catalyst at temperatures ranging from 380 to 500 ◦C. Ammonia was produced from renewable energy (hydro) by water hydrolyses in the 40s, but high production costs and the aging of the facilities originate production to stop on the 80s [106]. 24 of 33 16 of 40 Energies 2020, 13, 4086 E i 2020 13 FOR Synthetic gasoline (by the Fisher-Tropsch process) production requires 95.3 MJ/kg and its heating value is 42.5 MJ/kg, which means that it requires 2.25 times its energy content to be produced. In the case of ammonia (Haber-Bosch process plus H2 production and N2 separation), the production of 1 kg requires 43.2 MJ [107] and its heating value is 18.6 MJ/kg. Therefore, it requires 2.3 times its energy content to be produced, value similar to the F-T petrol production. Synthetic gasoline (by the Fisher-Tropsch process) production requires 95.3 MJ/kg and its heating value is 42.5 MJ/kg, which means that it requires 2.25 times its energy content to be produced. In the case of ammonia (Haber-Bosch process plus H2 production and N2 separation), the production of 1 kg requires 43.2 MJ [107] and its heating value is 18.6 MJ/kg. Therefore, it requires 2.3 times its energy content to be produced, value similar to the F-T petrol production. 15. Ammonia In terms of bio-production, it is necessary to use 2.72 kg of corn to produce 1 kg of ethanol, whereas it is necessary to use 3 kg of the same cereal to produce 1 kg of ammonia, using processes involving gasification and synthesis [108]. As ethanol has an energy density of 25 MJ/kg and ammonia only has 18.6 MJ/kg, in terms of energy, ammonia use is 1.5 worse than ethanol. And the production of ethanol from corn is a bad energetic efficiency when compared to the Brazilian production from sugar cane. In terms of bio-production, it is necessary to use 2.72 kg of corn to produce 1 kg of ethanol, whereas it is necessary to use 3 kg of the same cereal to produce 1 kg of ammonia, using processes involving gasification and synthesis [108]. As ethanol has an energy density of 25 MJ/kg and ammonia only has 18.6 MJ/kg, in terms of energy, ammonia use is 1.5 worse than ethanol. And the production of ethanol from corn is a bad energetic efficiency when compared to the Brazilian production from sugar cane. g For ammonia to be used as an energy carrier, we know that its production through the Haber-Bosch process (3H2 + N2 →2NH3 @ 500 ◦C and 300 bar) requires 43 MJ/kg [107], already including the production of hydrogen and separation of nitrogen from the air. If ammonia if burned in an IC engine with an efficiency of 40%, then the overall electricity-to-electricity efficiency will be 16%. The same study for liquid hydrogen (see in the hydrogen section) showed this efficiency to be 19.5%. p g For ammonia to be used as an energy carrier, we know that its production through the Haber- Bosch process (3H2 + N2  2NH3 @ 500 °C and 300 bar) requires 43 MJ/kg [107], already including the production of hydrogen and separation of nitrogen from the air. If ammonia if burned in an IC engine with an efficiency of 40%, then the overall electricity-to-electricity efficiency will be 16%. The same study for liquid hydrogen (see in the hydrogen section) showed this efficiency to be 19.5%. These examples show that the production and use of ammonia is not, as yet, energetically nor economically viable and it will require the development of more efficient processes. 16. Turpentine 16. Turpentine Spirit of turpentine or just turpentine is the resulting fluid from the distillation of pine resin. Traditionally it was used as a solvent (as in dry cleaners) and different trees produce slightly different compositions of the turpentine. Turpentine can also be produced straight from the wood through what is called destructive distillation, a kind of pyrolysis. Spirit of turpentine or just turpentine is the resulting fluid from the distillation of pine resin. Traditionally it was used as a solvent (as in dry cleaners) and different trees produce slightly different compositions of the turpentine. Turpentine can also be produced straight from the wood through what is called destructive distillation, a kind of pyrolysis. Although world annual pine resin production is low at less than a thousand tons [111], there is the development of genetically improved trees to produce high yields of resin [112,113]. However, only the small fraction of 20% of the resin may be transformed into turpentine [114]. Another source of turpentine is the black liquor, a by-product of the pulp and paper industry. , py y Although world annual pine resin production is low at less than a thousand tons [111], there is the development of genetically improved trees to produce high yields of resin [112,113]. However, only the small fraction of 20% of the resin may be transformed into turpentine [114]. Another source of turpentine is the black liquor, a by-product of the pulp and paper industry. Turpentine (C10H16) has been used in IC engines from 1824, when Samuel Morey [115] patented an atmospheric engine using it. But the better-known use of turpentine has been in the post-WWII by Soichiro Honda (founder of the Honda Motor Co), who produced motorcycles with small army-surplus engines (Figure 13) which run on turpentine, that he would sell himself after 1946, because of the lack of petrol in post-war Japan. p q , y p p p p p y Turpentine (C10H16) has been used in IC engines from 1824, when Samuel Morey [115] patented an atmospheric engine using it. But the better-known use of turpentine has been in the post-WWII by Soichiro Honda (founder of the Honda Motor Co), who produced motorcycles with small army- surplus engines (Figure 13) which run on turpentine, that he would sell himself after 1946, because of the lack of petrol in post-war Japan. Figure 13. Soichiro Honda motorcycle (1947 Honda A-type—adapted from world.honda.com). Figure 13. 15. Ammonia An off-shore wind project for the production of ammonia for use in vessels (Zero Emission Energy Distribution at Sea—ZEEDS [109]) plans water hydrolysis and nitrogen separation from air using the generated wind electricity. It is estimated that this ammonia would be three times more expensive than 3.5% sulphur heavy oil. Another interesting use of ammonia is the direct use in fuel cells [110]), that generally require pure hydrogen. These examples show that the production and use of ammonia is not, as yet, energetically nor economically viable and it will require the development of more efficient processes. An off-shore wind project for the production of ammonia for use in vessels (Zero Emission Energy Distribution at Sea—ZEEDS [109]) plans water hydrolysis and nitrogen separation from air using the generated wind electricity. It is estimated that this ammonia would be three times more expensive than 3.5% sulphur heavy oil. Another interesting use of ammonia is the direct use in fuel cells [110]), that generally require pure hydrogen. 17. Glycerine (or Glycerol) Before the intense production of biodiesel, the glycerin was a valuable substance for skin creams, lip sticks and as a food additive. However, the huge quantities of biodiesel produced worldwide generated large surpluses of glycerin, with its value plummeting, as there is no market for it. For each part of generated biodiesel, the transesterification process produces 10% of glycerin. Although glycerin (C3H8O3) may be burned, its atmospheric combustion (at temperatures lower than 300 ◦C) may produce toxic compounds such as the aldehyde acrolein. Acrolein is produced by the dehydration of glycerol and it is the black and sticky substance produced during the exposure at high temperature of vegetable oils, such as the deposits on the frying pans and responsible for their acrid smell. It has been associated to lung cancer [120], so its emission should be avoided. As a fuel, glycerin is a very difficult substance to burn in an engine. It solidifies at 18 ◦C, so it has a high viscosity and has to be injected hot (~100 ◦C) to enable sufficient atomization. Its auto-ignition temperature is 390 ◦C, so it is too high for straight use in compression ignition engines. Some researchers mixed it with diesel up to 20% [121], but the intake air needed to be heated up to 100 ◦C to sustain stable combustion. The power was slightly reduced, and the efficiency was slightly increased, whereas NOx and PM production were reduced, mostly at high power. One of the reported problems was the difficulty to produce and maintain stable mixtures of hydrocarbons and glycerol. However, at least one company has achieved the combustion of straight glycerin in a diesel engine (Aquafuel Research Ltd., Smarden, Kent, UK). The idea is to increase the intake temperature [122] up to a level that almost any fuel (even petrol) would burn in the diesel engine. So, the intake air should be heated (~200 ◦C) as well as the fuel (~100 ◦C), so the glycerin burns cleanly and efficiently. Formula E racing uses electric cars which batteries that have to be charged in the circuit garages. This company developed the electric generators to be used by the different teams to charge the car batteries. These generators are modified diesel engines (Cummins KTA50, 50 L, V16, turbocharged, capable of over 1 MW) that work on glycerol, because it is a clean biofuel. 16. Turpentine 16. Turpentine Soichiro Honda motorcycle (1947 Honda A-type—adapted from world.honda.com). Figure 13. Soichiro Honda motorcycle (1947 Honda A-type—adapted from world.honda.com). Figure 13. Soichiro Honda motorcycle (1947 Honda A-type—adapted from world.honda.com). 25 of 33 Energies 2020, 13, 4086 Turpentine (see Table 3) has a heating value higher than petrol or diesel and, as its density is also higher, its energy density (in volume or mass) is higher than the conventional fuels. Also, as its stoichiometric A/F (14.2) is lower than petrol, in fact the mixture air-turpentine has more energy than air-petrol and theoretically should produce higher torque and power from the same engine [116]. But its RON is lower than petrol, so the engines require less ignition advance when turpentine is added to petrol. Turpentine can also be added to diesel fuel, but its low cetane number (20 to 25) tends to reduce the engine efficiency [117], although some authors [118] report a 1–2% efficiency improvement when the mixtures are below 40%. It can be used in dual-fuel mode by fumigation, enabling the substitution of up to 75% of the diesel fuel, with noticeable reductions of smoke production [119]. 19 Conclusions 19. Conclusions 19. Conclusions In a time where the future use of internal combustion engines and/or fossil fuels for road transport is being put into question by many policy makers all over the world, this paper presents an overview of various solutions of alternative fuels that may be used to fuel car engines of the future in a sustainable way In a time where the future use of internal combustion engines and/or fossil fuels for road transport is being put into question by many policy makers all over the world, this paper presents an overview of various solutions of alternative fuels that may be used to fuel car engines of the future in a sustainable way. in a sustainable way. Some alternatives to the combination internal combustion engine—fossil fuels to propel vehicles exist, such as battery electric cars, fuel cell hybrid vehicles or just conventional vehicles fuelled with renewable and/or biofuels. The latter alternative seems to be particularly attractive, as liquid fuels have very high energy density and they are used in devices (IC engines) that have been developed for over a century. With this in mind, the authors discussed the various propositions for alternatives to fossil fuels. It seems that future liquid fuels will still be burned in IC engines, but the vehicles will be electrically assisted (hybrids) and the exhaust emissions, CO2 emissions and fuel consumption will b l th t d ’ f il f l Some alternatives to the combination internal combustion engine—fossil fuels to propel vehicles exist, such as battery electric cars, fuel cell hybrid vehicles or just conventional vehicles fuelled with renewable and/or biofuels. The latter alternative seems to be particularly attractive, as liquid fuels have very high energy density and they are used in devices (IC engines) that have been developed for over a century. With this in mind, the authors discussed the various propositions for alternatives to fossil fuels. It seems that future liquid fuels will still be burned in IC engines, but the vehicles will be electrically assisted (hybrids) and the exhaust emissions, CO2 emissions and fuel consumption will be lower than today’s fossil fuels. be lower than today’s fossil fuels. The various properties, applications and production processes of the various alternative fuels were presented and discussed, from the more conventional alcohols and biodiesel to the more unusual ammonia or turpentine. New concepts such as electrofuels and solar fuels were introduced and discussed. 18. Fage 18. Fage There are different processes to transform glycerin into usable fuels such as its reaction with dimethyl sulphate and or methanol (producing glycerol dimethoxy ether—[125]), etherification acetylification or anaerobic fermentation [126]. However, these processes are slow and economic unviable. But it is possible to produce FAGE (fatty acid glycerol formal ester) from a reaction between glycerine and other fats (vegetable or animal). FAGE has an LHV lower than biodiesel but is very dense (Table 3), so its energy density (by volume) is similar to biodiesel. But its boiling temperature is almost 300 ◦C and solidifies at 14 ◦C, which makes it very viscous. There are different processes to transform glycerin into usable fuels such as its reaction with dimethyl sulphate and or methanol (producing glycerol dimethoxy ether—[125]), etherification acetylification or anaerobic fermentation [126]. However, these processes are slow and economic unviable. But it is possible to produce FAGE (fatty acid glycerol formal ester) from a reaction between glycerine and other fats (vegetable or animal). FAGE has an LHV lower than biodiesel but is very dense (Table 3), so its energy density (by volume) is similar to biodiesel. But its boiling temperature is almost 300 °C and solidifies at 14 °C, which makes it very viscous. There is another way of transforming fats without the production of glycerol [126]. Instead of methanol, dimethoxymethane (DMM) is mixed with fat in a combined transesterification-trans-ketalization process producing fatty acid methyl ester (FAME) common biodiesel and FAGE. In overall, the process can be shown as Figure 14). y There is another way of transforming fats without the production of glycerol [126]. Instead of methanol, dimethoxymethane (DMM) is mixed with fat in a combined transesterification-trans- ketalization process producing fatty acid methyl ester (FAME) common biodiesel and FAGE. In overall, the process can be shown as Figure 14). Figure 14. The transesterification-transketalization process of FAME+FAGE production. Figure 14. The transesterification-transketalization process of FAME+FAGE production. Figure 14. The transesterification-transketalization process of FAME+FAGE production. Figure 14. The transesterification-transketalization process of FAME+FAGE production. 17. Glycerine (or Glycerol) Each generator can produce 850 kWe, enough for charging 40 car batteries in 50 min. These generators are also used by biodiesel producers, enabling them to use the by-product glycerin to produce electricity in their plants. The engines have a drop in power because the intake air density is reduced by the increased temperature. It seems that the high intake temperature is attained by reducing the heat removed in the inter-cooler after the turbo-charger. The emissions NOx and PM were reported as “virtually eliminated” [122], but these statements were reported in the company’s site. A paper reporting on the same company [123] refers 90 ◦C as the minimum intake temperature to enable stable combustion in a CI engine with glycerin, and 100 ◦C for petrol RON98. The diesel engine needs to be started on diesel and only can be run on glycerin after warm-up and needs to run again on diesel before being switched-off, to purge the injection system. In terms of laminar flame speed, glycerol is similar to petrol [124]. Energies 2020, 13, 4086 E i 2020 13 FOR 26 of 33 18 f 40 26 of 33 18 f 40 19 Conclusions 19. Conclusions These will be very important in the future, as the land used for the production of biofuels will be limited and restrained. These renewable fuels use significantly less resources in terms of land and water than conventional and second-generation biofuels, but they still have a huge problem of energy efficiency, requiring much more energy for its production than their energy content. These fuels may also be known as “energy carriers” a concept firstly used for hydrogen, whereas they “transport” the energy from where it is produced to where it is used. However, for the t t thi i till d ith t l l ffi i i The various properties, applications and production processes of the various alternative fuels were presented and discussed, from the more conventional alcohols and biodiesel to the more unusual ammonia or turpentine. New concepts such as electrofuels and solar fuels were introduced and discussed. These will be very important in the future, as the land used for the production of biofuels will be limited and restrained. These renewable fuels use significantly less resources in terms of land and water than conventional and second-generation biofuels, but they still have a huge problem of energy efficiency, requiring much more energy for its production than their energy content. These fuels may also be known as “energy carriers” a concept firstly used for hydrogen, whereas they “transport” the energy from where it is produced to where it is used. However, for the most part this is still done with extremely low energy efficiencies. most part this is still done with extremely low energy efficiencies. Some of these fuels are readily available and usable in IC engines with little or no modifications, but others require significant engine modifications and/or adaptations. However, it is possible to burn (in IC engines) unexpected fuels such as ammonia or glycerin which are currently used in large Some of these fuels are readily available and usable in IC engines with little or no modifications, but others require significant engine modifications and/or adaptations. However, it is possible to burn (in IC engines) unexpected fuels such as ammonia or glycerin, which are currently used in large diesel Energies 2020, 13, 4086 27 of 33 generators to charge the batteries of Formula E cars. Oxygenated fuels, for instance, are able to retain low particulate matter emissions due to the lack of carbon-carbon bonds. Author Contributions: Conceptualization, J.M.; methodology, J.M.; software, J.M.; formal analysis, J.M.; investigation, J.M., F.P.B.; resources, J.M., F.P.B.; data curation, J.M., F.P.B.; writing—original draft preparation, J.M., F.P.B.; writing—review and editing, F.P.B., J.M.; visualization, J.M., F.P.B.; supervision, J.M.; project administration, J.M.; funding acquisition, J.M. and F.P.B. All authors have read and agree to the published version of the manuscript. Acknowledgments: This work has been supported by FCT—Fundação para a Ciência e Tecnologia within the R&D Unit Project Scope UIDB/00319/2020 (MEtRICs—Mechanical Engineering and Resource Sustainability Center). Conflicts of Interest: The authors declare no conflict of interest. 19 Conclusions 19. Conclusions generators to charge the batteries of Formula E cars. Oxygenated fuels, for instance, are able to retain low particulate matter emissions due to the lack of carbon-carbon bonds. While synthetic fuels may more easily improve pollutant emissions to the degree that they can be custom manufactured, their greenhouse gas footprint will mostly depend on the sustainability level of its raw materials (fossil/renewable), the energy source and from which they are developed and the energy efficiency of the process. Author Contributions: Conceptualization, J.M.; methodology, J.M.; software, J.M.; formal analysis, J.M.; investigation, J.M., F.P.B.; resources, J.M., F.P.B.; data curation, J.M., F.P.B.; writing—original draft preparation, J.M., F.P.B.; writing—review and editing, F.P.B., J.M.; visualization, J.M., F.P.B.; supervision, J.M.; project administration, J.M.; funding acquisition, J.M. and F.P.B. All authors have read and agree to the published version of the manuscript. Acknowledgments: This work has been supported by FCT—Fundação para a Ciência e Tecnologia within the R&D Unit Project Scope UIDB/00319/2020 (MEtRICs—Mechanical Engineering and Resource Sustainability Center). Conflicts of Interest: The authors declare no conflict of interest Author Contributions: Conceptualization, J.M.; methodology, J.M.; software, J.M.; formal analysis, J.M.; investigation, J.M., F.P.B.; resources, J.M., F.P.B.; data curation, J.M., F.P.B.; writing—original draft preparation, J.M., F.P.B.; writing—review and editing, F.P.B., J.M.; visualization, J.M., F.P.B.; supervision, J.M.; project administration, J.M.; funding acquisition, J.M. and F.P.B. All authors have read and agree to the published version of the manuscript. F.P.B.; writing—review and editing, F.P.B., J.M.; visualization, J.M., F.P.B.; supervision, J.M.; project administration, J.M.; funding acquisition, J.M. and F.P.B. All authors have read and agree to the published version of the manuscript. Acknowledgments: This work has been supported by FCT—Fundação para a Ciência e Tecnologia within the R&D Unit Project Scope UIDB/00319/2020 (MEtRICs—Mechanical Engineering and Resource Sustainability Center). Acknowledgments: This work has been supported by FCT—Fundação para a Ciência e Tecnologia within the R&D Unit Project Scope UIDB/00319/2020 (MEtRICs—Mechanical Engineering and Resource Sustainability Center). Conflicts of Interest: The authors declare no conflict of interest. References 1. Kumar, R.R.; Alok, K. Adoption of electric vehicle: A literature review and prospects for sustainability. J. Clean. Prod. 2020, 253, 119911. [CrossRef] 1. Kumar, R.R.; Alok, K. Adoption of electric vehicle: A literature review and prospects for sustainability. J. Clean. Prod. 2020, 253, 119911. [CrossRef] 1. Kumar, R.R.; Alok, K. Adoption of electric vehicle: A literature review and prospects for sustainability. J. Clean. Prod. 2020, 253, 119911. [CrossRef] 2. 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Conclusions Glossary A/F Air-fuel ratio BEV Battery electric vehicle BTL Biomass to liquid fuel CI Compression ignition CN Cetane number CNG Compressed natural gas CO Carbon monoxide CO2 Carbon dioxide CR Compression ratio CTL Coal to liquid fuel DCL Direct coal liquefaction DEE Diethyl ether DMC Dimethyl carbonate DME Dimethyl ether DMF Dimethylfuran DMM Dimethoxymethane ETBE Ethyl tert-butyl ether ECU Electronic control unit EGR Exhaust gas recirculation FAGE Fatty acid glycerol formal ester FAME Fatty acid methyl ester F-T Fischer-Tropsch GTL Gas to liquid fuel HC Hydrocarbons HHV High heating value HTU Hydrothermal upgrading HV Heating value HVO Hydrogenated vegetable oils H/C Hydrogen to carbon ratio IC Internal combustion ICE Internal combustion engine ICL Indirect coal liquefaction IV Iodine value LHV Low heating value LNG Liquefied natural gas LPG Liquid petroleum gases Glossary A/F Air-fuel ratio BEV Battery electric vehicle BTL Biomass to liquid fuel CI Compression ignition CN Cetane number CNG Compressed natural gas CO Carbon monoxide CO2 Carbon dioxide CR Compression ratio CTL Coal to liquid fuel DCL Direct coal liquefaction DEE Diethyl ether DMC Dimethyl carbonate DME Dimethyl ether DMF Dimethylfuran DMM Dimethoxymethane ETBE Ethyl tert-butyl ether ECU Electronic control unit EGR Exhaust gas recirculation FAGE Fatty acid glycerol formal ester FAME Fatty acid methyl ester F-T Fischer-Tropsch GTL Gas to liquid fuel HC Hydrocarbons HHV High heating value HTU Hydrothermal upgrading HV Heating value HVO Hydrogenated vegetable oils H/C Hydrogen to carbon ratio IC Internal combustion ICE Internal combustion engine ICL Indirect coal liquefaction IV Iodine value LHV Low heating value LNG Liquefied natural gas LPG Liquid petroleum gases 28 of 33 Energies 2020, 13, 4086 MeFo Methyl formate MTBE Methyl tert-butyl ether NOx Oxides of nitrogen OEM Original equipment manufacturer PL Pyrolysis fuel liquids PM Particulate matter RON Research octane number SI Spark ignition TBA tert-Butyl alcohol USA United States of America VGO Vacuum gas oil WTW Well to wheel WWII Second World War MeFo Methyl formate MTBE Methyl tert-butyl ether NOx Oxides of nitrogen OEM Original equipment manufacturer PL Pyrolysis fuel liquids PM Particulate matter RON Research octane number SI Spark ignition TBA tert-Butyl alcohol USA United States of America VGO Vacuum gas oil WTW Well to wheel WWII Second World War MeFo Methyl formate MTBE Methyl tert-butyl ether NOx Oxides of nitrogen OEM Original equipment manufacturer PL Pyrolysis fuel liquids PM Particulate matter RON Research octane number SI Spark ignition TBA tert-Butyl alcohol USA United States of America VGO Vacuum gas oil WTW Well to wheel WWII Second World War References The End of the Fossil Fuel Car Is on the EU Agenda. 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The Impact of Urbanization and Human Mobility on Seasonal Influenza in Northern China
Viruses
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Citation: Yang, J.; Guo, X.; Zhang, T.; Wang, Q.; Zhang, X.; Yang, J.; Lai, S.; Feng, L.; Yang, W. The Impact of Urbanization and Human Mobility on Seasonal Influenza in Northern China. Viruses 2022, 14, 2563. https://doi.org/10.3390/v14112563 Citation: Yang, J.; Guo, X.; Zhang, T.; Wang, Q.; Zhang, X.; Yang, J.; Lai, S.; Feng, L.; Yang, W. The Impact of Urbanization and Human Mobility on Seasonal Influenza in Northern China. Viruses 2022, 14, 2563. https://doi.org/10.3390/v14112563 Keywords: seasonal influenza; human mobility; driver; China Academic Editor: Ayato Takada Academic Editor: Ayato Takada Article The Impact of Urbanization and Human Mobility on Seasonal Influenza in Northern China Jiao Yang 1, Xudong Guo 2 , Ting Zhang 1, Qing Wang 1, Xingxing Zhang 1, Jin Yang 1, Shengjie Lai 3, 1, Xudong Guo 2 , Ting Zhang 1, Qing Wang 1, Xingxing Zhang 1, Jin Yang 1, Shengjie Lai 3, eng 1,* and Weizhong Yang 1,* Jiao Yang 1, Xudong Guo 2 , Ting Zhang 1, Qing Wang 1, Xingxing Zhang 1, Jin Yang 1, Shengjie Lai 3, Luzhao Feng 1,* and Weizhong Yang 1,* 1 School of Population Medicine and Public Health, Chinese Academy of Medical Science (CAMS)/ Peking Union Medical College (PUMC), Beijing 100730, China g g ( ) j g 2 Department of Automation, Tsinghua University, Beijing 100084, China 3 WorldPop, School of Geography and Environmental Science, University of Southampton, Southampton SO171BJ, UK p J * Correspondence: fengluzhao@cams.cn (L.F.); yangweizhong@cams.cn (W.Y.) Abstract: The intensity of influenza epidemics varies significantly from year to year among regions with similar climatic conditions and populations. However, the underlying mechanisms of the temporal and spatial variations remain unclear. We investigated the impact of urbanization and public transportation size on influenza activity. We used 6-year weekly provincial-level surveillance data of influenza-like disease incidence (ILI) and viral activity in northern China. We derived the transmission potential of influenza for each epidemic season using the susceptible–exposed– infectious–removed–susceptible (SEIRS) model and estimated the transmissibility in the peak period via the instantaneous reproduction number (Rt). Public transport was found to explain approximately 28% of the variance in the seasonal transmission potential. Urbanization and public transportation size explained approximately 10% and 21% of the variance in maximum Rt in the peak period, respectively. For the mean Rt during the peak period, urbanization and public transportation accounted for 9% and 16% of the variance in Rt, respectively. Our results indicated that the differences in the intensity of influenza epidemics among the northern provinces of China were partially driven by urbanization and public transport size. These findings are beneficial for predicting influenza intensity and developing preparedness strategies for the early stages of epidemics. Citation: Yang, J.; Guo, X.; Zhang, T.; Wang, Q.; Zhang, X.; Yang, J.; Lai, S.; Feng, L.; Yang, W. The Impact of Urbanization and Human Mobility on Seasonal Influenza in Northern China. Viruses 2022, 14, 2563. https://doi.org/10.3390/v14112563 Academic Editor: Ayato Takada Received: 19 October 2022 Accepted: 17 November 2022 Published: 19 November 2022 viruses viruses viruses viruses viruses 1. Introduction In temperate regions, the peak influenza season occurs in the winter months [1], and the scale of seasonal influenza epidemics can vary greatly between provinces and years [2,3]. However, little is known about the drivers of this variation. A better understanding of the factors that govern epidemic intensity is necessary for the public health system to accurately and promptly prepare for seasonal influenza epidemics. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Climatic factors are important drivers of influenza epidemics in temperate regions. Experimental studies have shown that a reduction in relative humidity improves the viability and transmission of influenza virus aerosols [4,5]. Epidemiological evidence also indicates that a reduction in relative humidity is associated with a higher risk of influenza A in the population [6]. Urbanization and human mobility are also believed to be drivers of influenza epidemics [7–9]. A simulation-based investigation in Australia highlighted that the increased peak prevalence and faster spreading rate of influenza pandemics could partially be attributed to an increase in population fractions living in cities [7]. A study of weekly incidence data from the United States found that the size of the urban population was positively associated with the incidence of city-level influenza and further showed that the intensity of influenza epidemics was shaped by urbanization and humidity [10]. Empirical evidence revealed that airline volume was a significant predictor of the spread of Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/viruses Viruses 2022, 14, 2563. https://doi.org/10.3390/v14112563 Viruses 2022, 14, 2563 2 of 11 influenza between regions [8], and high mobility within countries (internal commuting) could accelerate epidemics [9]. However, these studies focused mainly on the impact of human mobility on interregional influenza epidemics. Evidence regarding the influence of human mobility on intracity or intraprovincial epidemics is limited. Recent studies on influenza epidemics have revealed unexplained differences between provinces with similar urbanization and climate conditions in China [2,3,11], suggesting that there are other unidentified factors driving the differences in influenza epidemics between provinces. China is a vast country that comprises provinces with different climatic and economic backgrounds. These factors have led to varying levels of heterogeneity regarding population structure and mobility. 1. Introduction Therefore, we assumed that the unexplained interprovince differences in influenza epidemic intensity may be caused by the hetero- geneity of population mobility in provinces with similar climates and urbanization levels. Higher human mobility inside a province increases close contact between people, and thus the transmission of the influenza virus among people may be enhanced. In the present study, we explored the above hypothesis by using 6 years (2012 to 2017) of data on weekly influenza-like disease and virus activity in 14 provinces in northern China. 2. Methods 2.1. Data The temperature of both the environment and the dew point for each province were obtained from the China Meteorological Administration to calculate the relative humidity, using the R package ‘humidity’ (R software, version 4.2.1). The approximating function can closely simulate relative humidity: r(t) = u × cos × (2 × π(t −5))/52 + m. Census data, including population size, urbanization, and public transportation data, were recovered from the China National Bureau of Statistics [12]. Weekly influenza-like disease incidence rate data (ILI) and viral detection positive rate data for each province were obtained from the Chinese National Influenza Surveillance Network. Referring to previous studies [13,14], proxy measures of the weekly incidence rate (referred to as the ‘incidence rate’) were obtained by multiplying the ILI percentage among patients visiting sentinel hospitals with the proportions of influenza-positive specimens. This proxy is considered a precise representation of the activity of influenza infection [15,16]. 2.3. GLM Model A generalized linear model (GLM) of the SEIRS model was further constructed to explore the patterns of influenza dynamics by fitting the incidence data. GLM avoids the defects associated with the nonlinearity of the SEIRS model. The corresponding generalized linear model is as follows: Yn+1,j = a · Wnj + b · Xnjrnj + c · XnjPnj + Qnj where Ynj = log[Inj], Inj indicate the number infected in week n of season j. We ob- tained Inj by multiplying the incidence rate (the ILI rate × the proportions of influenza- positive specimens) with the province population size. The parameter vector a is given by a = [log(g), log(g + σ1), . . . , log(g + σ6)], which is estimated from the SEIRS model. The design vector Wnj with seven elements indicates whether the data point associated with (n,j) is in the off-peak regime or in one of the six influenza seasons. b and c are parameter vectors with two elements, b = [ω1, ω2] and c = [ρ1, ρ2], where b is obtained by fitting the relationship between relative humidity and viral viability and c is obtained from the observed incidence data. Xnj is a design vector with two elements that indicate whether the point associated with (n,j) is in the off-peak or peak influenza season. Pnj indicates cu- mulative incidence, Pnj = 1 N Σn k=0Ikj. Onj is an offset term Onj = log(<S0j>) −log(N) + αYnj, where <S0j> = 0.9N refers to the expected population-level initial susceptibility each season, taken from Wang and colleagues’ study [19]. The influenza peak was defined as extending from 5 weeks before the peak incidence rate observed in each season to 5 weeks after [20]. A detailed explanation of the GLM model was provided in a previous study [10]. 2.2. SEIRS Model Referring to previous studies [3,10], we constructed a susceptible–exposed–infectious– removed–susceptible (SEIRS) compartmental model to work with province-level weekly incidence rate data (the ILI rate × the proportions of influenza-positive specimens). Suscep- tible (S) refers to individuals at risk of infection with influenza, representing approximately 90% of the total population. Exposed (E) refers to people in the latent period. Infectious (I) refers to people who have been infected. Removed (R) refers to people who have recovered or died. The SEIRS model consists of the following ordinary differential equations: dS/dt = −N−1βSI + δ(N −S −E −I) dE/dt = N−1βSI −εE dI/dt = εE −γI dR/dt = γI N = S + E + I + R dS/dt = −N−1βSI + δ(N −S −E −I) N = S + E + I + R where δ is the rate of reinfection, which is equal to 1/52; ε is the rate of infection after exposure, which is equal to 7; and γ is the rate of recovery from infection, which is equal to 7/2. The values of δ, ε, and γ were taken from Dalziel’s research [10]. The generation time was assumed to be 3 days. y After a certain period [17,18], the immunity of infected individuals weakens and these individuals enter the susceptible compartment. New infections are generated when a Viruses 2022, 14, 2563 3 of 11 susceptible individual comes into contact with an infected individual at a rate of βSI/N, where N refers to the size of the population. In a stable population, the incidence of new infections is governed by the transmission function β(t) = g+σ−ωr(t), where g refers to the maximum gain in the transmission potential at 0 relative humidity and ω refers to the rate of the loss of viral viability caused by relative humidity. The transmission function β(t) is composed of the sum of two parts: a seasonally invariant base transmission potential g, which refers to transmission between individuals under the same climatic conditions (in this case, the impact of climate is 0), and an additional transmission governed by relative humidity, σ−ωr(t), which increases with the decrease in relative humidity in Chinese provinces in winter and thus increases the risk of transmission between individuals under different climate conditions. 3. Results Annual data on population size, urbanization, and public transportation size are presented in Table 1. As shown in Figure 1, influenza incidence varies with urbanization, climate, and transportation size. The maximum and mean incidence of influenza at peak times tended to be higher in provinces with larger magnitudes of urbanization and larger transportation sizes (Figure 1A–D). Table 1. Characteristics of the fourteen provincial-level administrative divisions during 2012–2017. Year Province Beijing Tianjin Hebei Shanxi Inner Mongo- lia Liaoning Jilin Heilong jiang Shandong Henan Shaanxi Gansu Qinghai Ningxia Population size (mil- lions) 2012 20.69 14.13 72.88 36.11 24.90 43.89 27.50 38.34 96.85 94.06 37.53 25.78 5.73 6.47 2013 21.15 14.72 73.33 36.30 24.98 43.90 27.51 38.35 97.33 94.13 37.64 25.82 5.78 6.54 2014 21.52 15.17 73.84 36.48 25.05 43.91 27.52 38.33 97.89 94.36 37.75 25.91 5.83 6.62 2015 21.71 15.47 74.25 36.64 25.11 43.82 27.53 38.12 98.47 94.80 37.93 26.00 5.88 6.68 2016 21.73 15.62 74.70 36.82 25.20 43.78 27.33 37.99 99.47 95.32 38.13 26.10 5.93 6.75 2017 21.71 15.57 75.20 37.02 25.29 43.69 27.17 37.89 100.06 95.59 38.35 26.26 5.98 6.82 Urbanization (%) 2012 86.20 81.55 46.80 51.26 57.14 65.65 53.70 59.60 52.43 42.43 50.02 38.75 47.44 50.67 2013 86.30 82.01 48.12 52.66 58.71 66.45 54.20 57.40 53.75 43.80 51.31 40.13 48.51 52.01 2014 86.35 82.27 49.33 53.79 59.51 67.05 54.81 58.01 55.01 45.20 52.57 41.68 49.78 53.61 2015 86.50 82.64 51.33 55.03 60.30 67.35 55.31 58.80 57.01 46.85 53.92 43.19 50.30 55.23 2016 86.50 82.93 53.32 56.21 61.19 67.37 55.97 59.20 59.02 48.50 55.34 44.69 51.63 56.29 2017 86.50 82.93 55.01 57.34 62.02 67.49 56.65 59.40 60.58 50.16 56.79 46.39 53.07 57.98 Public trans- porta- tion size (mil- lions) 2012 7615.78 1299.51 2039.54 1248.38 963.49 4283.67 1705.61 2239.56 3982.68 2637.18 2545.99 1028.44 391.35 391.35 2013 8047.75 1609.27 2027.27 1563.64 1086.85 4356.33 1713.78 2381.56 4113.11 2661.57 2507.32 1107.11 417.60 417.60 2014 8158.48 1810.72 2053.42 1314.96 1070.99 4401.65 1768.67 2513.60 4038.54 2638.19 2692.46 1137.38 349.71 427.44 2015 7383.84 1858.13 1872.08 1321.32 1076.74 4347.10 1754.52 2574.76 3900.26 2570.70 2692.03 1098.23 379.09 423.77 2016 7349.53 1807.90 1860.48 1263.50 1145.88 4242.62 1713.69 2534.20 3911.36 2539.10 2694.06 1145.49 377.68 418.98 2017 7133.96 1732.79 1818.29 1271.83 1083.90 4328.24 1772.18 2550.84 3967.77 2594.21 2817.40 1249.46 367.10 407.44 The SEIRS model was used to fit the influenza incidence rate data to explore the reasons for the temporal and spatial differences in the intensity of the influenza epidemics. 2.4. Estimation of Transmissibility The weekly instantaneous reproduction number Rt was estimated according to the Bayesian framework applied to the branching process model proposed by Cori et al. [21], which is an extension of Fraser method [22]. Fraser proposed that the renewal estimation equation for the Rt of an epidemic could be written as: Rt = It ∑m s=0 wsIt−s (1) (1) where It refers to the number of reported cases (here, the incidence rate times a constant) between time t and time t + 1, and ws refers to the generation time distribution, such that ∑m s=0 ws = 1. The expected incidence at time t is Poisson distributed with a mean (Rt ∑m s=0 wsIt−s). The transmissibility is assumed to be constant over the time period [t −τ, t] and measured by R[t−τ,t]; then, the likelihood of It−τ, . . . . . . . . . , It given the reproduction number R[t−τ,t] and I0, . . . . . . . . . , It−τ−1 is as follows: P(It−τ, . . . . . . . . . , It I0, . . . . . . . . . , It−τ−1, w, R[t−τ,t]) = t ∏ s=t−τ e−R[t−τ,t]Λs (R[t−τ,t]Λs)Is Is! (2) (2) Viruses 2022, 14, 2563 4 of 11 where Λs = ∑m s=0 wsIt−s. The generation time distribution is a gamma distribution with a mean of 3 days (SD = 1.5 d) and is assumed to be constant throughout an epidemic. A Bayesian framework with a gamma-distributed prior with parameters (a, b) was developed for R[t−τ,t], and the posterior joint distribution of R[t−τ,t] can be derived as proportional to where Λs = ∑m s=0 wsIt−s. The generation time distribution is a gamma distribution with a mean of 3 days (SD = 1.5 d) and is assumed to be constant throughout an epidemic. A Bayesian framework with a gamma-distributed prior with parameters (a, b) was developed for R[t−τ,t], and the posterior joint distribution of R[t−τ,t] can be derived as proportional to R[t−τ,t](posterior) = R[t−τ,t] a+∑t s=t−τ Is−1 e−R[t−τ,t](∑t s=t−τ Λs+ 1 b ) t ∏ s=t−τ ΛsIs Is! (3) (3) Equation (3) indicates that the posterior distribution of R[t−τ,t] is a gamma distribution with the parameters (a + ∑t s=t−τ Is, (∑t s=t−τ Λs + 1 b)−1). 2.5. Regression Analysis with Transmissibility β and Rt of Each Influenza Season 2.5. Regression Analysis with Transmissibility β and Rt of Each Influenza Season A simple linear regression model was used to explore the relationship between driving factors and β. R-squared values (R2) were used to quantify the impact of individual drivers. To make the results more intuitive, we used the same method to further quantify the rela- tionship between each driving factor and the simulated Rt. Because public transportation is easily affected by population size and urbanization, for example, in provinces with larger population sizes and higher urbanization, the accessibility of public transport is higher and more people may use public transportation. Therefore, we can more accurately represent human mobility per unit density. A combined mobility index, h, was calculated using population size (PS), urbanization (U), and public transportation (PT) to represent human mobility more accurately: h = log  PS∗U PT  . A higher value of h indicates more frequent population mobility. Differences in Akaike information criteria (∆AIC) were used to esti- mate the relative quality of the GLM, where higher values indicate models with poorer relative support. 2.4. Estimation of Transmissibility Equation (3) indicates that the posterior distribution of R[t−τ,t] is a gamma distribution with the parameters (a + ∑t s=t−τ Is, (∑t s=t−τ Λs + 1 b)−1). 3. Results As described in the Methods section, the transmission potential of each influenza season in each province could be obtained using the SEIRS model. Influenza epidemics vary in intensity by year and province, indicating a difference in transmission potential. The SEIRS model is a common method for fitting influenza time series data. However, the model is nonlinear; thus, minor changes in input parameters can cause significant changes in the Table 1. Characteristics of the fourteen provincial-level administrative divisions during 2012–2017. The SEIRS model was used to fit the influenza incidence rate data to explore the reasons for the temporal and spatial differences in the intensity of the influenza epidemics. As described in the Methods section, the transmission potential of each influenza season in each province could be obtained using the SEIRS model. Influenza epidemics vary in intensity by year and province, indicating a difference in transmission potential. The SEIRS model is a common method for fitting influenza time series data. However, the model is nonlinear; thus, minor changes in input parameters can cause significant changes in the Viruses 2022, 14, 2563 5 of 11 prediction results. Therefore, a general function of the SEIRS model was constructed to work with province-level influenza incidence data. The results are shown in Figure 2. Ten fitted parameters (Supplementary Table S1) were obtained using province-level time series models. The results were obtained for the following three provinces randomly selected from the total of 14: Beijing, Heilongjiang, and Ningxia. Spearman’s r = 0.83 for the comparison of the observed and predicted influenza incidence (Figure 3). 99.51 2039.54 1248.38 963.49 4283.67 1705.61 2239.56 3982.68 2637.18 2545.99 1028.44 391.35 391.35 09.27 2027.27 1563.64 1086.85 4356.33 1713.78 2381.56 4113.11 2661.57 2507.32 1107.11 417.60 417.60 10.72 2053.42 1314.96 1070.99 4401.65 1768.67 2513.60 4038.54 2638.19 2692.46 1137.38 349.71 427.44 58.13 1872.08 1321.32 1076.74 4347.10 1754.52 2574.76 3900.26 2570.70 2692.03 1098.23 379.09 423.77 07.90 1860.48 1263.50 1145.88 4242.62 1713.69 2534.20 3911.36 2539.10 2694.06 1145.49 377.68 418.98 32.79 1818.29 1271.83 1083.90 4328.24 1772.18 2550.84 3967.77 2594.21 2817.40 1249.46 367.10 407.44 Figure 1. Bubble charts demonstrating the incidence rate in provinces with different levels of urban- ization, transport, and relative humidity (A–F). Provinces with higher max and mean incidence tended to have a higher magnitude of urbanization (A,B), lower relative humidity (C,D), and larger transportation size (E,F). Figure 1. 3. Results Bubble charts demonstrating the incidence rate in provinces with different levels of urbanization, transport, and relative humidity (A–F). Provinces with higher max and mean incidence tended to have a higher magnitude of urbanization (A,B), lower relative humidity (C,D), and larger transportation size (E,F). Figure 1. Bubble charts demonstrating the incidence rate in provinces with different levels of urban- zation, transport, and relative humidity (A–F). Provinces with higher max and mean incidence ended to have a higher magnitude of urbanization (A,B), lower relative humidity (C,D), and larger ransportation size (E,F). Figure 1. Bubble charts demonstrating the incidence rate in provinces with different levels of urbanization, transport, and relative humidity (A–F). Provinces with higher max and mean incidence tended to have a higher magnitude of urbanization (A,B), lower relative humidity (C,D), and larger transportation size (E,F). The SEIRS model was used to fit the influenza incidence rate data to explore the rea- sons for the temporal and spatial differences in the intensity of the influenza epidemics. As described in the Methods section, the transmission potential of each influenza season in each province could be obtained using the SEIRS model. Influenza epidemics vary in intensity by year and province, indicating a difference in transmission potential. The SEIRS model is a common method for fitting influenza time series data. However, the model is nonlinear; thus, minor changes in input parameters can cause significant changes in the prediction results. Therefore, a general function of the SEIRS model was constructed to work with province-level influenza incidence data. The results are shown in Figure 2. Ten fitted parameters (Supplementary Table S1) were obtained using province-level time series models. The results were obtained for the following three provinces randomly se- lected from the total of 14: Beijing, Heilongjiang, and Ningxia. Spearman’s r = 0.83 for the comparison of the observed and predicted influenza incidence (Figure 3). The early transmission potential obtained by SEIRS is only a mathematical value, and its practical significance is limited. In this regard, we further explored the factors that influenced the transmission potential. As shown in Figure 4, urbanization and public transportation size could explain 1.28% and 27.62% of the variation in the annual trans- mission potential, respectively. Close contact between individuals is a prerequisite for influenza transmission. The frequency of close contact can directly affect the transmis- sion potential of the influenza virus between persons. 3. Results A larger public transport system does not necessarily mean that contact between persons is more frequent. To meet the commuting needs of residents, public transportation may be more extensive in areas with large populations. The size of public transportation per unit population in urban areas can reduce the impact of population size to better represent the transmission potential of contact between people. Urbanization, population size, and public transportation size were used to calculate the combined index h, which indicated population mobility. The results showed a positive correlation between the combined index h and the annual transmission potential, R2 = 0.1349 (p < 0.05). Viruses 2022, 14, 2563 Viruses 2022, 14 6 of 11 Figure 2. Transmission potential and relative humidity predicted the observed differences in the intensity of the influenza epidemics in northern Chinese provinces (A–F). (A,C,E) simulated results of the SEIRS model in three provinces. (B,D,F) fitted results of the GLM model in three provinces. Figure 2. Transmission potential and relative humidity predicted the observed differences in the intensity of the influenza epidemics in northern Chinese provinces (A–F). (A,C,E) simulated results of the SEIRS model in three provinces. (B,D,F) fitted results of the GLM model in three provinces. Viruses 2022, 14, 2563 7 of 12 Figure 2. Transmission potential and relative humidity predicted the observed differences in the intensity of the influenza epidemics in northern Chinese provinces (A–F). (A,C,E) simulated results of the SEIRS model in three provinces. (B,D,F) fitted results of the GLM model in three provinces. Figure 2. Transmission potential and relative humidity predicted the observed differences in the intensity of the influenza epidemics in northern Chinese provinces (A–F). (A,C,E) simulated results of the SEIRS model in three provinces. (B,D,F) fitted results of the GLM model in three provinces. Viruses 2022, 14, 2563 7 of 12 Figure 3. Observed versus simulated influenza incidence in all provinces. Gray points show simu- lated influenza incidence rate. Blue line shows the fitted line between simulated influenza inci- dence and observed incidence. Th l l b d b SEIRS l h l l d Figure 3. Observed versus simulated influenza incidence in all provinces. Gray points show simu- lated influenza incidence rate. Blue line shows the fitted line between simulated influenza incidence and observed incidence. Figure 2. Transmission potential and relative humidity predicted the observed differences in the intensity of the influenza epidemics in northern Chinese provinces (A–F). 3. Results (A,C,E) simulated results of the SEIRS model in three provinces. (B,D,F) fitted results of the GLM model in three provinces. Figure 2. Transmission potential and relative humidity predicted the observed differences in the intensity of the influenza epidemics in northern Chinese provinces (A–F). (A,C,E) simulated results of the SEIRS model in three provinces. (B,D,F) fitted results of the GLM model in three provinces. 7 of 12 Figure 3. Observed versus simulated influenza incidence in all provinces. Gray points show simu- lated influenza incidence rate. Blue line shows the fitted line between simulated influenza inci- dence and observed incidence. Th l t i i t ti l bt i d b SEIRS i l th ti l l d Figure 3. Observed versus simulated influenza incidence in all provinces. Gray points show simu- lated influenza incidence rate. Blue line shows the fitted line between simulated influenza incidence and observed incidence. Figure 3. Observed versus simulated influenza incidence in all provinces. Gray points show simu- lated influenza incidence rate. Blue line shows the fitted line between simulated influenza inci- dence and observed incidence. Figure 3. Observed versus simulated influenza incidence in all provinces. Gray points show simu- lated influenza incidence rate. Blue line shows the fitted line between simulated influenza incidence and observed incidence. 7 of 11 of 12 Viruses 2022, 14, 2563 Viruses 2022, 14, 256 Figure 4. Urbanization, transportation size, and combined index estimated from census data pre- dicted transmission potential, maximum Rt, and mean Rt during peak period of influenza season (A–L). Gray points show transmission potential, maximum Rt, and mean Rt during peak period of influenza season estimated from the influenza incidence rate. Red lines refer to the prediction for transmission potential during peak period of influenza season (A–C). Purple lines refer to the pre- diction for maximum Rt during peak period of influenza season (D–F). Green lines refer to the prediction for mean Rt during peak period of the influenza season (G–L). Figure 4. Urbanization, transportation size, and combined index estimated from census data pre- dicted transmission potential, maximum Rt, and mean Rt during peak period of influenza season (A–L). Gray points show transmission potential, maximum Rt, and mean Rt during peak period of influenza season estimated from the influenza incidence rate. Red lines refer to the prediction for transmission potential during peak period of influenza season (A–C). 3. Results Purple lines refer to the prediction for maximum Rt during peak period of influenza season (D–F). Green lines refer to the prediction for mean Rt during peak period of the influenza season (G–L). Figure 4. Urbanization, transportation size, and combined index estimated from census data pre- dicted transmission potential, maximum Rt, and mean Rt during peak period of influenza season (A–L). Gray points show transmission potential, maximum Rt, and mean Rt during peak period of influenza season estimated from the influenza incidence rate. Red lines refer to the prediction for transmission potential during peak period of influenza season (A–C). Purple lines refer to the pre- diction for maximum Rt during peak period of influenza season (D–F). Green lines refer to the prediction for mean Rt during peak period of the influenza season (G–L). Figure 4. Urbanization, transportation size, and combined index estimated from census data pre- dicted transmission potential, maximum Rt, and mean Rt during peak period of influenza season (A–L). Gray points show transmission potential, maximum Rt, and mean Rt during peak period of influenza season estimated from the influenza incidence rate. Red lines refer to the prediction for transmission potential during peak period of influenza season (A–C). Purple lines refer to the prediction for maximum Rt during peak period of influenza season (D–F). Green lines refer to the prediction for mean Rt during peak period of the influenza season (G–L). 4. Discussion Weekly surveillance data on outpatient ILI and virus activity from 14 provinces in northern China revealed that the annual transmission potential was positively associated ith th i f bli t t ti Th i R d R f h i fl In addition, the association between urbanization, public transportation size, and the combined index h was also examined with the maximum Rt and mean Rt at the peak of each influenza season. Urbanization and public transportation size were significant drivers of max Rt and mean Rt during the peak period of each influenza season. season during 4. Discussion tively correlated with urbanization and the size of public transportation. The results pre- sented here suggest that, at least in northern China, the intensity of the influenza epidemic may be governed by urbanization and intra-city human mobility. Climate conditions, urbanization, and human mobility play a significant role in the spread of seasonal influenza. Relative humidity is an important environmental factor that affects the survival of influenza viruses in aerosols, and it is also a crucial driving factor Weekly surveillance data on outpatient ILI and virus activity from 14 provinces in northern China revealed that the annual transmission potential was positively associated with the size of public transportation. The maximum Rt and mean Rt of each influenza season during the peak period estimated from province-level incidence data were positively correlated with urbanization and the size of public transportation. The results presented here suggest that, at least in northern China, the intensity of the influenza epidemic may be governed by urbanization and intra-city human mobility. Viruses 2022, 14, 2563 8 of 11 8 of 11 Climate conditions, urbanization, and human mobility play a significant role in the spread of seasonal influenza. Relative humidity is an important environmental factor that affects the survival of influenza viruses in aerosols, and it is also a crucial driving factor for influenza seasonality [23]. In the current study, we controlled for the influence of climate on transmission by fitting approximate functions. Therefore, the annual transmission potential refers to the comprehensive influence of other factors, excluding climate conditions. The instantaneous reproduction number (Rt) is typically used to characterize real-time transmissibility. A higher Rt value indicates a higher transmission potential. The pathogen spreads when Rt > 1 and is under control when Rt < 1. We calculated the maximum Rt and the mean Rt of each influenza season for 14 provinces to quantify the transmissibility at peak times. Further analysis showed that the size of public transport was positively correlated with the yearly transmission potential. This was consistent with the results of a previous simulation study [24]. Globally, people traveling by air cause the transmission of pandemic and seasonal influenza viruses, especially the A/H3N2 viruses [25–30]. At the regional scale, the spatial transmission of influenza is dominated by patterns of human contact, including school closure times and commute patterns [2,31,32]. season during 4. Discussion In the current study, the maximum peak Rt and the mean peak Rt of the influenza season were positively associated with the size of public transport, which could explain the variations for more than one fifth of the maximum peak Rt variations and about one fifth of the peak mean Rt, respectively. These results provide new evidence for understanding the impact of human mobility on influenza epidemics. Previous studies have examined the impact of urbanization on the intensity or epi- demic patterns of influenza [2,7,10]. However, the definitions of urbanization vary between studies. For example, in Dalziel et al.’s study, urban population size is regarded as an indicator of urbanization [10]. In the studies by Lei and Zachreson, urbanization refers to the proportion of the total population living in urban areas [7,10]. In our study, different urbanization indicators were used to evaluate the relationship between urbanization and influenza transmission. Our results showed that the proportion of the total population liv- ing in urban areas was also positively correlated with the maximum peak Rt and the mean peak Rt of the influenza season. However, we did not find a consistent positive relationship between urban population density, urban population size, and influenza transmission (Supplementary Figures S2 and S3). Our findings suggest that the proportion of the total population living in urban areas may be a better indicator for studying the relationship between urbanization and influenza transmission in northern China compared with urban population size and urban population density. Two reasons may be responsible for this result. First, regarding infectious diseases, current explosive trends in urbanization mean that more people are concentrated in urban regions. Coupled with the spread of suburbs, this can lead to large hubs in the commuter in- teraction network, which can cause a faster spread of infectious diseases between work and home [33,34]. Second, public transportation (buses and subways) is a common means of traveling in many cities around the world; thus, if an infected person interacts closely with other users of public transportation on a bus or subway, combined with insufficient ventila- tion and overcrowded conditions, it can increase the risk of influenza for other uninfected people and lead to the spread of influenza among colleagues and family members [35]. season during 4. Discussion A higher transmission potential and Rt indicate that the number of infected cases will increase in a short period of time, requiring increased surge capacity in the public health system, including primary care facilities and clinical laboratories [36]. The significance of our study is that, when the influenza season arrives, it can help predict the intensity of the influenza epidemic according to urbanization and human mobility to prepare for its medical and social impact in advance. Additionally, obtaining information on transmissibility at peak times is beneficial for mitigating influenza spread by vaccination and taking non- pharmaceutical interventions (NPIs) in the early stages of epidemics [37,38]. Viruses 2022, 14, 2563 9 of 11 9 of 11 A potential limitation of our study was that school holidays were not included in our model. Previous studies have emphasized the importance of children in the spread of in- fluenza, and the impact of school holidays and school closures on transmissibility [16,39,40]. Additionally, we did not have information on the impact of antigen drift and host immu- nity on epidemics. However, a study based on the city-level analysis of the subtypes and antigenical characteristics of the influenza virus in Australia demonstrated that antigenic novelty has limited effects on epidemic size. It suggested that other factors drive influenza epidemics apart from host immunity at the local scale in temperate areas [41]. 5. Conclusions In conclusion, urbanization and human mobility were positively associated with the intensity of influenza. Increased commuting by public transport (including buses and subways) can accelerate the spread of influenza. Monitoring flows for public transport may be conducive to early detection and response to influenza epidemics. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/v14112563/s1. Figure S1: The map indicating the provinces studied in northern China. The color indicates the climatic domain: cold- temperate (black); mid- temperate (blue); warm-temperate (green); Table S1: Summary statistics on the means of fitted model parameters across provinces; Figure S2: The association for urban population density with transmission potential (A), maximum Rt (B) and mean Rt (C) in peak time of influenza season; Figure S3: The association for urban population size with transmission potential (A), maximum Rt (B) and mean Rt (C) in peak time of influenza season. Author Contributions: Conceptualization, W.Y., L.F. and S.L.; formal analysis, J.Y. (Jiao Yang), S.L. and X.G.; data curation, T.Z. and Q.W.; writing—original draft preparation, J.Y. (Jiao Yang), X.G., L.F. and W.Y.; writing—review and editing, J.Y. (Jin Yang), X.Z., J.Y. (Jiao Yang), L.F., S.L. and Q.W.; supervision, W.Y. and L.F. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences under Grants 2020-I2M-1-001 and 2021-I2M-1-044, the non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences under Grant 2021-RC330-002 and the National Institute for Health under Grant MIDAS Mobility R01AI160780. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Due to the potentially sensitive information included, the original dataset is not public and is available from the corresponding author upon reasonable request. Data Availability Statement: Due to the potentially sensitive information included, the original dataset is not public and is available from the corresponding author upon reasonable request. Acknowledgments: For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. Acknowledgments: For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. Conflicts of Interest: The authors declare no competing interests. g 6. Peci, A.; Winter, A.L.; Li, Y.; Gnaneshan, S.; Liu, J.; Mubareka, S.; Gubbay, J.B. Effects of Absolute Humidity, Relative Humidity, Temperature, and Wind Speed on Influenza Activity in Toronto, Ontario, Canada. Appl. Environ. Microbiol. 2019, 85, e02426-18. [CrossRef] p , J g J yg , , [ Lowen, A.C.; Mubareka, S.; Steel, J.; Palese, P. Influenza virus transmission is dependent on relative humi PLoS Pathog. 2007, 3, 1470–1476. [CrossRef] 1. Tamerius, J.; Nelson, M.I.; Zhou, S.Z.; Viboud, C.; Miller, M.A.; Alonso, W.J. 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[CrossRef] [PubMed] y of Statistics of China. Available online: http://www.stats.gov.cn/tjsj/ndsj/ (accessed on 16 September 202 Wang, X.; Xie, Y.; Du, X.; Chen, T.; Yang, L.; Wang, D.; Shu, Y. Nonpharmaceutical Interventions Used to Con 12. 35. Zhu, S.; Srebric, J.; Spengler, J.D.; Demokritou, P. An advanced numerical model for the assessment of airborne transmission of influenza in bus microenvironments. Build Environ. 2012, 47, 67–75. [CrossRef] [PubMed] 36. Crawford, J.M.; Stallone, R.; Zhang, F.; Gerolimatos, M.; Korologos, D.D.; Sweetapple, C.; De Geronimo, M.; Dlugacz, Y.; Armellino, D.M.; Ginocchio, C.C. Laboratory surge response to pandemic (H1N1) 2009 outbreak, New York City metropolitan area, USA. Emerg. Infect. Dis. 2010, 16, 8–13. [CrossRef] g g g f 40. Wu, J.T.; Cowling, B.J.; Lau, E.H.; Ip, D.K.; Ho, L.M.; Tsang, T.; Chuang, S.K.; Leung, P.Y.; Lo, S.V.; Liu, and mitigation of pandemic (H1N1) 2009, Hong Kong. Emerg. Infect. Dis. 2010, 16, 538–541. [CrossRef] 41. 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Infect. Dis. 2008, 14, 1660–1662. [CrossRef] Cowling, B.J.; Lau, E.H.; Lam, C.L.; Cheng, C.K.; Kovar, J.; Chan, K.H.; Peiris, J.S.; Leung, G.M winter influenza season, Hong Kong. Emerg. Infect. Dis. 2008, 14, 1660–1662. [CrossRef] J p , , [ ] 38. Feng, L.; Zhang, T.; Wang, Q.; Xie, Y.; Peng, Z.; Zheng, J.; Qin, Y.; Zhang, M.; Lai, S.; Wang, D.; et al. Impact of COVID-19 39. Cowling, B.J.; Lau, E.H.; Lam, C.L.; Cheng, C.K.; Kovar, J.; Chan, K.H.; Peiris, J.S.; Leung, G.M. Effects of school closures, 2008 winter influenza season, Hong Kong. Emerg. Infect. Dis. 2008, 14, 1660–1662. [CrossRef] 38. Feng, L.; Zhang, T.; Wang, Q.; Xie, Y.; Peng, Z.; Zheng, J.; Qin, Y.; Zhang, M.; Lai, S.; Wang, D.; et outbreaks and interventions on influenza in China and the United States. Nat. Commun. 2021, 12, 3249 , , [ ] 39. Cowling, B.J.; Lau, E.H.; Lam, C.L.; Cheng, C.K.; Kovar, J.; Chan, K.H.; Peiris, J.S.; Leung, G.M. Effects of school closures, 2008 winter influenza season, Hong Kong. Emerg. Infect. 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Feng, L.; Zhang, T.; Wang, Q.; Xie, Y.; Peng, Z.; Zheng, J.; Qin, Y.; Zhang, M.; Lai, S.; Wang, D.; et al. Impact of COVID-19 outbreaks and interventions on influenza in China and the United States. Nat. Commun. 2021, 12, 3249. [CrossRef] 39. Cowling, B.J.; Lau, E.H.; Lam, C.L.; Cheng, C.K.; Kovar, J.; Chan, K.H.; Peiris, J.S.; Leung, G.M. Effects of school closures, 2008 winter influenza season, Hong Kong. Emerg. Infect. Dis. 2008, 14, 1660–1662. [CrossRef] g g g f 40. Wu, J.T.; Cowling, B.J.; Lau, E.H.; Ip, D.K.; Ho, L.M.; Tsang, T.; Chuang, S.K.; Leung, P.Y.; Lo, S.V.; Liu, S.H.; et al. School closure and mitigation of pandemic (H1N1) 2009, Hong Kong. Emerg. Infect. Dis. 2010, 16, 538–541. [CrossRef] 41. Lam, E.K.S.; Morris, D.H.; Hurt, A.C.; Barr, I.G.; Russell, C.A. The impac virus epidemics in Australia. Nat. Commun. 2020, 11, 2741. [CrossRef] 41. Lam, E.K.S.; Morris, D.H.; Hurt, A.C.; Barr, I.G.; Russell, C.A. 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Reducing the rate and duration of Re-ADMISsions among patients with unipolar disorder and bipolar disorder using smartphone-based monitoring and treatment – the RADMIS trials: study protocol for two randomized controlled trials
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Faurholt-Jepsen et al. Trials (2017) 18:277 DOI 10.1186/s13063-017-2015-3 Faurholt-Jepsen et al. Trials (2017) 18:277 DOI 10.1186/s13063-017-2015-3 Open Access * Correspondence: maria@faurholt-jepsen.dk 1Psychiatric Center Copenhagen, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark 2Psychiatric Center Copenhagen, Rigshospitalet, University Hospitalet of Copenhagen, Copenhagen, Denmark Full list of author information is available at the end of the article Reducing the rate and duration of Re- ADMISsions among patients with unipolar disorder and bipolar disorder using smartphone-based monitoring and treatment – the RADMIS trials: study protocol for two randomized controlled trials Maria Faurholt-Jepsen1,2*, Mads Frost3, Klaus Martiny1,2, Nanna Tuxen1,2, Nicole Rosenberg1,2, Jonas Busk4, Ole Winther4, Jakob Eyvind Bardram4,5 and Lars Vedel Kessing1,2 Background Unipolar disorder and bipolar disorder are common men- tal diseases with a lifetime prevalence of 15–20% and 1– 2%, respectively [1, 2]. Unipolar disorder and bipolar dis- order combined account for nearly half of all morbidity and mortality due to mental and substance use disorders [3] and burden society with the highest health care costs of all psychiatric and neurological disorders [4]. Treatment of unipolar disorder and bipolar disorder applies a variety of methods, including antidepressants, mood stabilizers, psychoeducation, and cognitive behav- ioral psychotherapy (CBT). y g Although psychiatric treatment internationally, and in Denmark, has shifted more from inpatient treatment to outpatient treatment during recent decades, costs due to psychiatric hospitalization are still a major burden com- prising two thirds of all direct costs in the five Regions of Denmark [5]. Patients with affective disorders are more frequently hospitalized than any other patient group, counting more than 10,800 patients in 2013 among the en- tire Danish population of 5.4 million people, and 20% of all psychiatric hospitalizations in Denmark [6]. The cost of these hospitalizations alone is estimated to be DKK648 million (approximately €8.7 million) per year in Denmark (10,800 hospitalizations with a mean of 20 days’ stay for a cost of 3000 DKK/day (€400)). The discharge period after hospitalization is a high-risk period with a more than 300 times increased risk of suicide during the first week [7] and a high risk of re-admission [8, 9]. Patients are often afraid of leaving the hospital, feeling alone with the prospect of an appointment with a physician 1 or more months ahead. A pilot study from our group in which 45 patients dis- charged from psychiatric hospital self-monitored symp- toms using a daily computer-based self-monitoring system emphasized the pivotal importance of continued contact with a clinician such as a nurse [10]. Smartphones com- prise a unique platform for the monitoring and treatment of depression and mania. Depression and mania are associ- ated with changes in several behavioral components (e.g., reduction in activity level, change in speech) and motivational states (e.g., anhedonia), some of which may be detectable using readily available smartphone sensors [11–13]. Prior work done by our group has developed and tested a unique smartphone-based system for the treatment of bipolar disorder (the MONARCA system) [14, 15]. (Continued from previous page) Discussion: If the smartphone-based monitoring system proves effective in reducing the rate and duration of re- admissions, there will be basis for using a system of this kind in the treatment of unipolar and bipolar disorder in general and on a larger scale. Trial registration: ClinicalTrials.gov, ID: NCT03033420. Registered 13 January 2017. Ethical approval has been obtained. daily contact with a psychiatric hospital nurse using a bidirectional feedback system combined with objective smartphone-based early warning signs, assessed using a smartphone app, will reduce the risk of re-admission and the risk of relapse of depressive and (hypo)manic symptoms. Abstract Background: Unipolar and bipolar disorder combined account for nearly half of all morbidity and mortality due to mental and substance use disorders, and burden society with the highest health care costs of all psychiatric and neurological disorders. Among these, costs due to psychiatric hospitalization are a major burden. Smartphones comprise an innovative and unique platform for the monitoring and treatment of depression and mania. No prior trial has investigated whether the use of a smartphone-based system can prevent re-admission among patients discharged from hospital. The present RADMIS trials aim to investigate whether using a smartphone-based monitoring and treatment system, including an integrated clinical feedback loop, reduces the rate and duration of re-admissions more than standard treatment in unipolar disorder and bipolar disorder. Methods: The RADMIS trials use a randomized controlled, single-blind, parallel-group design. Patients with unipolar disorder and patients with bipolar disorder are invited to participate in each trial when discharged from psychiatric hospitals in The Capital Region of Denmark following an affective episode and randomized to either (1) a smartphone- based monitoring system including (a) an integrated feedback loop between patients and clinicians and (b) context-aware cognitive behavioral therapy (CBT) modules (intervention group) or (2) standard treatment (control group) for a 6-month trial period. The trial started in May 2017. The outcomes are (1) number and duration of re-admissions (primary), (2) severity of depressive and manic (only for patients with bipolar disorder) symptoms; psychosocial functioning; number of affective episodes (secondary), and (3) perceived stress, quality of life, self-rated depressive symptoms, self-rated manic symptoms (only for patients with bipolar disorder), recovery, empowerment, adherence to medication, wellbeing, ruminations, worrying, and satisfaction (tertiary). A total of 400 patients (200 patients with unipolar disorder and 200 patients with bipolar disorder) will be included in the RADMIS trials. (Continued on next page) © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Faurholt-Jepsen et al. Trials (2017) 18:277 Page 2 of 13 (Continued from previous page) Objectives To investigate in two individual, randomized controlled, single-blind, parallel-group trials whether the use of a smartphone-based monitoring and treatment system, in- cluding an integrated feedback loop, on both subjective and automatically generated behavioral data on mea- sures of illness activity (phone usage, social activity, physical activity, mobility, voice recognition and sleep) and context-aware CBT modules, the Monsenso system, reduces the rate and duration of re-admissions more than treatment-as-usual in adult patients with unipolar disorder or bipolar disorder, respectively. The MONARCA system, developed and tested by our group [14, 15], has been proved highly usable and useful by patients with bipolar disorder with a high self- assessment adherence (87–95%), and was considered to help patients to better manage their disease [32]. A study investigating the effect of the MONARCA system (daily self-monitoring including a two-level feedback loop to cli- nicians) in a RCT found no overall effect in primary ana- lyses but secondary analyses suggested that the system was able to reduce manic symptoms, but may sustain de- pressive symptoms in patients with bipolar disorder [33]. These results are in accordance with the findings that psy- chological interventions for bipolar disorder in general have more effect on manic than depressive symptoms and more effect on preventing that treating acute depression [23–25]. Consequently, emphasis on depressive symptoms should be a high priority including considerations on mechanisms to reduce the negative processing bias and depressive ruminations in patients with unipolar disorder and bipolar disorder [23, 33]. Further, to investigate if the Monsenso system reduces the severity of clinically rated affective symptoms and the num- ber of affective episodes, improves psychosocial functioning, quality of life, severity of self-assessed affective symptoms, recovery, empowerment, adherence to medication, well- being, and satisfaction with care, and reduces perceived stress, rumination and worrying more than standard treat- ment without a smartphone-based system in adult patients with unipolar disorder or bipolar disorder, respectively. Data will be collected and analyzed separately accord- ing to psychiatric diagnosis (unipolar disorder and bipo- lar disorder). Finally, research by our group found that automatically generated objective smartphone data reflect illness activ- ity in patients with bipolar disorder. Background The MONARCA system is capable of collecting subjective self-assessment data and automatically generated objective smartphone data from patients on a daily basis while allowing simple bidirectional communication be- tween clinicians and the patients [15]. We find it likely that CBT has proven efficacious in the treatment and pre- vention of depressive episodes in unipolar disorder [16], but the effect in relation to bipolar disorder is controver- sial [17]. Depressive symptoms dominate bipolar dis- order as 80% of episodes are depressive [18], but bipolar depression is difficult to treat as only a few drugs have proven effects [19] and psychological interventions have proven ambient effects on depressive symptoms reveal- ing positive effects in some studies [20–22], but not in others [23–25]. Specifically targeting depressive rumin- ation, rumination-focused cognitive therapy and con- creteness training (a facilitated self-help intervention intended to increase specificity of processing in patients with depression) have shown encouraging results in the treatment of residual depression [26]. However, the number of CBT therapists is very low compared with the huge demand for CBT. Internet- and computer- based CBT systems exist and have been proved effective in relation to depression [27]. Recently, CBT has been suggested for smartphones [28] but the effect has never been tested in a randomized controlled trial (RCT). In the RADMIS trial we will develop a smartphone- based CBT program that includes methods to relieve de- pressive ruminations [23, 29]. The smartphone-based system used in the RADMIS trials is inspired by the MONARCA system (see below), and is called the Mon- senso system. By using the sensing, computational and communication capabilities of smartphones, it is possible to continuously monitor an individual’s context includ- ing physical activity, location, and environment. Thus, smartphones hold significant promise as a platform to monitor behavioral and environmental indicators of de- pression and mania and for treatment, facilitating early intervention and smartphone-based CBT. Prior research on using smartphones in the detection of unipolar disorder and bipolar disorder has shown Faurholt-Jepsen et al. Trials (2017) 18:277 Page 3 of 13 Page 3 of 13 promising but preliminary results. A small study on eight patients with depression (not including a control group) found that sensor data from smartphones could detect so- cial patterns [30]. Another study found that sensors from smartphones were effective at detecting social and sleep behaviors among patients with depression [31]. Background of re-admission more than standard treatment in adult pa- tients with unipolar disorder or bipolar disorder, respect- ively, being discharged from hospital. Objectives Data on physical ac- tivity (the number of changes in cell tower ID/day), social activity (the number and duration of phone calls/ day and the number of text messages/day) [11–13] and voice features collected during phone calls [34] corre- lated significantly with the severity of clinically rated de- pressive and manic symptoms. Although these findings are encouraging and innovative, there is a need to inte- grate self-monitored smartphone data with automatically generated objective smartphone data on physical and so- cial activity, as well as speech and sleep, into a compos- ite smartphone-generated electronic measure. This composite measure should be modeled to have a high correlation with depressive and manic symptoms, and a high predictive ability to identify upcoming affective epi- sodes for the individual patient in order to facilitate early intervention. Trial design and study organization The RADMIS trials are randomized controlled, single- blind, parallel-group trials with a balanced allocation ratio (1:1) of adult patients with unipolar disorder or bipolar dis- order stratified according to the psychiatric center from where the patients are discharged and the number of former hospitalizations (three or less or more than three). The included patients are randomized separately according to psychiatric diagnosis (unipolar disorder or bipolar dis- order) to either active use of the Monsenso system (inter- vention group) or to standard treatment (control group). Methods The present trial protocol is reported according to the CONsolidated Standards Of Reporting Trials (CONSORT) Statement and Standard Protocol Items: Recommenda- tions for Interventional Trials (SPIRIT) [35–37] (Fig. 1, Additional file 1). The trial protocol describes two individual, random- ized controlled, single-blind, parallel-group trials, the RADMIS trials, investigating the effect of using the Monsenso system on a daily basis, this including an inte- grated feedback loop and context-aware CBT modules, compared with standard treatment in adult patients with unipolar disorder and bipolar disorder, respectively. Hypotheses Using a smartphone-based monitoring and treatment sys- tem for daily electronic self-monitoring, including an inte- grated feedback loop, on both subjective and automatically generated behavioral data on measures of illness activity (phone usage, social activity, physical activity, mobility, voice recognition and sleep) and context-aware CBT mod- ules, the Monsenso system, reduces the rate and duration Faurholt-Jepsen et al. Trials (2017) 18:277 Page 4 of 13 Fig. 1 Schedule of enrolment, interventions, and outcome assessments in the RADMIS trials (unipolar disorder or bipolar disorder) to either the intervention group or the control group for a 6-month trial period. Thus, the RADMIS trials consists of two separate RCTs with two patient groups investigating the effect of the same intervention (the Monsenso system). The flow diagram of the RADMIS trials is presented in Fig. 2. The trails are con- ducted at the Psychiatric Center Copenhagen, Rigshospita- let, Copenhagen, Denmark. No changes in study design or methods have been made after trial commencement. Thus, the RADMIS trials consists of two separate RCTs with two patient groups investigating the effect of the same intervention (the Monsenso system). The flow diagram of the RADMIS trials is presented in Fig. 2. The trails are con- ducted at the Psychiatric Center Copenhagen, Rigshospita- let, Copenhagen, Denmark. No changes in study design or methods have been made after trial commencement. An overview of the outcome assessments conducted by researchers blinded to intervention group is pre- sented in Table 1. Information regarding the primary outcome, re-admissions and duration of re-admissions, will be obtained after completion of the two RCTs by linkage of the unique personal identification number (CPR) which is assigned to all 5.4 million persons living in Denmark [39] with the Danish Psychiatric Central Register [40]. The smartphones In the RADMIS trials, the Monsenso system is available for smartphones capable of collecting subjective and automatically generated behavioral data on measures of illness activity. All patients randomized to the interven- tion group are offered the loan of an Android smart- phone free of charge for the 6-month trial period. The patients in the intervention group are encouraged to use the Monsenso system for daily electronic self- monitoring. Economic costs from data traffic due to the RADMIS trials are refunded to all participants regardless the choice of smartphone type. Exclusion criteria: patients who are pregnant and those who lack Danish language skills are excluded, since these factors may influence the possible effect of the RADMIS intervention. Participants and settings Inclusion criteria: all patients over the age of 18 years with a unipolar disorder or bipolar disorder diagnosis, according to the International Classification of Diseases, version 10 (ICD-10) using Schedules for Clinical Assess- ments in Neuropsychiatry (SCAN) [38], who are dis- charged from a psychiatric hospital in The Capital Region of Denmark during the period March 2017 to October 2018 following an affective episode (depression or mania) are invited to participate in the RADMIS tri- als. This corresponds to approximately 1200 patients with unipolar disorder and 600 patients with bipolar dis- order per year. The mental health services in The Cap- ital Region of Denmark covers a recruitment area of the Capital Region, Denmark, corresponding to 1.4 million people. The intervention group All included patients have been discharged from a psy- chiatric hospital at The Capital Region of Denmark dur- ing the period from May 2017 to October 2018 following an affective episode (depression or mania). All included patients continue standard treatment-as-usual at a community psychiatric centre, a private psychiatrist, a general practitioner or outpatient treatment at a hos- pital during the trial period. Study procedure Potential participants are invited to participate in the RADMIS trials by contact with the staff during hospitalization. All potential participants who accept to meet with the RADMIS staff for further trial information are screened by trained researcher to make sure that they fulfil the criteria for participation, and are then in- cluded in the RADMIS trials. Following inclusion, base- line assessments are performed on all patients, and after these assessments the numbered opaque allocation enve- lopes are distributed by a research secretary (HGN) to the RADMIS study nurses. The included patients are randomized separately according to psychiatric diagnosis Subjective (self-monitored) measures of illness activity in the intervention group The patients ran- domized to the intervention group, regardless the choice of smartphone are, on a daily basis, prompted by an alarm in the Monsenso system at a self-chosen time during the day to evaluate subjective measures of illness activity. Faurholt-Jepsen et al. Trials (2017) 18:277 Page 5 of 13 Fig. 2 Flow diagram of the RADMIS trials Fig. 2 Flow diagram of the RADMIS trials Fig. 2 Flow diagram of the RADMIS trials Table 1 Outcome assessments during the RADMIS trial SCAN and background information Rating scales Questionnaires Clinical information Baseline x x x x Randomization to (1) smartphone-based monitoring and treatment (the intervention group) or (2) treatment-as-usual (the control group) 3-month follow-up x x x 6-month follow-up x x x SCAN Schedules for Clinical Assessment in Neuropsychiatry interview Rating scales: Hamilton Depression Rating Scale 17-item (HDRS-17); Young Mania Rating Scale (only for patients with a bipolar disorder diagnosis); Psychosocial functioning test (Functional Assessment Short Test (FAST)) Questionnaires: perceived stress according to Cohen’s Perceived Stress Scale; quality of life according to the WHO Quality of Life-BREF (WHOQOL-BREF); self-rated depressive symptoms according to Beck’s Depressive Inventory (BDI); self-rated depressive symptoms according to the Hamilton Depression Self-rating Scale 6-item (HDRS-6); self-rated manic symptoms according to the Altman Self-rating Scale for Mania (ASRM) (only for patients with a bipolar disorder diagnosis); recovery according to the Recovery Assessment Scale; empowerment according to Rogers’ Empowerment Scale; adherence to medication according to the Medicine Adherence Rating Scale; wellbeing according to the WHO (five) Wellbeing Index; rumination according to the Rumination Response Scale (RRS); worrying according to the Penn State Worry Questionnaire (PSWQ); satisfaction according to the Verona Satisfaction Scale-Affective Disorder (VSS-A). Study procedure The HDRS-6, the ASRM and the WHO (five) are filled out every month during the study 6-month period Clinical information: re-admissions and duration of re-admissions (register data); number of affective episodes; number of contacts with clinicians and psychiatric emergency rooms; hospitalizations; medication, etc Fig. 2 Flow diagram of the RADMIS trials Table 1 Outcome assessments during the RADMIS trial SCAN and background information Rating scales Questionnaires Clinical information Baseline x x x x Randomization to (1) smartphone-based monitoring and treatment (the intervention group) or (2) treatment-as-usual (the control group) 3-month follow-up x x x 6-month follow-up x x x SCAN Schedules for Clinical Assessment in Neuropsychiatry interview Rating scales: Hamilton Depression Rating Scale 17-item (HDRS-17); Young Mania Rating Scale (only for patients with a bipolar disorder diagnosis); Psychosocial functioning test (Functional Assessment Short Test (FAST)) Questionnaires: perceived stress according to Cohen’s Perceived Stress Scale; quality of life according to the WHO Quality of Life-BREF (WHOQOL-BREF); self-rated depressive symptoms according to Beck’s Depressive Inventory (BDI); self-rated depressive symptoms according to the Hamilton Depression Self-rating Scale 6-item (HDRS-6); self-rated manic symptoms according to the Altman Self-rating Scale for Mania (ASRM) (only for patients with a bipolar disorder diagnosis); recovery according to the Recovery Assessment Scale; empowerment according to Rogers’ Empowerment Scale; adherence to medication according to the Medicine Adherence Rating Scale; wellbeing according to the WHO (five) Wellbeing Index; rumination according to the Rumination Response Scale (RRS); worrying according to the Penn State Worry Questionnaire (PSWQ); satisfaction according to the Verona Satisfaction Scale-Affective Disorder (VSS-A). The HDRS-6, the ASRM and the WHO (five) are filled out every month during the study 6-month period Clinical information: re-admissions and duration of re-admissions (register data); number of affective episodes; number of contacts with clinicians and psychiatric emergency rooms; hospitalizations; medication, etc Faurholt-Jepsen et al. Study procedure Trials (2017) 18:277 Page 6 of 13 The following subjective (self-monitored) measures of ill- ness activity are available for daily evaluation for patients with unipolar disorder as well as bipolar disorder: sleep duration (number of hours slept per night, measured in half-hour intervals), time of falling asleep the previous evening (hh:mm), time woken up the following morning (hh:mm), medicine intake (taken as prescribed/taken with changes (if changes, the patients are asked to specify these)/not taken), anxiety (scored from “not present,” “present to some degree” or “present” on a scale from 0, 1, 2), cognitive problems (scored from “not present,” “present to some degree” or “present” on a scale from 0, 1, 2), alco- hol consumption (number of units consumed per day, 0 to +10 scale), stress (scored from “not present,” “present to some degree” or “present” on a scale from 0, 1, 2), menstru- ation for women (yes/no), individual early warning signs (yes/no), a number (unlimited) of personal parameters (cre- ated by the patients themselves), and a free-text note. Fig. 3 The Monsenso self-assessment system. Screenshot of the smartphone-based self-assessment Fig. 3 The Monsenso self-assessment system. Screenshot of the smartphone-based self-assessment all patients in the intervention group (can be obtained by contacting the corresponding author). Suicidal thoughts will not be evaluated as part of the self-monitoring due to ethical concerns and since deteri- oration, leading to suicidal thoughts, will be reflected on other of the self-monitored parameters in the system. Suicidal patients will be treated using standard care and taken care of by the usual clinician. Automatically generated behavioral data (objective data) on measures of illness activity Smartphones are capable of collecting automatically generated behavioral data on measures of illness activity on a daily basis (ob- jective data) during the 6-month trial period. In addition to these items, the following self-monitored measures of illness activity are available for daily evaluation specifically for patients with unipolar disorder: mood (scored from “euthymic” to “depressive” on a scale from 0, −0.5, −1, −2, −3), activity level (scored from “normal” to “very low” on a scale from 0, −1, −2, −3), irritability (scored from “not present,” “present to some degree” or “present” on a scale from 0, 1, 2). Study procedure The smartphone automatically transfers the self- monitored subjective measures and, for some smart- phones, also the automatically generated behavioral data on measures of illness activity to servers at the hospital through secure connections (I-suite number RHP-2011-03). By giving informed consent to partici- pate in the RADMIS, the patients allow for the RAD- MIS study nurses and their health care provider to access the monitored data through a secure web interface. The RADMIS study nurses go through the collected data approximately two to three times a week, or more often on patients where it is deemed necessary. A personal homepage is set up on a server allowing the patients to access all their own data through a similar secure web interface. sent/received, (3) physical activity measured by the step counter in the smartphones, and (4) mobility based on the location estimation available in the smartphones (which again rely on, e.g., GPD or GSM cell tower infor- mation depending on specific circumstances). Further- more, speech activity is collected by extraction of different voice features during phone calls. Voice feature extraction will take place directly on the smartphones and no recording of the actual speech/conversation will take place. The intention is to synthesize these automat- ically generated behavioral data into one aggregated ob- jective composite measure. Smartphone-based CBT modules The smartphone- based CBT modules include four modules: psychoeduca- tion, behavioral activation, cognitive restructuring and rumination-focused CBT. The psychoeducation and mood-monitoring system include strategies for detecting and intervening with early signs of relapse. Patients are encouraged to use the module in an individualized and sequential way according to their condition, affective state, and insight and following guidance from the study nurse if wanted. The behavioral activation module was inspired by Martell et al. (2010) and Lejuez et al. (2001) [41, 42]. It includes activity monitoring and activity scheduling and focus on regulation of sleep and daily routines. Several studies have established the efficacy for behavioral activation for treating depression [43–46] in- cluding a few studies of behavioral activation using smartphones; however, not including a control standard treatment arm [47, 48]. Further, regulating patterns of activity and sleep are helpful in reducing both depressive and manic symptoms [43, 49]. 1. The feedback loop on subjective measures: regardless the type of smartphone a feedback loop on the subjective measures is established. Study procedure A standard of scoring thresholds for when the RADMIS study nurses initially should react was made. For example, the RADMIS study nurses react if the patients register ≥−2 on the mood item for 2 days or more, or if the patients register changes in their sleep patterns of 1 h or more for more than 3 days. Following a run-in phase of approximately 2 to 4 weeks of self- monitoring, the patients and the RADMIS study nurses individualize the thresholds for when reac- tion should be made. Also the RADMIS study nurses and the patients agree on a concordance status in (a) the patients’ most important items for identifying prodromal symptoms of depression as well as (hypo)mania (only for patients with a bipolar disorder diagnosis), (b) the threshold for future early warning signs, and (c) actions to be taken in case of depression or (hypo)mania (only for patients with a bipolar disorder diagnosis) The cognitive restructuring includes simple techniques to help patients to identify and modify dysfunctional auto- matic thoughts. Cognitive restructuring is a key element of CBT and is included in a number of studies that show the efficiency of CBT in the treatment of depression including some studies of Internet-based CBT [50, 51], and a few studies including patients with bipolar disorder [20, 22]. A main purpose of the smartphone-based CBT modules was to help patients to reduce depressive ruminations. Therefore, a specific rumination-focused CBT strategy, based on the theory and practice described by Watkins et al. [25], was included. Rumination-focused CBT has shown encouraging results in the treatment of residual de- pression [25] and the Monsenso system includes the iden- tification and reduction of unhelpful ruminations. p p g 2. The integrated feedback loop on subjective and automatically generated behavioral data on measures of illness activity: a feedback loop integrating subjective and automatically generated behavioral data on measures of illness activity is established. Study procedure Similarly, the following additional self-monitored measures of illness activity are available for daily evaluation specifically for patients with bipolar dis- order: mood (scored from “depressive” to “manic” on a scale from −3, −2, −1, −0.5, 0, +0.5, +1, +2, +3), activity level (scored from “very low” to “very high” on a scale from −3, −2, −1, 0, +1, +2, +3), mixed mood (yes/no), irritability (scored from “not present,” “present to some degree” or “present” on a scale from 0, 1, 2). Examples of some of the automatically generated be- havioral data on measures of illness activity collected by the smartphones: (1) phone usage measured as the num- ber of times and amount of time the smartphones screen are turned on/off, battery usage, ambient light, and “proximity” detection, (2) social activity measured as the number of in- and outgoing phone calls and text mes- sages, the duration of in- and outgoing phone calls, the length of the text messages, and the time of the day when the phone calls and/or text messages are made/ Fig. 4 The Monsenso self-assessment system. Screenshot of the smartphone-based self-assessment of mood After midnight, the entered subjective (self-monitored) measures of illness activity are “locked” and further changes cannot be made. If the patients wish to change their subjective evaluation they can enter a second evaluation in addition to the initial one, and both of the subjective evaluations are then visible for the patient and the health care provider when logging on to in the Mon- senso system. If the patients forget to evaluate the sub- jective measures it is possible to enter and evaluate retrospectively for up to 2 days. It is then noted in the Monsenso system that the subjective measures are col- lected retrospectively. Screenshots from the Monsenso system are presented in Figs. 3 and 4. A user’s guide for the Monsenso system was developed and handed out to Fig. 4 The Monsenso self-assessment system. Screenshot of the smartphone-based self-assessment of mood Faurholt-Jepsen et al. Trials (2017) 18:277 Page 7 of 13 Page 7 of 13 for the integrated feedback loop. Patients allocated to the intervention group of the RADMIS trials will have the Monsenso application installed on a smartphone. Study procedure The feedback loop integrates both subjective and automatically generated behavioral data on measures of illness activity in a flexible and adjustable model (a learning system) resulting in prediction analyses of the collected data providing messages for both the patients and the RADMIS study nurses such as: “You are invited to contact your RADMIS study nurse.” The integrated feedback loop between patients and clinicians Study nurses with experience with unipolar disorder and bipolar disorder are assigned to the pa- tients allocated to the intervention group of the RAD- MIS trials. The RADMIS study nurses are responsible Page 8 of 13 Page 8 of 13 Faurholt-Jepsen et al. Trials (2017) 18:277 Faurholt-Jepsen et al. Trials (2017) 18:277 Beck’s Depressive Inventory (BDI) [56–58], self-rated de- pressive symptoms according to the Hamilton Depres- sion Self-rating Scale 6-item (HDRS-6) [59], self-rated manic symptoms according to Altman Self-rating Scale for Mania (ASRM) (only for patients with a bipolar dis- order diagnosis) [34], recovery according to the Recovery Assessment Scale [60], empowerment according to Rog- ers’ Empowerment Scale [61], adherence to medication according to the Medicine Adherence Rating Scale [62], wellbeing according to the WHO (five) Wellbeing Index [63], rumination according to the Ruminative Response Scale (RRS), worrying according to the Penn State Worry Questionnaire (PSWQ) [64], and satisfaction ac- cording to the Verona Satisfaction Scale-Affective Dis- order (VSS-A) [65]. The patients are asked to fill out HDRS-6, ASRM (only for patients with a bipolar diagno- sis) and WHO (five) every month during the 6-month trial period in both groups and in both trials. Actions taken by the RADMIS study nurses, as part of the feedback loop in the intervention group, in case of signs of deterioration of a patient in the intervention group: 1. Contact the patient and give advice on how to handle the situation 2. If the first action is not enough, the study nurses ask the patient to contact the usual physician or other clinician 3. If the above actions are not enough, or if contact with the patient is not possible, the study nurses contact the patient’s usual physician or other clinician themselves 4. Outcomes Primary outcomes  Rate and duration of hospital re-admissions of patients with unipolar disorder and bipolar disorder, respectively, as according to data retrieved from the Danish Psychiatric Central Register [40] Study procedure In case of acute deterioration and/or severe symptoms, the study nurses recommend the patient to contact the psychiatric emergency service in Copenhagen, Denmark The Monsenso system was designed in an interactive process between patients with unipolar disorder, bipolar disorder, IT researchers, clinicians, and clinical re- searchers before the beginning of the RADMIS trial. Assessments Researchers blinded to the patients’ allocation of interven- tion group (MFJ and one other physician) who are not in- volved in the treatment of the patients carry out all outcome assessments. The unipolar disorder or bipolar disorder diagnoses according to ICD-10 are confirmed by a SCAN interview before inclusion of the patients. The patients are, regardless of randomization group, enrolled for a 6-month trial period and invited for outcome assess- ments by researchers blinded to intervention at baseline, after 3 months and after 6 months (Table 1). Researchers blinded to the patients’ allocation of interven- tion group (MFJ and one other physician) who are not in- volved in the treatment of the patients carry out all outcome assessments. The unipolar disorder or bipolar disorder diagnoses according to ICD-10 are confirmed by a SCAN interview before inclusion of the patients. The patients are, regardless of randomization group, enrolled for a 6-month trial period and invited for outcome assess- ments by researchers blinded to intervention at baseline, after 3 months and after 6 months (Table 1). y  Psychosocial functioning according to the Functional Assessment Short Test (FAST)  Number of depressive episodes and manic episodes (manic episodes only for patients with a bipolar disorder diagnosis) defined as HDRS-17 > 13 and YMRS >13, respectively  Automatically generated behavioral data measured as an aggregated composite measure At each visit with the researchers, the assessments in- clude the following: The control group Patients allocated to the control group continue with their standard treatment-as-usual and are asked to con- tinue communicating as usual using their mobile phone (smartphone). Secondary outcomes  Severity of depressive symptoms and manic symptoms (manic symptoms only for patients with a bipolar disorder diagnosis) measured using the HDRS-17 and the YMRS, respectively Statistical power and sample size calculation Statistical power and sample size calculation The statistical power and sample size was calculated using http://stat.ubc.ca/~rollin/stats/ssize/n2.html. The statistical power and sample size was calculated using http://stat.ubc.ca/~rollin/stats/ssize/n2.html. The study will use a stratified design, where patients are stratified according to the psychiatric centres where patients are discharged from and according to the num- ber of previous hospitalizations (three or less or more than three). The statistical analyses will adjusted for the two stratification variables, and also age and sex as pos- sible prognostic variables. Further, in analyses on con- tinuous variables, potential differences in baseline score on the outcome in question will be included as a poten- tial confounder. If there are no statistically significant main effects of age and sex, these variables will be ex- cluded from the final statistical analyses. g p The primary outcomes are differences in the number and duration of re-admissions to a psychiatric hospital be- tween the intervention group and the control group −ana- lyzed separately according to psychiatric diagnosis (unipolar disorder or bipolar disorder). Power calculations are similar for the two RCTs/patient groups (unipolar dis- order or bipolar disorder). Based on prior findings aiming to reduce re-hospitalization due to recurrent depression [66] and bipolar disorder [9], combined with presump- tions of positive effects of the immediate early contact with patients during the vulnerable period following dis- charge from a psychiatric hospital, regardless of diagnosis, we expect a reduction in re-admissions from 30% to 15% in the intervention group using smartphone-based treat- ment. Thus, anticipating a hazard ratio (HR) of 0.50 in the comparison of the intervention group with the control group on the primary outcome, a two-sided risk of type 1 error, α of 0.05, a type 2 error risk, β, a statistical power of 80%, the sample size (N) is calculated as N = 200 (100 pa- tients in each arm of each RCT) per RCT and, thus, a total of 400 patients (200 patients with unipolar disorder and 200 patients with bipolar disorder) is estimated to be re- quired for the RADMIS trial. Cumulated durations of re- hospitalizations comprise similar power calculations and sample sizes. Allocation concealment and implementation p The allocation sequence is concealed from the re- searchers (e.g., MFJ) enrolling and assessing the patients and from the RADMIS study nurses. Allocation is con- cealed in numbered, opaque and sealed envelopes. Tertiary outcomes 1. Clinician-administrated rating scales: the severity of depressive and manic symptoms (manic symptoms only assessed for patients with a bipolar disorder diagnosis) is measured using the Hamilton Depression Rating Scale 17-item (HDRS-17) [52] and the Young Mania Rating Scale (YMRS) [34], respectively, and psychosocial func- tioning is measured using the Functioning Assessment Short Test (FAST) [53]  Perceived stress according to Cohen’s Perceived Stress Scale [54], quality of life according to the WHO Quality of Life-BREF (WHOQOL-BREF) [55], self-rated depressive symptoms according to Beck’s Depressive Inventory (BDI) [56–60], self-rated depressive symptoms according to the Hamilton Depression Self-rating Scale 6-item (HDRS-6) [59], self-rated manic symptoms according to the Altman Self-rating scale for Mania (ASRM) (only for patients with a bipolar disorder diagnosis) [34], 2. Questionnaires: perceived stress according to Cohen’s Perceived Stress Scale [54], quality of life ac- cording to the WHO Quality of Life-BREF (WHOQOL- BREF) [55], self-rated depressive symptoms according to Page 9 of 13 Faurholt-Jepsen et al. Trials (2017) 18:277 Statistical power and sample size calculation Regarding the secondary outcomes, the clin- ically relevant difference in depressive or manic symptoms is defined as a minimum of three scores on the HDRS-17 or on the YMRS, respectively, and the standard deviation (SD) is set at 7 with a mean score of 12 versus 15 in the intervention group and the control group, respectively. The statistical power to detect a three-score difference in the areas under the curves between the intervention and the control groups on the HDRS-17 or the YMRS, re- spectively, is 80% with α = 0.05 for a two-sample compari- son of means including a total of 200 patients (100 patients in each arm) in each of the two RADMIS RCTs. Randomization recovery according to the Recovery Assessment Scale [60], empowerment according to Roger’s Empowerment Scale [61], adherence to medication according to the Medicine Adherence Rating Scale [62], wellbeing according to the WHO (five) Well- being Index [63], rumination according to the Ruminative Response Scale (RRS), worrying according to the Penn State Worry Questionnaire (PSWQ) [64], and satisfaction according to the Verona Satisfaction Scale-Affective Disorder (VSS-A) [65] Sequence generation A computer-generated list of random allocation num- bers using Pharma Consulting Group (http://www.phar- maconsultinggroup.com) will be generated. Patients included in the trial are randomized with a balanced al- location ratio of 1:1 to (1) using the Monsenso system, including the integrated feedback loop, based on a com- bination of subjective self-monitored measures and auto- matically generated behavioral data on measures of illness activity) including context-aware CBT modules (the intervention group) or to (2) standard treatment-as- usual (the control group) (Fig. 1). No changes in trial outcomes have been made after trial commencement. Since the RADMIS trial is single-blinded, random block sizes are used to help preserve unpredictability [67, 68]. The RADMIS study nurses are unaware of the range of numbers in the block sizes. Statistical methods randomized, controlled trials with clearly defined clinical outcome measures. randomized, controlled trials with clearly defined clinical outcome measures. Data from all randomized patients are collected until drop- out or the end of the trial period. Analysis will be carried out with an intention-to-treat (ITT) approach. The primary outcomes are differences in time to re-admission and dur- ation of re-admissions during the 6-month trial period be- tween the intervention group and the control group. Time to the first re-admission will be estimated using a Kaplan- Meier plot with reasons for censoring being date of death or end of study. The differences in cumulated prevention of re-admission in the intervention group and the control group will tested using a log-rank test. Analysis of second- ary and tertiary outcomes will be done employing a linear mixed-effects model with random intercept for each par- ticipant and a fixed effect of visit. Differences in outcomes between the intervention group and the control group will be analyzed, firstly in an unadjusted model (except for dif- ferences in baseline values of the outcome variable in ana- lyses on continuous variables) and then in models adjusted for the two stratification variables (1) psychiatric center and (2) the number of prior hospitalizations, and also for age and sex as possible prognostic variables. If there are no sta- tistically significant main effects of age and sex, these vari- ables will be excluded from the final analyses. Furthermore, subanalyses will be done employing a linear mixed-effects model with random intercept for each participant and a fixed effect of visit on differences in the HDRS-17 and the YMRS in patients with the presence of depressive and manic symptoms (defined as HDRS-17 > 0 or YMRS >0, re- spectively) at a given time point during the trial period be- tween the intervention group and the control group. Additionally, in addition to analyses of the secondary out- comes, we will analyze differences in depressive and manic episodes defined as HDRS ≥14 and YMRS ≥14, respectively, during the trial period. Potential interactions between randomization group and visit number on any specific out- come variable in the analyses will be investigated and re- ported accordingly. The statistical threshold for significance is p ≤0.05 (two-tailed). Data will be managed by MFJ and entered using Epidata®. Advantages First, the RADMIS trials’ use a pragmatic design with few exclusion criteria and the results of the trials will be generalizable to patients who are going to be discharged from psychiatric hospitals in general with a diagnosis of unipolar disorder or bipolar disorder and have clinical rele- vance. The patients are recruited at a critical time point at discharge from hospital at which they still suffer from some affective symptoms and have a need for continued treat- ment. Following hospitalization, patients often experience a service gap between inpatient and outpatient services, and the RADMIS smartphone-based monitoring and treatment system is developed among others to fill out this gap. Second, it is a major advantage that clinical information on the primary outcome will be available for all included patients (100%), regardless of whether they drop out of the RADMIS trials or not, as data on date of re-admission and duration of re-hospitalizations routinely are reported nationwide to the Danish Psychiatric Central Register [40]. Also, the information on re-admissions and duration of re-hospitalizations are collected without the risk of unblinding of the researcher and not based on the pa- tients’ subjective evaluations. Third, information on the secondary outcome mea- sures are based on standardized clinical rating scales often used as the “gold standard,” and are conducted by trained researchers blinded to intervention groups. In contrast to this, the tertiary outcomes are based on the patients’ subjective and unblinded evaluations and are, thus, at risk of performance bias. Fourth, the intervention used in the RADMIS trials is specifically designed to address the needs of the patients when being discharged from a psychiatric hospitalization. During the design phase of the trial the software was designed in a close collaboration between patients with uni- polar disorder and bipolar disorder, IT researchers, clini- cians, and clinical researchers during an interactive process. Fifth, automatically generated behavioral data collected by the smartphones will be combined in an aggregated combined measure of illness activity that will be reported as an additional outcome measure. These data are object- ive and have been shown to correlate with the severity of depressive and manic symptoms in studies by our group. However, using these data as outcome measures is still ex- perimental and will be developed further during the RAD- MIS trials. Statistical methods All analyses will be done using SPSS, version 22.0 (IBM, New York, NY, USA) and STATA version 12 (StataCorp LP, College Station, TX, USA). Blinding g Owing to the type of intervention in the RADMIS trials, the patient, the patients’ health care provider and the RADMIS study nurses are aware of the allocated randomization group. The researchers responsible for outcome assessments, data entry, data analyses, inter- pretation of analyses and writing of papers are kept blinded to allocation at all times during the trial period, data analyses and interpretation of analyses. The trials are, therefore, single-blinded. The RADMIS study nurses do not collect any outcome measures. At each visit with the researchers, all patients are thoroughly instructed not to mention anything about randomization allocation. In case of unblinding of the included patients’ allocation status, one of the other researchers blinded to interven- tion in the RADMIS trials will conduct the outcome assessments. Page 10 of 13 Faurholt-Jepsen et al. Trials (2017) 18:277 Page 10 of 13 Advantages Consequently, the aggregated smartphone- based measure of illness activity is not included as a pre- defined outcome measure in this study protocol. Trial status The trial is ongoing. Recruitment begins March 2017. All potential participants are invited to receive information about the RADMIS trial on an individual basis where the information is given in a quiet and undisturbed room. All information is presented in both written and verbal form and participants can bring a friend or relative to the introductory conversation. Participants are informed that participation is voluntary and that consent can be withdrawn at any time during the trial without this having any consequences for future treatment possibilities. All included patients sign a Consent Form and are given a copy of this together with their rights as a participant in a clinical trial. All included patients are offered to loan a smartphone free of charge during the trial period, and economic costs due to data traffic from the Monsenso system are refunded. Participants do not receive other economic compensation for participating in the RADMIS trials. Authors’ contributions MFJ, LVK, KM, and JB conceived the trial and authored the first draft of the trial protocol. OW, NR, NT, JB, MF, and JB have been revising and optimizing the trial protocol. All authors contributed to, and approved, the final version of the manuscript. Acknowledgements Acknowledgements Not applicable. Additional file Additional file 1: SPIRIT checklist for the RADMIS trial. (DOC 115 kb) The intervention group The RADMIS trials are designed to investigate the effect of the entire Monsenso system, including self-monitoring, automatically generated behavioral data, integrated feed- back loop and context-aware CBT modules. Thus, we will not be able to distinguish the effects of the individual components of the intervention. Consent for publication The secondary outcome measures are differences in clinic- ally rated depressive and manic symptoms as assessed with standardized rating scales. Since the patients are aware of their allocation status these outcome measures are single blinded. The tertiary outcomes are questionnaires evaluated by the patients themselves and are, therefore, unblinded. Patients will consent to the publication of data. Written informed consent will be obtained from the patients for publication of their individual details and accompanying images in this manuscript. The Consent Form is held by the authors and is available for review by the Editor-in-Chief. Competing interests MFJ declares that she has no competing interests. MF and JB are co-founders and shareholders in Monsenso ApS. LVK has, within the last 3 years, been a consultant for Lundbeck and AstraZeneca. JoB, KM, NT, NR, and OW declare that they have no competing interests. MFJ declares that she has no competing interests. MF and JB are co-founders and shareholders in Monsenso ApS. LVK has, within the last 3 years, been a consultant for Lundbeck and AstraZeneca. JoB, KM, NT, NR, and OW declare that they have no competing interests. Ethical approval and consent to participate The usual health care provider/physician/clinician of the patients randomized to the intervention group is contacted by letter at the beginning of the trial and informed about the trial and that the RADMIS study nurses might contact them if there are signs of patient deterioration. All i l i i i i d i i f i b h Ethical approval and consent to participate pp p p Ethical permission for the RADMIS trials was obtained from the Regional Ethics Committee in The Capital Region of Denmark and The Data Agency, Capital Region of Copenhagen (H-16046093). The law on handling of personal data will be respected. The patients’ health care journals will be read to confirm information regarding the patients’ clinical history. The trial was registered at ClinicalTrials.gov as NCT03033420 on 13 January 2017. All positive, neutral and negative findings of the trial will be published according to the CONSORT guidelines [36]. All electronically monitored data are stored at a secure server at Capital Region, Copenhagen, Denmark. The usual health care provider/physician/clinician of the patients randomized to the intervention group is contacted by letter at the beginning of the trial and informed about the trial and that the RADMIS study nurses might contact them if there are signs of patient deterioration. All potential participants are invited to receive information about the RADMIS trial on an individual basis where the information is given in a quiet and undisturbed room. All information is presented in both written and verbal form and participants can bring a friend or relative to the introductory conversation. Participants are informed that participation is voluntary and that consent can be withdrawn at any time during the trial without this having any consequences for future treatment possibilities. All included patients sign a Consent Form and are given a copy of this together with their rights as a participant in a clinical trial. All included patients are offered to loan a smartphone free of charge during the trial period, and economic costs due to data traffic from the Monsenso system are refunded. Participants do not receive other economic compensation for participating in the RADMIS trials. Ethical permission for the RADMIS trials was obtained from the Regional Ethics Committee in The Capital Region of Denmark and The Data Agency, Capital Region of Copenhagen (H-16046093). The law on handling of personal data will be respected. The patients’ health care journals will be read to confirm information regarding the patients’ clinical history. The trial was registered at ClinicalTrials.gov as NCT03033420 on 13 January 2017. All positive, neutral and negative findings of the trial will be published according to the CONSORT guidelines [36]. All electronically monitored data are stored at a secure server at Capital Region, Copenhagen, Denmark. Perspectives If the Monsenso system proves effective in reducing the rate and duration of re-admissions in patients with uni- polar disorder and bipolar disorder in the present trial, there will be basis for using a system of this kind in psy- chiatric treatment in general and on a larger scale. Discussion Monitoring of illness activity using smartphones seems promising as an intervention in unipolar disorder and bipolar disorder, but few studies using rigorous method- ology have been published. No study investigating the ef- fect of a smartphone-based intervention on rate and duration of re-admissions in both unipolar disorder and bipolar disorder has been published. The RADMIS trials aim to investigate the effect of a smartphone-based monitoring and treatment system on the risk of psychi- atric hospitalization, clinical function, and wellbeing in Sixth, the Monsenso system will be available for both Android and iPhones. Thus, patients are not excluded due to preference of smartphone type and/or operating Page 11 of 13 Page 11 of 13 Page 11 of 13 Faurholt-Jepsen et al. Trials (2017) 18:277 system. Patients randomized to the intervention group are offered to loan a smartphone free of charge during the RADMIS trials and, thus, there are no requirements for smartphone access. RCT: Randomized controlled trial; RRS: The Ruminative Response Scale; SCAN: Schedules for Clinical Assessments in Neuropsychiatry; The MONARCA system: Smartphone-based monitoring system for patients with bipolar disorder; The Monsenso system: Smartphone-based monitoring system including CBT modules for patients with both unipolar disorder and bipolar disorder (inspired by the MONARCA system); The RADMIS trial: Reducing the rate and duration of Re-ADMISsions among patients with unipolar disorder and bipolar disorder using smartphone-based monitoring and treatment; VSS-A: The Verona Satisfaction Scale-Affective Disorder; WHOQOL-BREF: The WHO Quality of Life-BREF; YMRS: The Young Mania Rating Scale Availability of data and materials Not applicable. Funding Funding The RADMIS trial is funded by the Innovation Foundation (5164-00001B). 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Int J Methods Psychiatr Res. 2003;12(2):85–91. 64. Meyer TJ, Miller ML, Metzger RL, Borkovec TD. Development and validation of the Penn State Worry Questionnaire. Behav Res Ther. 1990;28(6):487–95. 65. Kessing LV, Hansen HV, Ruggeri M, Bech P. Satisfaction with treatment among patients with depressive and bipolar disorders. Soc Psychiatry Psychiatr Epidemiol. 2006;41(2):148–55. 66. Hansen HV, Christensen EM, Dam H, Gluud C, Wetterslev J, Kessing LV. The effects of centralised and specialised intervention in the early course of severe unipolar depressive disorder: a randomised clinical trial. PloS One. 2012;7(3):e32950. doi:10.1371/journal.pone.0032950. 67. Schulz KF, Grimes DA. Allocation concealment in randomised trials: defending against deciphering. Lancet. 2002;359(9306):614–8. 68. Schulz KF, Grimes DA. Unequal group sizes in randomised trials: guarding against guessing. Lancet. 2002;359(9310):966–70. 64. Meyer TJ, Miller ML, Metzger RL, Borkovec TD. Development and validation of the Penn State Worry Questionnaire. Behav Res Ther. 1990;28(6):487–95. 65. Kessing LV, Hansen HV, Ruggeri M, Bech P. Satisfaction with treatment among patients with depressive and bipolar disorders. Soc Psychiatry Psychiatr Epidemiol. 2006;41(2):148–55. References Schizophr Res. 2000;42(3):241–7. 62. Thompson K, Kulkarni J, Sergejew AA. Reliability and validity of a new Medication Adherence Rating Scale (MARS) for the psychoses. Schizophr Res. 2000;42(3):241–7.
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Policy analysis for administrative role based access control without separate administration
Journal of computer security
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Policy Analysis for Administrative Role Based Access Control without Separate Administration Ping Yang, Mikhail Gofman, Zijiang Yang To cite this version: Ping Yang, Mikhail Gofman, Zijiang Yang. Policy Analysis for Administrative Role Based Access Control without Separate Administration. 27th Data and Applications Security and Privacy (DBSec), Jul 2013, Newark, NJ, United States. pp.49-64, ￿10.1007/978-3-642-39256-6_4￿. ￿hal-01490717￿ To cite this version: Ping Yang, Mikhail Gofman, Zijiang Yang. Policy Analysis for Administrative Role Based Access Control without Separate Administration. 27th Data and Applications Security and Privacy (DBSec), Jul 2013, Newark, NJ, United States. pp.49-64, ￿10.1007/978-3-642-39256-6_4￿. ￿hal-01490717￿ Distributed under a Creative Commons Attribution 4.0 International License Policy Analysis for Administrative Role Based Access Control without Separate Administration Ping Yang1 and Mikhail Gofman2 and Zijiang Yang3 1 Dept. of Computer Science, State University of New York at Binghamton, NY, USA 2 Dept. of Computer Science, California State University at Fullerton, CA, USA 3 School of Computer Science and Engineering, Xi’an University of Technology, China Dept. of Computer Science, Western Michigan University, MI, USA Abstract. Access control is widely used in large systems for restricting resource access to authorized users. In particular, role based access control (RBAC) is a generalized approach to access control and is well recognized for its many ad- vantages in managing authorization policies. This paper considers user-role reachability analysis of administrative role based access control (ARBAC), which defines administrative roles and specifies how members of each administrative role can change the RBAC policy. Most exist- ing works on user-role reachability analysis assume the separate administration restriction in ARBAC policies. While this restriction greatly simplifies the user- role reachability analysis, it also limits the expressiveness and applicability of ARBAC. In this paper, we consider analysis of ARBAC without the separate administration restriction and present new techniques to reduce the number of ARBAC rules and users considered during analysis. We also present a number of parallel algorithms that speed up the analysis on multi-core systems. The exper- imental results show that our techniques significantly reduce the analysis time, making it practical to analyze ARBAC without separate administration. HAL Id: hal-01490717 https://inria.hal.science/hal-01490717v1 Submitted on 15 Mar 2017 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1 Introduction Access control is widely used for restricting resource access to authorized users. In par- ticular, role based access control (RBAC) [2] is broadly recognized as a generalized approach to access control that has many advantages in performing authorization man- agement. An RBAC policy is a tuple ⟨U, R, P, UA, PA⟩where U, R and P are finite sets of users, roles, and permissions, respectively. UA ⊆U × R represents the user- role assignment relation and PA ⊆P × R represents the permission-role assignment relation. RBAC also supports role hierarchy: r1 ⪰r2 specifies that r1 is senior to r2 (or r2 is junior to r1), which implies that every member of r1 is also a member of r2, and every permission assigned to r2 is also available to members of r1. Administrative role-based access control (ARBAC’97) [17] defines administrative roles and specifies how members of each administrative role can change the RBAC policy. ARBAC specifies user-role administration which controls the way changes are made to the user-role assignments. This control is enforced by two types of rules: (1) can assign(ra, c, rt) that grants an administrative role ra permission to assign a target role rt to any user who satisfies the precondition c, and (2) can revoke(ra, rt) that grants an administrative role ra permission to revoke a target role rt from a user. The precondition c is a conjunction of literals, where each literal is either r (positive precondition) or ¬r (negative precondition) for some role r. ARBAC’97 requires sep- arate administration [22], i.e., administrative roles cannot be target roles in can assign and can revoke rules or appear in the preconditions. In the rest of this paper, we repre- sent the precondition c as P ∧¬N where P contains all positive preconditions and N contains all negative preconditions in c. The correctness of ARBAC policies is critical to system security because any design flaws and human specification errors in ARBAC may result in the leak of confidential data to unauthorized users. Large organizations may have large ARBAC policies. In such organizations, manual inspection of ARBAC policies for correctness can be im- practical because actions performed by different administrators may interfere with each other in subtle ways. Thus, automated analysis algorithms are essential to ensure that an ARBAC policy conforms to the desirable correctness properties. 1 Introduction Contributions: This paper presents a number of reduction techniques that improve the scalability of the algorithm in [22]. Our main contributions are summarized below. – We propose two static reduction techniques – optimized slicing (Section 3.1) and hierarchical rule reduction (Section 3.4) – to reduce the number of ARBAC rules considered during analysis. – We develop a user equivalent set reduction technique that reduces the number of users considered during analysis (Section 3.2). – We propose a lazy reduction technique that delays performing unnecessary transi- tions (Section 3.3). – We present several parallel algorithms, which speed up the analysis on multi-core or multi-processor platforms (Section 4). – We evaluate the effectiveness of our reduction techniques and our parallel algo- rithms on an ARBAC policy representing a university administration. The experi- mental results show that our techniques significantly reduce the analysis time. Organization: The rest of the paper is organized as follows. Section 2 describes the user-role reachability analysis algorithm for ARBAC without separate administration developed in [22]. Sections 3 and 4 present our reduction techniques and our parallel algorithms, respectively. The experimental results are given in Section 5, followed by a discussion of related research in Section 6. Section 7 concludes the paper. 1 Introduction This paper considers the user-role reachability analysis of ARBAC [22], which asks “given an RBAC policy φ, an ARBAC policy ψ, a set of users U, a target user ut, and a set of roles (called the “goal”), is it possible for users in U ∪{ut} to assign ut to roles in the goal”? Since many security analysis problems, such as user-role availability [16], role containment [16], and weakest precondition [22], can be reduced to this problem, user-role reachability is crucial for ARBAC analysis. Researchers have shown that user-role reachability analysis is intractable even un- der various restrictions on the ARBAC policy [16, 18]. Most existing research on user- role reachability analysis [9, 8, 14] follows the definition of ARBAC’97 that assumes separate administration. By disallowing an administrative role to serve as the target role in any of the ARBAC rules, it is sufficient to consider the user-role assignments of only the target user. However, in practice, the separate administration restriction does not always hold. For example, a university ARBAC policy may specify that the role DeptChair can assign a member of role Faculty to role AdmissionComittee, which can in turn assign any user to role Student. Formally, this specification translates to the rules can assign(DeptChair, Faculty, AdmissionComittee) and can assign(AdmissionCommittee, true, Student), which do not satisfy the sepa- rate administration restriction. Analysis of ARBAC without separate administration is significantly more challeng- ing because we need to consider administrative actions that change the role member- ships of all users, not only the target user. For example, a non-target user u may assign another non-target user u1 to an administrative role, which can in turn change the role assignments of the target user. Stoller et al. [22] tackled this problem by developing an algorithm that is fixed parameter tractable with respect to the number of users and mixed roles. That is, the algorithm is exponential to the number of users and mixed roles, but is polynomial to the size of the policy when the number of users and mixed roles is fixed. However, since the number of users is usually large in large organizations, the algorithm does not scale well when analyzing ARBAC policies in such organizations. For example, we have applied this algorithm to analyze a university ARBAC policy containing 150 users and the program failed to terminate within 12 hours for 3 out of the 10 randomly generated queries. 2 Preliminaries: User-Role Reachability Analysis of ARBAC User-role reachability analysis of ARBAC [22] asks: “given an RBAC policy φ, an ARBAC policy ψ, a set of users U, a target user ut, and a set of roles (called the “goal”), is it possible for users in U ∪{ut} to assign ut to all roles in the goal”? Let UA0 be a set of all user-role assignments in φ. The user-role reachability analysis instance is represented as a tuple I = ⟨UA0, ut, ψ, goal⟩. ⟨ ⟩ Stoller et al. [22] presented an algorithm for analyzing ARBAC without separate administration, which is formalized in Algorithm 1. The algorithm is fixed parameter tractable with respect to the number of users and mixed roles. A role is negative if it appears negatively in some precondition in the policy; other roles are non-negative. A role is positive if it appears in the goal, appears positively in some precondition in the policy, or is an administrative role; other roles are non-positive. A role that is both neg- ative and positive is a mixed role. Note that their algorithm is applied to ARBAC with- out role hierarchy; ARBAC with role hierarchy can be converted to the corresponding non-hierarchical policy using the algorithm in [18]. Let I = ⟨UA0, ut, ψ, goal⟩be a user-role reachability analysis problem instance. The algorithm works as follows. First, the algorithm performs a slicing transformation (function slicing in Line 3), which back-chains along the ARBAC rules to identify roles and rules relevant to the goal, and then eliminates the irrelevant ones. Function slicing takes into account whether a role appears positively or negatively in the policy, and computes a set Rel+ of positive roles and a set Rel−of negative roles that are relevant to the goal. A set RelRule of relevant rules is computed as a collection of all can assign rules whose Algorithm 1 The User-Role Reachability Analysis Algorithm in [22]. 2 Preliminaries: User-Role Reachability Analysis of ARBAC 1: Processed = Rel+ = Rel−= ∅; RelRule = ∅; 2: procedure analysis(UA0, ut, ψ, goal) 3: (Rel+, Rel−, RelRule) = slicing(UA0, ψ, goal); W = Reached = {closure(UA0)}; 4: if goal ⊆{r | (ut, r) ∈closure(UA0)} then return true; end if 5: while W ̸= ∅do 6: remove a state s from W; 7: for all can assign(ra, P ∧¬N, r) ∈RelRule do 8: for all (user u ∈U) do 9: if (r ∈(Rel+ ∩Rel−), (u, r) ̸∈s, P ⊆{r | (u, r) ∈s}, N ∩{r | (u, r) ∈s} = ∅, and (u′, ra) ∈s for some user u′) 10: then s′ = closure(s ∪{(u, r)}); add transition s ua(ra,u,r) → s′ to G; 11: if goal ⊆{r|(ut, r) ∈s′} then return true; end if 12: if s′ ̸∈Reached then W = W ∪{s′}; Reached = Reached ∪{s′} end if 13: end if end for end for 14: for all (can revoke(ra, r) ∈RelRule) 15: for all (user u ∈U) 16: if ((u, r) ∈s and (u′, ra) ∈s for some user u′) 17: then s′ = closure(s \ {(u, r)}); add transition s ur(ra,u,r) → s′ to G; 18: if goal ⊆{r | (ut, r) ∈s′} then return true; end if 19: if s′ ̸∈Reached then W = W ∪{s′}; Reached = Reached ∪{s′} end if 20: end if end for end for 21: end while 22: return false; 23: procedure slicing(UA0, ψ, goal) 24: if goal = ∅then return (∅, ∅, ∅) end if 25: Processed = Processed ∪goal; R+ = goal; R−= ∅; Rule = ∅; 26: for all can assign(ra, P ∧¬N, r) ∈ψ where r ∈goal do 27: (R1, R2, R3) = slicing(UA0, ψ, ({ra} ∪P) \ Processed); R+ = R+ ∪R1; 28: R−= R−∪N ∪R2; Rule = Rule ∪{can assign(ra, P ∧N, r)} ∪R3; 29: end for 30: RelRev = {can revoke(ra, r) ∈ψ | r ∈R−}; Rule = Rule ∪RelRev; 31: for all can revoke(ra, r) ∈RelRev where ra ̸∈Processed do 32: (R4, R5, R6) = slicing(UA0, ψ, {ra}); R+ = R+ ∪R4; 33: R−= R−∪R5; Rule = Rule ∪R6 34: end for 35: return (R+, R−, Rule) 36: procedure closure(s) 37: s1 = s; 38: for all can assign(ra, P ∧¬N, r) ∈RelRule do 39: for all user u ∈U do 40: if (r ∈(Rel+ \ Rel−), (u, r) ̸∈s, P ⊆{r | (u, r) ∈s}, N ∩{r | (u, r) ∈s} = ∅, and (u′, ra) ∈s for some user u′) 41: then s1 = s1 ∪(u, r); end if end for end for 42: if s == s1 then return s1; else return closure(s1); Algorithm 1 The User-Role Reachability Analysis Algorithm in [22]. 2 Preliminaries: User-Role Reachability Analysis of ARBAC Algorithm 1 The User-Role Reachability Analysis Algorithm in [22]. Algorithm 1 The User-Role Reachability Analysis Algorithm in [22]. 2 Preliminaries: User-Role Reachability Analysis of ARBAC 1: Processed = Rel+ = Rel−= ∅; RelRule = ∅; 2: procedure analysis(UA0, ut, ψ, goal) 3: (Rel+, Rel−, RelRule) = slicing(UA0, ψ, goal); W = Reached = {closure(UA0)}; 4: if goal ⊆{r | (ut, r) ∈closure(UA0)} then return true; end if 5: while W ̸= ∅do 6: remove a state s from W; 7: for all can assign(ra, P ∧¬N, r) ∈RelRule do 8: for all (user u ∈U) do 9: if (r ∈(Rel+ ∩Rel−), (u, r) ̸∈s, P ⊆{r | (u, r) ∈s}, N ∩{r | (u, r) ∈s} = ∅, and (u′, ra) ∈s for some user u′) 10: then s′ = closure(s ∪{(u, r)}); add transition s ua(ra,u,r) → s′ to G; 11: if goal ⊆{r|(ut, r) ∈s′} then return true; end if 12: if s′ ̸∈Reached then W = W ∪{s′}; Reached = Reached ∪{s′} end if 13: end if end for end for 14: for all (can revoke(ra, r) ∈RelRule) 15: for all (user u ∈U) 16: if ((u, r) ∈s and (u′, ra) ∈s for some user u′) 17: then s′ = closure(s \ {(u, r)}); add transition s ur(ra,u,r) → s′ to G; 18: if goal ⊆{r | (ut, r) ∈s′} then return true; end if 19: if s′ ̸∈Reached then W = W ∪{s′}; Reached = Reached ∪{s′} end if 20: end if end for end for 21: end while 22: return false; 23: procedure slicing(UA0, ψ, goal) 24: if goal = ∅then return (∅, ∅, ∅) end if 25: Processed = Processed ∪goal; R+ = goal; R−= ∅; Rule = ∅; 1: Processed = Rel+ = Rel−= ∅; RelRule = ∅; 2: procedure analysis(UA0, ut, ψ, goal) 5: while W ̸= ∅do 23: procedure slicing(UA0, ψ, goal) 23: procedure slicing(UA0, ψ, goal) 24: if goal = ∅then return (∅, ∅, ∅) end if 25: Processed = Processed ∪goal; R+ = goal; R−= ∅; Rule = ∅; 26: for all can assign(ra, P ∧¬N, r) ∈ψ where r ∈goal do 27: (R1, R2, R3) = slicing(UA0, ψ, ({ra} ∪P) \ Processed); R+ = R+ ∪R1; 28: R−= R−∪N ∪R2; Rule = Rule ∪{can assign(ra, P ∧N, r)} ∪R3; 29: end for 30: RelRev = {can revoke(ra, r) ∈ψ | r ∈R−}; Rule = Rule ∪RelRev; 31: for all can revoke(ra, r) ∈RelRev where ra ̸∈Processed do 32: (R4, R5, R6) = slicing(UA0, ψ, {ra}); R+ = R+ ∪R4; 33: R−= R−∪R5; Rule = Rule ∪R6 34: end for 35: return (R+, R−, Rule) 36: procedure closure(s) 37: s1 = s; 38: for all can assign(ra, P ∧¬N, r) ∈RelRule do 39: for all user u ∈U do 40: if (r ∈(Rel+ \ Rel−), (u, r) ̸∈s, P ⊆{r | (u, r) ∈s}, N ∩{r | (u, r) ∈s} = ∅, and (u′, ra) ∈s for some user u′) 41: then s1 = s1 ∪(u, r); end if end for end for 42: if s == s1 then return s1; else return closure(s1); 24: if goal = ∅then return (∅, ∅, ∅) end if 24: if goal = ∅then return (∅, ∅, ∅) end if 25: Processed = Processed ∪goal; R+ = goal; R−= ∅; Rule = ∅; 26: for all can assign(ra, P ∧¬N, r) ∈ψ where r ∈goal do 27: (R1, R2, R3) = slicing(UA0, ψ, ({ra} ∪P) \ Processed); R+ = R+ ∪R1; { ( )} 28: R−= R−∪N ∪R2; Rule = Rule ∪{can assign(ra, P ∧N, r)} ∪R3; 29: end for 30: RelRev = {can revoke(ra, r) ∈ψ | r ∈R−}; Rule = Rule ∪RelRev; 31: for all can revoke(ra, r) ∈RelRev where ra ̸∈Processed do 32: (R4, R5, R6) = slicing(UA0, ψ, {ra}); R+ = R+ ∪R4; 33: R−= R−∪R5; Rule = Rule ∪R6 34: end for 35: return (R+, R−, Rule) 36: procedure closure(s) 37: s1 = s; 38: for all can assign(ra, P ∧¬N, r) ∈RelRule do 39: for all user u ∈U do 40: if (r ∈(Rel+ \ Rel−), (u, r) ̸∈s, P ⊆{r | (u, r) ∈s}, N ∩{r | (u, r) ∈s} = ∅, and (u′, ra) ∈s for some user u′) 41: then s1 = s1 ∪(u, r); end if end for end for 42: if s == s1 then return s1; else return closure(s1); targets are in Rel+ and all can revoke rules whose targets are in Rel−; only rules in RelRule need to be applied during analysis. 2 Preliminaries: User-Role Reachability Analysis of ARBAC Next, the algorithm constructs a reduced transition graph G using rules in RelRule. Each state in G is a set of user-role assignments and each transition describes an allowed change to the state defined by the ARBAC policy ψ. A transition is either ua(ra, u, r) which specifies that an administrative role ra adds user u to role r, or ur(ra, u, r) which specifies that an administrative role ra revokes user u from role r. The follow- ing reductions are applied: (1) Transitions that revoke non-negative roles (i.e., roles in Rel+ \ Rel−) or add non-positive roles (i.e., Rel−\ Rel+) are prohibited because they do not enable any other transitions; (2) Transitions that add non-negative roles or revoke non-positive roles are invisible; such transitions will not disable any other transitions. Transitions that add or revoke mixed roles are visible. The invisible transitions together with a visible transition form a single composite transition. The graph G is constructed as follows. First, the algorithm computes closure(UA0), which is the largest state that is reachable from UA0 by performing all invisible tran- sitions enabled from UA0 (function closure in Line 3). The algorithm then computes a set of all states reachable from closure(UA0) (Lines 5–21), and returns true iff there exists a state s in G such that goal ⊆{r | (ut, r) ∈s} (Lines 4, 11, and 18). In [22], they have also identified a condition called the hierarchical role assignment (HRA), under which analysis of ARBAC without separate administration can be reduced to analysis of ARBAC with separate administration. An ARBAC policy satisfies HRA if, for all can assign(ra, P ∧¬N, r) where r is an administrative role, ra ⪰r. Example 1 Consider the following ARBAC policy ψ and the reachability analysis problem for this policy with the initial RBAC policy UA0 = {(u1, r1), (u1, r3), (u2, r2), (u2, r8), (u3, r2), (u3, r8), (ut, r6)}, the target user ut, and the goal {r5}. 1. can assign(r1, {r2} ∧¬∅, r3) 2. can assign(r6, {r4, r3} ∧¬∅, r5) 3. can assign(r1, {r6} ∧¬{r3}, r4) 4. can assign(r2, {r8, r1} ∧¬∅, r6) 5. can assign(r2, {r6} ∧¬∅, r7) 6. can revoke(r1, r2) 7. can revoke(r1, r3) 8. 2 Preliminaries: User-Role Reachability Analysis of ARBAC can revoke(r1, r4) Example 1 Consider the following ARBAC policy ψ and the reachability analysis problem for this policy with the initial RBAC policy UA0 = {(u1, r1), (u1, r3), (u2, r2), (u2, r8), (u3, r2), (u3, r8), (ut, r6)}, the target user ut, and the goal {r5}. 3. can assign(r1, {r6} ∧¬{r3}, r4) 4. can assign(r2, {r8, r1} ∧¬∅, r6) 5. can assign(r2, {r6} ∧¬∅, r7) 6. can revoke(r1, r2) 7. can revoke(r1, r3) 8. can revoke(r1, r4) This policy does not satisfy the separate administration restriction, because role r6 is both an administrative role in rule 2 and a target role in rule 4. This policy does not satisfy the separate administration restriction, because role r6 is both an administrative role in rule 2 and a target role in rule 4. First, the algorithm performs slicing to compute a set Rel+ of positive relevant roles and a set Rel−of negative relevant roles as follows. Initially, Rel+ contains all roles in the goal, i.e. r5. Since the target role of rule 2 is r5, the algorithm adds positive precon- ditions and administrative role of rule 2, i.e. r4, r3, and r6, to Rel+. The algorithm then processes rules 1 and 3, whose target roles are r4 and r3, respectively, adds their posi- tive preconditions and administrative roles, i.e. r2, r6, and r1, to Rel+, and adds their negative preconditions, i.e. r3, to Rel−. Repeat this process until all roles in Rel+ are processed, which results in Rel+ = {r1, r2, r3, r4, r5, r6, r8} and Rel−= {r3}. The set of mixed roles is Rel+ ∩Rel−= {r3}; other roles are both positive and non-negative. RelRule contains rules 1, 2, 3, 4, and 7. Next, the algorithm computes the initial state closure(UA0). Since rule 3 is enabled from UA0 and r4 is a non-negative role, (ut, r4) is added to UA0 through an invisible transition. 2 Preliminaries: User-Role Reachability Analysis of ARBAC The algorithm then computes all states reachable from closure(UA0) using ur(r1,u1,r3) ua(r1,u2,r3) (u1,r1),(u1,r3), (u2,r2),(u2,r8), (u3,r2),(u3,r8), ( ) ( ) (u1,r1), (u2,r2),(u2,r8), (u3,r2),(u3,r8), ( ) ( ) (u1,r1),(u2,r2), (u2,r8),(u2,r3), (u3,r2),(u3,r8), ( ) ( ) ur(r1,u2,r3) (ut,r6), (ut,r4) (ut,r6), (ut,r4) (ut,r6), (ut,r4) ua(r1,u3,r3) (u1,r1),(u2,r2), (u2 r8) (u3 r3) ur(r1,u3,r3) ua(r1,u2,r3) (u1,r1),(u1,r3), (u2,r2)(u2,r8), ua(r1,u3,r3) ur(r1,u2,r3) (u1,r1),(u1,r3), ur(r1,u3,r3) ur(r1,u3,r3) ua(r1,u3,r3) ur(r1,u3,r3) ua(r u r ) (u2,r8),(u3,r3), (u3,r2),(u3,r8), (ut,r6), (ut,r4) ( 2, 2)( 2, 8), (u2,r3),(u3,r2), (u3,r8),(ut,r6), (ut,r4) 1 1 1 3 (u2,r2)(u2,r8), (u3,r3),(u3,r2), (u3,r8),(ut,r6), (ut,r4) (u1,r1),(u1,r3), (u2,r2),(u2,r8), (u2,r3), (u3,r3) (u3,r2),(u3,r8), (u r ) (u r ) ur(r1,u2,r3) ua(r1,u3,r3) ur(r1,u3,r3) (u1,r1),(u2,r2), (u2,r8),(u2,r3), (u3,r3),(u3,r2), (u3,r8),(ut,r6), (ut,r4) ua(r1,u2,r3) ur(r1,u2,r3) (ut,r6), (ut,r4) Fig. 1. Graph constructed in Example 1 using the algorithm in [22]. ur(r1,u3,r3) Fig. 1. Graph constructed in Example 1 using the algorithm in [22]. rules in RelRule. The resulting graph is given in Figure 1. Because the graph does not contain (ut, r5), the goal is not reachable. 2 rules in RelRule. The resulting graph is given in Figure 1. Because the graph does not contain (ut, r5), the goal is not reachable. 2 3 Reduction Techniques The analysis algorithm described in Section 2, although simple, does not scale well for policies containing a large number of users. Let I = ⟨UA0, ut, ψ, goal⟩be a user-role reachability analysis problem instance. In this section, we present a number of tech- niques for reducing the number of users and ARBAC rules considered during analysis. 12: end for 13: RelRev = {can revoke(ra, r) | r ∈R−}; Rule = Rule ∪RelRev; 13: RelRev = {can revoke(ra, r) | r ∈R−}; Rule = Rule ∪RelRev; 15: if ra ̸∈Processed ∧(ra is negative ∨ra is a non-negative role not assigned to any user in UA0) then 16: (R4, R5, R6) = slicing(ψ, {ra}); 17: R+ = R+ ∪R4; R−= R−∪R5; Rule = Rule ∪R6 18: end if 19: end for 20: return (R+, R−, Rule); 15: if ra ̸∈Processed ∧(ra is negative ∨ra is a non-negative role not assigned to any user in UA0) then 17: R+ = R+ ∪R4; R−= R−∪R5; Rule = Rule ∪R6 the target user. In addition, since a negative role may become non-negative after slicing, to further reduce the number of relevant rules computed, we perform slicing multiple times until the set of negative roles remains unchanged. For non-target users, it is sufficient to apply only rules that assign such users to administrative roles, which have permission to assign the target user ut to the goal. The pseudocode is given in Algorithm 2. The reduction is given in Lines 6–10 and 15–16 of Algorithm 2. For every can assign(ra, P ∧¬N, r) where r ∈goal, we check if ra is a nonnegative or irrevocable role assigned to a user in UA0. If so, we do not slice ra; otherwise, we apply function slicing defined in Algorithm 1 to slice ra (Lines 6–8). This is different from Algorithm 1, in which ra is always sliced. Next, we compute a set S of all nonnegative or irrevocable roles in P that are assigned to the target user in UA0, and for every rule whose target role is in P \ S, we recursively call function optslicing to slice the administrative roles of such rules (Lines 9–10). Note that we do not slice roles in P for non-target users, while Algorithm 1 does. Finally, for every can revoke(ra, r), if ra is a nonnegative or irrevocable role assigned to some user in UA0, we do not slice ra (Lines 15–16). Example: Consider the ARBAC policy and the query in Example 1. First, we com- pute a set of relevant roles and rules for the target user ut using our optimized slicing mechanism. Since r6 is a non-negative role assigned to ut in UA0, we do not slice r6. 3.1 Optimized Slicing In this section, we present an approach to reduce the number of roles processed during slicing, and hence reduce the number of relevant rules computed. We say that a role is irrevocable if there does not exist a can revoke rule that revokes the role. For the target user ut, we apply function slicing defined in Algorithm 1 to perform slicing, except that Line 27 in the algorithm is replaced with the following: S = {r | r ∈(P ∪{ra}) ∧(r is nonnegative or irrevocable) ∧(ut, r) ∈UA0}; (R1, R2, R3) = slicing(UA0, ψ, (({ra} ∪P) \ S) \ Processed); Similarly, Line 32 of Algorithm 1 is replaced with the following: Similarly, Line 32 of Algorithm 1 is replaced with the following: S = {ra | (ra is nonnegative or irrevocable) ∧(ut, ra) ∈UA0}; (R4, R5, R6) = slicing(UA0, ψ, ({ra} \ S)); R+ = R+ ∪R4; Basically, prior to slicing, we collect a set of nonnegative and irrevocable roles in the ARBAC policy. During slicing, we do not slice nonnegative or irrevocable roles assigned to the target user in the initial policy UA0. This is safe because such roles will not be revoked during the analysis and hence we do not need to reassign such roles to Algorithm 2 An Optimized Slicing Algorithm for Non-Target Users. Algorithm 2 An Optimized Slicing Algorithm for Non-Target Users. 2: procedure optslicing(UA0, ψ, goal) 3: if (goal == ∅) then return (∅, ∅, ∅); end if 4: Processed = Processed ∪goal; R+ = goal; R−= ∅; Rule = ∅; 4: Processed = Processed ∪goal; R+ = goal; R−= ∅; Rule = ∅; 5: for all can assign(ra, P ∧¬N, r) where (ut, r) ∈goal do 5: for all can assign(ra, P ∧¬N, r) where (ut, r) ∈goal do 6: if ((u, ra) ∈UA0 for some user u and (ra is non-negative or irrevocable)) then 7: R1 = R2 = R3 = ∅; 6: if ((u, ra) ∈UA0 for some user u and (ra is non-negative or irrevocable)) then 7: R1 = R2 = R3 = ∅; 8: else (R1, R2, R3) = slicing(UA0, ψ, {ra} \ Processed); end if 9 S { | P ( i i i bl ) ( ) UA } ( ) ( { } \ ) 9: S = {r | r ∈P ∧(r is non-negative or irrevocable) ∧(ut, r) ∈UA0}; { | ( ( ) 10: (R′ 1, R′ 2, R′ 3) = optslicing(UA0, ψ, (P \ S) \ Processed); 10: (R′ 1, R′ 2, R′ 3) = optslicing(UA0, ψ, (P \ S) \ Processed); ( ) ( ( \ ) \ ) 11: R+ = R+ ∪S ∪R1 ∪R′ 1; R−= R−∪N ∪R2 ∪R′ 2; Rule = Rule ∪R3 ∪R′ 3; 12: end for ( ) ( ( \ ) \ ) 11: R+ = R+ ∪S ∪R1 ∪R′ 1; R−= R−∪N ∪R2 ∪R′ 2; Rule = Rule ∪R3 ∪R′ 3; 3.2 User Equivalent Set Reduction In this section, we show that from each state it is sufficient to perform visible transitions for the target user and non-target users assigned distinct sets of roles. Our technique is based on a notion of user equivalent set defined below. Definition 1 The user equivalent set w.r.t a state s is defined as ue(s) = {(Uset1, Rset1), . . . , (Usetn, Rsetn)} where Rset1 ̸= . . . ̸= Rsetn, Uset1 ∪. . . ∪Usetn = {u|(u, r) ∈s}, and for every u ∈Useti, Rseti = {r|(u, r) ∈s}. The user equivalent set w.r.t a state s is basically an alternative representation of s, in which all users assigned the same set of roles are grouped together. Let Gue be the transition graph constructed using the user equivalent set representation. There is a transition ue(s) α→ue(s′) in Gue if and only if there is a transition s α→s′ in G. The goal is reachable in Gue if and only if there exists a state sg ∈Gue and (Uset, Rset) ∈ sg such that ut ∈Uset, and goal ⊆Rset. g Our user equivalent set reduction works as follows. For every state s and every (Uset, Rset) ∈s, we compute only transitions for the target user and transitions for one randomly selected non-target user in Uset, if Uset contains such users. This is different from Algorithm 1, which computes transitions for all users in Uset. Intuitively, the user equivalent set reduction is correct because transitions performed on all users in Uset are the same, and transitions performed on one user in Uset do not disable transitions performed on other users in Uset. We use Gredue to represent the transition graph constructed with the user equivalent set reduction. The correctness of the reduction is formalized in Theorem 1. Given two states s1 and s2, we say that s1 ≡s2 if there exists a substitution δ = {u1/u′ 1, . . . , un/u′ n}, where u1 ̸= . . . ̸= un ̸= ut and u′ 1 ̸= . . . ̸= u′ n ̸= ut, such that s1δ = s2. For example, {({u1, ut}, {r1, r2}), ({u2}, {r2})} ≡{({u2, ut}, {r1, r2}), ({u1}, {r2})} holds because there exists a substitution δ = {u1/u2, u2/u1} such that {({u1, ut}, {r1, r2}), ({u2}, {r2})}δ = {({u2, ut}, {r1, r2}), ({u1}, {r2})}. 12: end for Therefore, for the target user, Rel+ = {r1, r2, r3, r4, r5, r6}, Rel−= {r3}, and RelRule = {1, 2, 3, 7}. Next, we compute a set of relevant roles and rules for non- target users using our optimized slicing mechanism. Only administrative roles that have permissions to assign the target user to the goal, i.e., r6 and r1, need to be sliced. Since r6 and r1 are non-negative roles assigned to ut and u1 in UA0, respectively, we do not slice these two roles. As a result, for non-target users, Rel+ = {r1, r6}, Rel−= {r3}, and RelRule = ∅. This means that there is no need to assign roles to non-target users. The transition graph constructed with the optimized slicing con- tains only one state {(u1, r1), (u1, r3), (u2, r2), (u2, r8), (u3, r2), (u3, r8), (ut, r6), (ut, r4)}. 3.2 User Equivalent Set Reduction Theorem 1 Let I = ⟨UA0, ut, ψ, goal⟩be a user-role reachability analysis instance, and Gredue and Gue be transition graphs constructed for I with and without using the user equivalent set reduction. The goal is reachable in Gue iff the goal is reachable in Gredue. Example: Consider the user-role reachability analysis instance in Example 1. Since non-target users u2 and u3 are assigned the same set of roles in the initial state, we ur(r1,u1,r3) ua(r1,u2,r3) ({u1},{r1,r3}), ({u2, u3},{r2,r8}), ({u1},{r1}), ({u u } {r r }) ({u1},{r1}), ({u3} {r2 r8}) ({u2, u3},{r2,r8}), ({ut},{r6,r4}) ur(r1,u2,r3) ( ) ur(r u r ) ( ) ( ) ({u2,u3},{r2,r8}), ({ut},{r6,r4}} ({u3},{r2,r8}), ({u2},{r2,r3,r8}), ({ut},{r6,r4}) ua(r1,u2,r3) ur(r1,u2,r3) ua(r1,u3,r3) ua(r1,u3,r3) ur(r1,u3,r3) {({u1},{r1,r3}), ({ } { }) ({u1},{r1}), {({u1},{r1,r3}), ur(r1,u3,r3) ({u3},{r2,r8}), ({u2},{r2,r3,r8}), ({ut},{r6,r4}) ({u2,u3},{r2,r3,r8}), ({ut},{r6,r4}) {({u1},{r1,r3}), ({u2,u3},{r2,r3,r8}), ({ut},{r6,r4}) Fig. 2. The transition graph constructed with the user equivalent set reduction. {({u1},{r1,r3}), {({u1},{r1,r3}), ({u2,u3},{r2,r3,r8}), ({ut},{r6,r4}) Fig. 2. The transition graph constructed with the user equivalent set reduction. need to perform only transitions for u2 or u3, but not both, from the initial state. This is different from Algorithm 1, which performs transitions for both u2 and u3 from the initial state. The graph constructed with the user equivalent set reduction is given in Figure 2, which contains 6 states and 9 transitions, i.e., 25% reduction on states and 47% on transitions. Optimization: We can reduce the size of the state by replacing Uset in (Uset, Rset) with a pair (counter, target), where counter records the number of non-target users in Uset, and target is either 1 (ut ∈Uset) or 0 (ut ̸∈Uset). 3.3 Delayed Revocation In this section, we propose to reduce the size of the transition graph by delaying tran- sitions that can neither enable new transitions in s nor be disabled by any transitions. ur(r u r) Formally, a transition s ur(ra,u,r) → s′ is not performed from s (i.e. is delayed) if Formally, a transition s ur(ra,u,r) → s′ is not performed from s (i.e. is delayed) if 1. trans(s) = trans(s′) ∪{ur(ra, u, r)} where trans(s) and trans(s′) are sets of all visible transitions enabled from s and s′, respectively, 2. s \ s′ = {(u, r)}, 2. s \ s′ = {(u, r)}, 3. ra is non-negative or irrevocable. Rules 1 and 2 specify that s ur(ra,u,r) → s′ does not enable new visible and invisible transitions, respectively. Rule 3 specifies that s ur(ra,u,r) → s′ cannot be disabled by other transitions. Rules 1 and 2 specify that s ur(ra,u,r) → s Given a state s, we compute transitions that can be delayed in s as follows. First, we perform all ua transitions that assign users in s to roles. Next, for every ur transition that is enabled in s, we check if the transition enables any transition. If so, we perform the transition from s. Otherwise, we add the transition to a set Delayed. Since performing ur transitions may enable new ua and ur transitions, after all can revoke rules are processed, we compute new transitions and check if any transitions in Delayed enable other transitions. If so, such transitions are performed from s and are removed from Delayed. Repeat the above process until no new transitions are computed. The correctness of the delayed revocation reduction is formalized in Theorem 2. Theorem 2 Let I = ⟨UA0, ut, ψ, goal⟩be a user-role reachability analysis instance, s0 = closure(UA0), and Gdr and G be transition graphs constructed for I with and without the delayed revocation reduction. The goal is reachable in G iff the goal is reachable in Gdr. Theorem 2 Let I = ⟨UA0, ut, ψ, goal⟩be a user-role reachability analysis instance, s0 = closure(UA0), and Gdr and G be transition graphs constructed for I with and without the delayed revocation reduction. The goal is reachable in G iff the goal is reachable in Gdr. Example: Consider the user-role reachability analysis instance in Example 1. 3.4 Hierarchical Rule Reduction Hierarchical rule reduction avoids considering rules whose administrative precondi- tions are junior to non-negative or irrevocable administrative roles in UA0. This is safe because senior roles inherit all administrative permissions of their junior roles, and non-negative/irrevocable roles are never revoked during analysis. This reduction does not reduce the size of the transition graph, but may reduce the analysis time since fewer rules are applied during analysis. Consider the user-role reachability analysis problem instance in Example 1 and the role hierarchy r1 ⪰r2. The following three rules are added after the pol- icy is transformed into the non-hierarchical one: can assign(r1, {r8, r1} ∧¬∅, r6), can assign(r1, {r6} ∧¬∅, r7), and can assign(r1, {r1} ∧¬∅, r3). Since r1 is a non- negative role, r1 will never be revoked during analysis. As a result, rules 4 and 5 in Example 1 are not useful for reaching the goal (since administrative roles of these two rules are r2, which is junior to r1), and hence will not be applied during analysis. 3.3 Delayed Revocation Since transition ur(r1, u1, r3) does not enable new transitions from the initial state and r1 is non-negative, with delayed revocation reduction, this transition is not performed from the initial state. The transition graph constructed contains 4 states and 8 transitions, i.e., 50% reduction on the number of states and 47% reduction on the number of transitions. 4 Parallel Analysis Algorithm || start(tn); 7: procedure start(ti) 8: while !done do 9: if( W == ∅and all threads are idle) then done = 1; end if 10: while (W ̸= ∅) 11: lock(W); remove a state s from W; unlock(W); 12: for all transitions s ua(ra,u,r) → s′ 13: if goal ⊆{r | (ut, r) ∈s′} then return true; end if 14: lock(Reached(h(s′)); 15: if (Reached(h(s′)) does not exist) 16: Reached(h(s′)) = {s′}; unlock(Reached(h(s′))); 17: lock(W); W = W ∪{s′}; unlock(W); 18: else if (s′ ̸∈Reached(h(s′))) 19: Reached(h(s′)) = Reached(h(s′)) ∪{s′}; unlock(Reached(h(s′))); 20: lock(W); W = W ∪{s′}; unlock(W); 21: else unlock(Reached(h(s′))); end if 22: end if end for end while 23: end while 24: return false; Hash value :h1 S11 S12 S1k …… Reached(h1) Hash value: h2 S21 S22 S2r …… Reached(h2) …… Hash value: h m Sm1 Sm2 Smt …… Reached(h ) Hash value: h m Sm1 Sm2 Smt …… Reached(hm) Fig. 3. Implementation of the set of reachable states Reached. Hash value :h1 S11 S12 S1k …… Reached(h1) Hash value: h2 S21 S22 S2r …… Reached(h2) …… Hash value: h m Sm1 Sm2 Smt …… Reached(h ) Hash value: h m Sm1 Sm2 Smt …… Reached(hm) Fig. 3. Implementation of the set of reachable states Reached. Fig. 3. Implementation of the set of reachable states Reached. Fig. 3. Implementation of the set of reachable states Reached. Algorithm 3 User-Role Reachability Analysis Algorithm in [22]. 1: Reached = W = Rel+ = Rel−= ∅; RelRule = ∅; done = 0; 2: procedure mcanalysis(UA0, ut, ψ, goal) 3: (Rel+, Rel−, RelRule) = slicing(UA0, ψ, goal); init = closure(UA0); 4: if goal ⊆{r | (ut, r) ∈init} then return true; end if 5: W = Reached(h(init)) = {init}; 6: start(t1) || . . . 4 Parallel Analysis Algorithm Multi-core processors are becoming pervasive. In order for software applications to benefit from the continued exponential throughput advances in new computer systems, it is important to parallelize the applications. In this section, we extend Algorithm 1 to perform analysis in parallel. The pseudocode of our parallel algorithm is given in Algorithm 3. First, we perform slicing to eliminate irrelevant roles, as we do in Algorithm 1. We then compute the initial state init of the transition graph and add init to a workset W (Line 5). Next, we create n threads t0, . . ., tn (Line 6; || represents the concur- rent execution of threads). Finally, each thread ti removes one state from W, com- putes transitions enabled from the state using Lines 7 –10 and 14–17 of Algorithm 1, and adds the target states to W and the set of reachable state Reached if the tar- get states are not already in Reached (Lines 11–20). Since multiple threads may ac- cess Reached at the same time, Reached needs to be protected by locks in order to ensure the correct execution of the program. Obviously, locking and unlocking Reached every time a thread accesses Reached imposes high overhead. To reduce the time spent on waiting for locks to access Reached, we implemented Reached Hash value :h1 S11 S12 S1k …… Reached(h1) Hash value: h2 S21 S22 S2r …… Reached(h2) …… Hash value: h m Sm1 Sm2 Smt …… Reached(h ) Hash value: h m Sm1 Sm2 Smt …… Reached(hm) Fig. 3. Implementation of the set of reachable states Reached. Algorithm 3 User-Role Reachability Analysis Algorithm in [22]. 1: Reached = W = Rel+ = Rel−= ∅; RelRule = ∅; done = 0; 2: procedure mcanalysis(UA0, ut, ψ, goal) 3: (Rel+, Rel−, RelRule) = slicing(UA0, ψ, goal); init = closure(UA0); 4: if goal ⊆{r | (ut, r) ∈init} then return true; end if 5: W = Reached(h(init)) = {init}; 6: start(t1) || . . . 4 Parallel Analysis Algorithm || start(tn); 7: procedure start(ti) 8: while !done do 9: if( W == ∅and all threads are idle) then done = 1; end if 10: while (W ̸= ∅) 11: lock(W); remove a state s from W; unlock(W); 12: for all transitions s ua(ra,u,r) → s′ 13: if goal ⊆{r | (ut, r) ∈s′} then return true; end if 14: lock(Reached(h(s′)); 15: if (Reached(h(s′)) does not exist) 16: Reached(h(s′)) = {s′}; unlock(Reached(h(s′))); 17: lock(W); W = W ∪{s′}; unlock(W); 18: else if (s′ ̸∈Reached(h(s′))) 19: Reached(h(s′)) = Reached(h(s′)) ∪{s′}; unlock(Reached(h(s′))); 20: lock(W); W = W ∪{s′}; unlock(W); 21: else unlock(Reached(h(s′))); end if 22: end if end for end while 23: end while 24: return false; as a hashtable shown in Figure 3. The hashtable is partitioned into multiple regions Reached(h1), . . . , Reached(hm); Reached(hi) stores a set of states whose hash val- ues are hi. Once a thread computes a transition s α→s′, it computes the hash value h(s′) of s′, locks Reached(h(s′)), adds s′ to Reached(h(s′)) if s′ is not already in Reached(h(s′)), and unlocks Reached(h(s′)). The above approach enables two threads to access two different regions in Reached simultaneously. Our experimental results show that locking Reached(h(s)) instead of Reached significantly improves the performance. This is because threads access Reached very frequently and checking if a state is in Reached is relatively expensive. The algorithm terminates if the goal is reached, or if W is empty and all threads are not performing any computation. It is also possible to reduce the time spent on waiting for locks to access the workset W by having each thread to have its own workset. Below, we present three approaches to minimizing (or completely removing) the number of operations performed on lock- ing/unlocking W. – NoLock: In this approach, each thread is not allowed to access other threads’ work- sets. Every time a thread computes a transition, it stores the target state in its own workset, if the target state is not already in Reached. This approach eliminates the requirement for locking, but may result in idle threads (due to empty workset). – FullLock: In this approach, a thread is allowed to access other threads’ workset to retrieve a state to process, if the thread’s workset is empty. 4 Parallel Analysis Algorithm This approach en- sures that all threads will be approximately equally busy, but it requires to lock the workset every time the workset is accessed. – PartialLock: In this approach, whenever a thread ti computes a new transition, it checks if thread t(i−1) mod n is idle. If so, it locks the workset of t(i−1) mod n, adds the target state to the workset, unlocks the workset, and starts t(i−1) mod n. The advantage of this approach is that locking is only needed when ti adds a state to t(i−1) mod n’s workset. This approach has limitation that each thread ti has to frequently check if t(i−1)modn is sleeping. Discussion: Two threads can safely access the same region in Reached simultaneously if neither thread adds a state to or removes a state from the same region. Thus, in some cases, it may be possible to improve the performance by replacing mutual exclusion locks on Reached with reader-writer locks. Unlike a mutual exclusion lock, which prevents all concurrent accesses to a critical region, a reader-writer lock allows multiple threads performing read operations to enter critical region. Our experiments, however, show that such optimization does not yield performance improvement (in fact, it often causes performance degradation). This is because multiple threads rarely access the same region in Reached simultaneously during analysis, and reader-writer locks, due to their complexity, impose greater overheads than mutual exclusion locks. 5 Performance Results This section evaluates the effectiveness of our reduction techniques and our parallel algorithms using the university ARBAC policy developed in [22] and the university RBAC policy developed in [7]. All reported data were obtained on a 2.4GHz 2 Quad- Core AMD Opteron Processor with 16GB RAM running Ubuntu 3.2.0. The university RBAC and ARBAC policies contain 845 users, 32 roles, 329 can assign rules, and 78 can revoke rules, after being converted to the corresponding non-hierarchical policies. The policies include rules for assignment of users to vari- ous student and employee roles. Student roles include undergraduate student, graduate student, teaching assistant, research assistant, honors student, etc. Employee roles in- clude president, provost, dean, department chair, faculty, honor program director, etc. A sample can assign rule is: the honors program director can assign an undergraduate 50 non-target users NoReduct OptSlice DelayedRev UserEquivSet AllReduct State 111 45 54 15 4 Transition 620 264 278 61 9 Time 0.97 0.41 0.57 0.13 0.09 75 non-target users NoReduct OptSlice DelayedRev UserEquivSet AllReduct State 24909 245 6393 273 5 Transition 214165 2168 42718 3222 10 Time 34.30 6.18 10.99 0.15 0.09 100 non-target users NoReduct OptSlice DelayedRev UserEquivSet AllReduct State 12706 7552 7855 39 6 Transition 145115 99520 107323 225 11 Time 2363 2029.26 2166.81 0.81 0.1 Table 1. Performance of analysis algorithms without reduction, with a single reduction, and with all reductions. Table 1. Performance of analysis algorithms without reduction, with a single reduction, and with all reductions. Table 1. Performance of analysis algorithms without reduction, with a single reduction, and with all reductions. student to the honors student role. A sample user-role reachability problem instance is: can a user who is a member of the department chair role and a user who is a member of the undergraduate student role assign the latter user to the honor student role? The university ARBAC policy does not satisfy the separate administration restric- tion. In addition, the policy has hierarchical role assignment w.r.t all administrative roles except those for assigning users to roles honor student and graduate student. This means that if the goal contains these two roles, then we cannot directly apply the algo- rithm for analyzing ARBAC with separate administration to carry out analysis. In our experiments, we randomly select one target user ut, one role r, and n non-target users {u1, . . . , un}. 5 Performance Results We then apply analysis algorithms to check if users in {u1, . . . , un, ut} together can assign ut to both honor student role and role r. Effectiveness of Reduction Techniques Table 1 gives the the size of the transition graph and the execution time for three sets of experiments with different numbers of randomly chosen non-target users (50, 75 or 100). Each data point reported in the table is an average over 8 randomly generated queries. The five columns represent reduction techniques applied during the experiments: with no reduction (NoReduct), with opti- mized slicing (OptSlice), with user equivalent set (UserEquivSet), with delayed revoca- tion (DelayedRev), and with all reductions (AllReduct). Note that we do not include the hierarchical rule reduction in the table as it is not effective in our experiments. This is because all administrative roles in the university policy that have junior roles are mixed roles and remain mixed after applying all reductions. We observe that, while all reduction techniques improve the performance, their ef- fectiveness varies under different queries. UserEquivSet performs the best for all three sets of experiments and DelayedRev is the least effective. Integrating all reductions leads to a very effective solution. When the problem becomes difficult for the baseline algorithm to solve, AllReduct achieves an improvement of four orders of magnitude in execution time. In addition, when the number of non-target users is 150, NoReduct fails to complete 3 of the 8 queries within 12 hours, whereas the average analysis time of AllReduct is only 0.1 seconds. 50 non-target users 15 threads 30 threads NoReduct SharedWorkset NoLock FullLock PartialLock SharedWorkset NoLock FullLock PartialLock Time 0.97 0.33 0.40 0.32 0.52 0.32 0.43 0.34 0.45 75 non-target users 15 threads 30 threads NoReduct SharedWorkset NoLock FullLock PartialLock SharedWorkset NoLock FullLock PartialLock Time 34.30 6.58 6.60 5.85 6.74 5.82 7.04 5.80 6.54 100 non-target users 15 threads 30 threads NoReduct SharedWorkset NoLock FullLock PartialLock SharedWorkset NoLock FullLock PartialLock Time 2363 1059.73 517.09 436.87 513.46 776.34 537.68 407.53 531.83 Table 2. Performance of the parallel algorithm without reduction. Performance Results of Parallel Algorithms Table 2 gives the execution time of four parallel analysis algorithms without reductions – SharedWorkset (Algorithm 3), NoLock, PartialLock, and FullLock – with 15 and 30 threads. The results show that, on average, FullLock performs the best, followed by PartialLock, NoLock, and Shared- Workset. 5 Performance Results FullLock and SharedWorkset with 30 threads outperform those with 15 threads, because the threads often wait for locks to access the worksets in FullLock and SharedWorkset, and hence more CPU cores are utilized with 30 threads than 15 threads. NoLock and PartialLock with 15 threads outperform those with 30 threads, be- cause the threads do not or only occasionally wait for locks in NoLock and PartialLock, and hence the CPU cores are mostly utilized with 15 threads. 6 Related Work A number of researchers have considered analysis of fixed security policy [13, 15, 10, 11], analysis of a single change to a fixed policy, or analysis of differences between two fixed policies [15, 6]. However, none of them consider analysis of ARBAC. 7 Conclusion and Future Work This paper considers the user-role reachability analysis without the separate administra- tion restriction, which was shown to be PSPACE-complete in general. We present new analysis techniques with the goal of finding a practical solution to the problem. Our techniques focus on reducing the number of ARBAC rules and users considered during analysis and delaying unnecessary computations. We have also presented a number of parallel algorithms that speed up the analysis on multi-core systems. The experimental results on a university ARBAC policy show that our techniques significantly reduce the analysis time. In the future, we plan to develop symbolic analysis algorithms to implic- itly search the state space with a potential to further improve the performance of the user-role reachability analysis. Acknowledgement: This work was supported in part by NSF Grant CNS-0855204. We thank Kyoung-Don Kang for providing feedbacks on parallel algorithms and Dulcinea Chau for her contribution to the implementation of parallel algorithms. 6 Related Work A number of researchers have studied user-role reachability analysis of ARBAC. Schaad et al. [20] applied the Alloy analyzer [12] to check the separation of duty prop- erties for ARBAC97; they did not consider preconditions for any operations. Li et al. [16] presented algorithms and complexity results for various analysis problems for two restricted versions of ARBAC97, called AATU and AAR; they did not consider negative preconditions. Jayaraman et al. [14] presented an abstraction refinement mechanism for detecting errors in ARBAC policies. Alberti et. al [1] developed a symbolic backward algorithm for analyzing Administrative Attribute-based RBAC policies, in which the policy and the query are encoded into a Bernays-Shonfinkel-Ramsey first order logic formulas. Becker [3] proposed a language DYNPAL for specifying dynamic autho- rization policies, which is more expressive than ARBAC, and presented techniques for analyzing DYNPAL. Sasturkar et al. [19] showed that user-role reachability analysis of ARBAC is PSPACE-complete, and presented algorithms and complexity results for ARBAC analysis subject to a variety of restrictions. Stoller et al. [21] presented algo- rithms for analyzing parameterized ARBAC. Gofman et al. [9] presented algorithms for analyzing evolving ARBAC. Uzun et al. [23] developed algorithms for analyzing temporal role-based access control models. However, none of the above works consider analysis of ARBAC without separate administration. Several researchers have considered analysis of ARBAC without separate admin- istration. Stoller et al. [22] provided fixed-parameter tractable algorithms for ARBAC with and without the separate administrative restriction. Their algorithm for analyzing ARBAC without separate administration is exponential to the number of users in the policy, which is usually large in practice. Our work significantly improved the scala- bility of their algorithm by reducing the number of ARBAC rules and users considered during analysis. Ferrara et al [4] converted ARBAC policies to imperative programs and applied abstract-interpretation techniques to analyze the converted programs. However, if the goal is reachable, their approach cannot produce a trace which shows how the goal is reachable. Later, the same authors showed that if the goal is reachable in an AR- BAC policy, then there exists a run of S with at most |administrative roles| + 1 users in which the goal is reachable [5]. However, their algorithm and reduction techniques are different from ours. Their techniques can be combined with ours to further reduce the analysis time. In addition, none of the above works present parallel analysis algorithms. 3. M. Y. Becker. Specification and analysis of dynamic authorisation policies. In 22nd IEEE Computer Security Foundations Symposium (CSF), 2009. References 1. F. Alberti, A. Armando, and S. Ranise. Efficient symbolic automated analysis of admin- istrative attribute-based rbac-policies. In ACM Symposium on Information, Computer and Communications Security, pages 165–175, 2011. 2. A. N. S. I. (ANSI). Role-based access control. ANSI INCITS Standard 359-2004, Feb. 2004. 3. M. Y. Becker. Specification and analysis of dynamic authorisation policies. In 22nd IEEE Computer Security Foundations Symposium (CSF), 2009. 4. A. L. Ferrara, P. Madhusudan, and G. Parlato. Security analysis of role-based access control through program verification. In Computer Security Foundations Symposium, pages 113– 125, 2012. 5. A. L. Ferrara, P. Madhusudan, and G. Parlato. Policy analysis for self-administrated role- based access control. In to appear, International Conference on Tools and Algorithms for the Construction and Analysis of Systems, 2013. 6. K. Fisler, S. Krishnamurthi, L. A. Meyerovich, and M. C. Tschantz. Verification and change- impact analysis of access-control policies. In International Conference on Software Engi- neering (ICSE), pages 196–205, 2005. 7. M. Gofman, R. Luo, J. He, Y. Zhang, and P. Yang. Incremental information flow analysis of role based access control. In International Conference on Security and Management, pages 397–403, 2009. 8. M. Gofman, R. Luo, A. Solomon, Y. Zhang, P. Yang, and S. Stoller. Rbac-pat: A policy analysis tool for role based access control. In Tools and Algorithms for the Construction and Analysis of Systems, pages 46–49, 2009. 9. M. Gofman, R. Luo, and P. Yang. User-role reachability analysis of evolving administrative role based access control. In European Symposium on Research in Computer Security, 2010. 10. J. D. Guttman, A. L. Herzog, J. D. Ramsdell, and C. W. Skorupka. Verifying information flow goals in Security-Enhanced Linux. Journal of Computer Security, 13(1):115–134, 2005. 11. K. Irwin, T. Yu, and W. H. Winsborough. On the modeling and analysis of obligations. In ACM Conference on Computer and Communications Security, pages 134–143, 2006. 12. D. Jackson, I. Schechter, and I. Shlyakhter. Alcoa: the alloy constraint analyzer. pages 730–733, June 2000. 13. S. Jajodia, P. Samarati, and V. S. Subrahmanian. A logical language for expressing autho- rizations. In Symposium on Security and Privacy, pages 31–42, 1997. 14. K. Jayaraman, V. Ganesh, M. Tripunitara, M. Rinard, and S. Chapin. Automatic error find- ing for access control policies. In Proceedings of 18th ACM Conference on Computer and Communications Security (CCS), 2011. 15. S. Jha and T. Reps. Model-checking SPKI-SDSI. Journal of Computer Security, 12:317–353, 2004. 16. N. Li and M. References V. Tripunitara. Security analysis in role-based access control. ACM Transac- tions on Information and System Security, 9(4):391–420, Nov. 2006. 17. R. Sandhu, V. Bhamidipati, and Q. Munawer. The ARBAC97 model for role-based ad- ministration of roles. ACM Transactions on Information and Systems Security (TISSEC), 2(1):105–135, Feb. 1999. 18. A. Sasturkar, P. Yang, S. D. Stoller, and C. Ramakrishnan. Policy analysis for administrative role based access control. In IEEE Computer Security Foundations Workshop, 2006. 19. A. Sasturkar, P. Yang, S. D. Stoller, and C. Ramakrishnan. Policy analysis for administrative role based access control. Theoretical Computer Science, 412(44):6208–6234, 2011. 20. A. Schaad and J. D. Moffett. A lightweight approach to specification and analysis of role- based access control extensions. In ACM Symposium on Access Control Models and Tech- nologies, pages 13–22, 2002. 21. S. D. Stoller, P. Yang, M. I. Gofman, and C. Ramakrishnan. Symbolic reachability anal- ysis for parameterized administrative role-based access control. Journal of Computers & Security, pages 148–164, 2011. 22. S. D. Stoller, P. Yang, C. R. Ramakrishnan, and M. I. Gofman. Efficient policy analysis for administrative role based access control. In 14th ACM Conference on Computer and Communications Security (CCS), pages 445–455, 2007. 23. E. Uzun, V. Atluri, S. Sural, J. Vaidya, G. Parlato, A. L. Ferrara, and M. Parthasarathy. Analyzing temporal role based access control models. 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MR findings of primary ovarian granulosa cell tumor with focus on the differentiation with other ovarian sex cord-stromal tumors
Journal of ovarian research
2,018
cc-by
6,397
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: To describe magnetic resonance imaging (MRI) features of ovarian granulosa cell tumors (OGCTs) and compare with other sex cord-stromal tumors (OSCs) in ovary. Methods: MR findings of 18 patients with surgically confirmed ovarian granulosa cell tumor were retrospectively reviewed by two radiologists with consensus reading. All MR examinations were prospectively performed within one month. Clinical and imaging characteristics of OGCTs were evaluated and compared with OSCs (control group). Results: In 18 patients, 20 ovarian granulosa cell tumors were detected on MRI. Sixteen tumors appeared as solid or mostly solid mass (16/20), while 4 tumors as cystic mass. Pathological pelvic fluid was detected in 1 OGCT (1/18) and 11 OSCs (11/34) (p = 0.031).On T2 weighted imaging (T2WI), most of OGCTs displayed hyperintense signal and mixed signal (19/20); on T1 weighted imaging (T1WI), 11 OGCTs (11/20) displayed similar signal as on T2WI imaging. The lesion signal between OGCT and OSC differed significantly on both T1WI (p = 0.017) and T2WI (p = 0.002). Tumoral bleeding was detected in 6 OGCTs on MRI. On diffusion weighted imaging (DWI) images, OGCTs mostly appeared as high signal (16/20). Average apparent diffusion coefficient (ADC) value derived from DWI images in the OGCT group (0.84 ± 0.26× 10−3 mm2/s was less than the control group (1.22 ± 0.47 × 10−3 mm2/s) with statistical difference (p = 0.002). Conclusions: MRI could provide important information in OGCT diagnosis. ADC value might be useful in differentiating OGCT from OSC. Conclusions: MRI could provide important information in OGCT diagnosis. ADC value might be useful in differentiating OGCT from OSC. Keywords: Ovarian granulosa cell tumor, Sex-cord tumor, MRI, Diagnostic imaging with stages II–IV granulosa cell tumor [4–7]. Owing to the superb soft-tissue resolution and free radiation, magnetic resonance imaging (MRI) is widely used as a problem-solving modality in assessment of complex adnexal masses that are indeterminate on ultrasonog- raphy (US) or computed tomography (CT) [8]. Till now, most of reported OGCTs in the literatures are published as case report and no detailed MRI knowl- edges of OGCTs have been comprehensively described [6–13]. In this study, by evaluating OGCTs in our single institution, we aimed to: (1) thoroughly evalu- ate the MRI appearances of OGCTs in a large cohort of samples and record ADC values for each lesion; (2) compare these features with OSCs. MR findings of primary ovarian granulosa cell tumor with focus on the differentiation with other ovarian sex cord-stromal tumors He Zhang1†, Hongyu Zhang2†, Shouxin Gu1, Yanyu Zhang1, Xuefen Liu1 and Guofu Zhang1,3* Zhang et al. Journal of Ovarian Research (2018) 11:46 https://doi.org/10.1186/s13048-018-0416-x Zhang et al. Journal of Ovarian Research (2018) 11:46 https://doi.org/10.1186/s13048-018-0416-x Background Ovarian granulosa cell tumor (OGCT) is a rare sex cord-stromal tumor in ovary, accounting only 2–3% of all ovarian tumors [1]. Pathologically, OGCTs are classi- fied into two subtypes: adult and juvenile form, in which adult type occupying 95% of all OGCTs [2]. Despite OGCT have a favorable prognosis, an incidence of 25–30% metastases or recurrences make it as a low malignant potential ovarian tumor [3]. Chemotherapy is recommended as adjuvant treatment for patients * Correspondence: dr.zhanghe@yahoo.com †He Zhang and Hongyu Zhang contributed equally to this work. 1Department of Radiology, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, People’s Republic of China 3Institute of functional and molecular medical imaging, Fudan University, Shanghai 200040, People’s Republic of China Full list of author information is available at the end of the article * Correspondence: dr.zhanghe@yahoo.com †He Zhang and Hongyu Zhang contributed equally to this work. 1Department of Radiology, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, People’s Republic of China 3Institute of functional and molecular medical imaging, Fudan University, Shanghai 200040, People’s Republic of China Full list of author information is available at the end of the article Page 2 of 9 Page 2 of 9 Zhang et al. Journal of Ovarian Research (2018) 11:46 the lesion; and presence of capsule, pelvic-free fluid and lymph node were also noted. On T1WI, hypo-, iso-, and hyperintensity were similar for the pelvic fluid, pelvic wall muscle, and fat signal; on T2WI, hypo-, iso-, and hyperin- tensity were similar for the pelvic bone, pelvic wall muscle, and fat signal; on b = 800 mm−2/s DWI images, the low, intermediate, and high-signal intensity were similar for the pelvic bone, myometrium, and endometrium. After the intravenous injection of the contrast medium, the le- sion enhancement type was graded as follows: 1, minor enhancement (clearly less than the myometrium); 2, mild enhancement (less than the myometrium); 3, moderate enhancement (similar to the myometrium); or 4, avid enhancement (more than the myometrium). ADCs were measured manually on post-processing workstation (Leonardo, Siemens, Germany) by one reviewer (H.Z.). Two observers (S. X. G. and H.Z., with 6 and 10 years of experience in gynecological imaging, respectively), who were blinded to the histological results independently, analyzed MRI datasets of each participant. At the end of the study, two observers were also required to determine the tumor etiology (benign or malignant) according to previous established criteria [14–16]. Results The histological results revealed 20 OGCTs in 18 pa- tients (24–79 years of age; average age, 48.2 ± 15.1 years), including 17 adult types and 1 juvenile type (Fig. 1). Laparotomy was performed in 13 patients while others with laparoscopic surgery. Nine patients had regular or irregular menses, while menopause in nine patients. According to the international federation of obstetrics and gynecology (FIGO) staging system [17], twelve patients were classified as Ia, 4 as Ic, 1 as IIIa and 1 as IIIc. All primary tumors were solitary lesion detected on MRI, except for three primary lesions in one patient at initial evaluation (Fig. 2). Most of OGCTs at presenta- tion appeared as the large mass with the average diam- eter of 9.33 ± 5.43 mm. Among them, five patients were Image acquisition MRI was performed using a 1.5-T MR system (Magnetom Avanto, Siemens, Erlangen, Germany) with a phased-array coil. The routine MRI protocols used for assessment of pelvic masses included axial turbo spin-echo (TSE) T1WI, sagittal TSE T2WI, and axial/sagittal TSE fat-suppressed T2WI (FS T2WI). For axial images, the transverse plane was perpendicular to the long axis of uterine body; for sagittal images, the longitudinal plane was parallel to the main body of uterus. DWI using an echo-planar imaging two-dimensional (EP2D) se- quence performed in the axial plane with parallel acquisi- tion technique by using b value = 0, 100, and 800 s/mm2. Contrast-enhanced pelvic imaging was acquired at mul- tiple phases of contrast medium enhancement in both sa- gittal and axial planes. Statistical analyses Continuous variables were expressed as the means ± standard deviation (S.D.) and compared with the un- paired t test if normally distributed or the Mann–Whit- ney test if not normally distributed. A nonparametric test (Mann–Whitney) was used to test other nonpara- metric variables within each group. The area under the receiver operating characteristic (ROC) curve (AUC) was calculated for ADCs to discriminate OGCTs from OSCs. SPSS (version 13.0, SPSS, Chicago, USA) was used to perform statistical analyses. P values ≤0.05 were considered statistically significant. Background For interobserver discrepancies in the evaluation of uterine lesions, consen- sus was achieved. Study subjects Between December 2009 and December 2015, 1217 con- secutive patients with clinically suspected adnexal disease prospectively underwent 1.5 T MRI examinations before pelvic or laparoscopic surgery at our institution. The time interval between the MRI evaluation and surgery was less than one month (2–27 days; mean, 5 ± 12 days). Among them, 18 patients with histologically proven OGCT (24–79 years of age; average age, 45.9 ± 15.3 years) were included in this study when we retrospectively re- trieved the database on the Picture Archiving and Com- munication System (PACS). Two recurrent OGCTs were excluded for further analysis because the primary imaging data was evaluated in another hospital. Thirty four patients with OSCs, including histologically proven scler- osing stroma tumors (SST, n = 4), fibrothecomas (n = 21), and fibromas (n = 9), were included as the comparative group. Patients with any previous pelvic surgery or radiation history were arbitrarily excluded because the inherent structure of the uterus may has been altered. Details of the samples studied are summarized in Table 1. aindicates 20 lesions in 18 patients Image analysis The location, size (the largest dimension in two orthog- onal planes), margin (regular or irregular); visibility of hemorrhagic component (high signal on T1WI) within Table 1 Summaries of histological results in 54 ovarian sex-cord lesions detected on MRI in 52 patients Pathology diagnosis Numbers Granulosa cell tumor 20a Fibrothecoma 21 Sclerosing stroma tumor 4 Fibroma 9 aindicates 20 lesions in 18 patients Table 1 Summaries of histological results in 54 ovarian sex-cord lesions detected on MRI in 52 patients Pathology diagnosis Numbers Granulosa cell tumor 20a Fibrothecoma 21 Sclerosing stroma tumor 4 Fibroma 9 aindicates 20 lesions in 18 patients Table 1 Summaries of histological results in 54 ovarian sex-cord lesions detected on MRI in 52 patients Page 3 of 9 Zhang et al. Journal of Ovarian Research (2018) 11:46 Fig. 1 A 32-year-old woman histologically proved OGCT in juvenile type (IIIc). On sagittal FS-T2WI image (a), the giant mass with irregular margin (arrow) occupy the majority of pelvis with the uterus being pushed forwardly (arrow head). (b) On contrast-enhanced images, the mass show homogeneously moderate enhancement; the necrotic area do not show enhancement (star) Fig. 1 A 32-year-old woman histologically proved OGCT in juvenile type (IIIc). On sagittal FS-T2WI image (a), the giant mass with irregular margin (arrow) occupy the majority of pelvis with the uterus being pushed forwardly (arrow head). (b) On contrast-enhanced images, the mass show homogeneously moderate enhancement; the necrotic area do not show enhancement (star) Accordingly, OSCs mainly displayed as isointense (12/34) and hyperintense signal intensity (10/34) on T2WI images. also accompanied by other ovarian etiologies, including endometrial polyps (n = 2), uterine fibroids (n = 2), fol- licular cyst (n = 1), Brenner tumor (n = 1) and fibroid and mucinous cystadenoma (n = 1). Vaginal discharge was recorded in one OG patient. The details of base- line characteristics for all studied samples are summa- rized in Table 2. Neoplastic bleeding can be seen in six OGCTs, appear- ing as the patchy high signal intensity on T1WI images in the tumor body (Fig. 3), which was not identified in OSC group (p = 0.000). Seventeen OGCTs were round or oval masses with regular margin (17/20) and intact capsule (16/20), while 33 (33/34) and 34(34/34) observed in OSCs, respectively (p = 0.015). Regarding the tumor component, OGCTs mostly appeared as the solid or mostly solid component (16/20, Fig. Image analysis 4), while OSCs al- ways showed purely solid component (28/34) (p = 0.000). On the post-contrast images, fourteen lesions (14/20) in OGCT group displayed mild enhancement and six MRI characteristics In this studied samples, OGCTs showed varying signal intensities on both T1WI and T2WI images. On T1WI, OGCTs showed various signal from low to mixed signal, which was different to OSCs mostly appearing as hypoin- tense and isointense mass (p = 0.017). On T2WI, OGCTs mainly displayed as high and mixed signal (19/20). Fig. 2 A 61-year-old woman with primary adult type OGCT (Ia). There are oval, solid masses on right adnexal region (a, b) and right iliac fossa (c, d). Two lesions appears as similar signals with intermediate signal on both T1WI (a, c) and FS-T2WI (b, d) with intact capsule. On DWI image (e), the mass at the right iliac fossa shows relatively high signal (arrow) and low signal on the ADC map (f). The gross specimen reveals the yellow, solid mass with smooth capsule and a thin septa (g). Photomicrograph, hematoxylin and eosin stained section (× 40) shows the oval cells arrange closely with pleomorphic nuclei, prominent nucleoli and scanty cytoplasm. Note, the small vessels embed among the intercellular space (arrow head) Fig. 2 A 61-year-old woman with primary adult type OGCT (Ia). There are oval, solid masses on right adnexal region (a, b) and right iliac fossa (c, d). Two lesions appears as similar signals with intermediate signal on both T1WI (a, c) and FS-T2WI (b, d) with intact capsule. On DWI image (e), the mass at the right iliac fossa shows relatively high signal (arrow) and low signal on the ADC map (f). The gross specimen reveals the yellow, solid mass with smooth capsule and a thin septa (g). Photomicrograph, hematoxylin and eosin stained section (× 40) shows the oval cells arrange closely with pleomorphic nuclei, prominent nucleoli and scanty cytoplasm. Note, the small vessels embed among the intercellular space (arrow head) Zhang et al. Journal of Ovarian Research (2018) 11:46 Page 4 of 9 showed moderate enhancement. In OSC group, most of fibromas showed minor enhancement and 4 SSTs ap- peared inhomogeneously avid enhancement (Fig. 5). On DWI images, 80 % of OGCTs (16/20, 80.0%) showed high signal intensities in comparison with 47% (16/34, 47.1%) in OCS group. The average ADC value (817 ± 144 × 10−3 s/m2) in OGCT group was obviously less than OSC group (1223 ± 473 × 10−3 s/m2, p = 0.002) and fibrothecoma (1209 ± 437 × 10−3 s/m2, p = 0.001, Fig. 6). MRI characteristics When use the ADC cutoff value as 619 × 10−3 s/m2, MRI could yield a sensitivity of 79.4% and a specificity of 60.0% for diagnosis of OGCT, respectively; the AUC is 0.784(95% CI:0.658–0.910) (Fig. 7). Enlarged lymph nodes were not observed in all OGCTs at MRI images. Pathological fluid was only noted in one OGCT, obviously less than 11 cases in OSCs group. At multi- variate analysis, neoplastic hemorrhagic contents (OR: 3.429), component (OR: 2.50) and no pathological fluid (OR: 8.130) are more indicative of OGCT diagnosis (Table 2). On MRI, two readers accurately determined the malignant condition in 17 cases. If combining ADC values, then they could yield a sensitivity of 95.0% and a specificity of 94.1% for OGCT diagnosis. Three lesions were misdiagnosed as uterine fibroids and two lesion as fibrothecoma before invasive procedures. The overall diagnostic performance of MRI for diagnosing OGCT is listed as Table 3. Discussion OGCTs account for less than 5% of all malignant ovar- ian tumors, representing the most common malignant sex cord–stromal tumor in ovary origin; clinically, it may require additional chemotherapy after tumor re- moval surgery [13]. Radiological knowledge of this rare ovarian tumor is still limited in the reported literature, especially focusing on MR acquisition. MRI characteristics Herein, we col- lected 20 OGCTs in 18 patients with prospective MR Table 2 Basic and imaging characteristics of OGCTs OGCTs OSCs P value ORa Imaging findings T1 signals 20 34 0.017 hypointensity 4 8 isointensity 5 26 hyperintensity 5 mixed 6 T2 signals 0.002 hypointensity 5 isointensity 1 12 hyperintensity 12 10 mixed 7 7 DWI signals 0.003 hypointensity 1 7 isointensity 3 11 hyperintensity 16 16 mixed Margin 0.326 0.172 Regular/Iregular 17/3 33/1 Capsule 0.015 0.800 Present/Absent 16/4 34/0 Hemorrhage 0.001 3.429 Present/Absent 6/14 0/34 Component 0.000 2.50 Solid(80–100% solid component) 8 28 Cyst (80–100% cystic component) 4 1 Solid with cystic changes (others) 8 5 Enhancement (homogeneous/inhomogeneous) 0.034 minor 16(16/0) mild 14(12/2) 10(7/3) moderate 6(5/1) 4(4/0) avid 4(1/3) Maximum diameter (mm) 9.33 ± 5.43 8.5 ± 8.9 0.753 < 5 6 17 5–10 9 12 > 10 5 5 ADC values(×10−3 s/m2) 817 ± 144 (558–1120) 1223 ± 473 (460–2230) 0.002 Septa 0.121 Present/Absent 6/14 5/29 Lymph node 0.913 Present/Absent 0/18 0/34 Table 2 Basic and imaging characteristics of OGCTs (Continued) OGCTs OSCs P value ORa Pelvic fluid 0.031 8.130 Physiological/ Pathological 17/1 23/11 Clinical findings Age(years) 48.2 ± 15.1 (24–79) 54.8 ± 15.0 (21–81) 0.139 Vaginal discharge/bleeding 1 0 0.106 Menstruation Regular(irregular) 9(4) 18(5) 0.607 Menopause 9 16 Accompanying lesions 0.280 Yes/ No 7/12 9/25 aIndicates odds ratio Discussion OGCTs account for less than 5% of all malignant ovar- ian tumors, representing the most common malignant sex cord–stromal tumor in ovary origin; clinically, it may require additional chemotherapy after tumor re- moval surgery [13]. Radiological knowledge of this rare ovarian tumor is still limited in the reported literature, especially focusing on MR acquisition. Herein, we col- lected 20 OGCTs in 18 patients with prospective MR Page 5 of 9 Zhang et al. Journal of Ovarian Research (2018) 11:46 Fig. 3 A 54 -year-old woman with primary adult type OGCT (Ic). The mass shows as the purely cystic lesion with mostly high signal on T1WI (a) and T2WI (b). Note, the hemorrhagic contents locates on the left side of the tumor, representing the relatively high signal on T1WI and low signal on T2WI (arrowhead) and high signal on DWI (c). After injection of contrast medium, the cystic wall shows minor enhancement (d) Fig. 3 A 54 -year-old woman with primary adult type OGCT (Ic). The mass shows as the purely cystic lesion with mostly high signal on T1WI (a) and T2WI (b). Note, the hemorrhagic contents locates on the left side of the tumor, representing the relatively high signal on T1WI and low signal on T2WI (arrowhead) and high signal on DWI (c). After injection of contrast medium, the cystic wall shows minor enhancement (d) Fig. 4 A 50-year-old woman with primary adult type OGCT (IIIa). (a) The spongelike changes (arrowhead) are observed in the interior of the tumor appearing as the mostly solid mass (b). The cystic contents (arrowhead) give the low signal on DWI (c) and relatively high signal on ADC map (d), while the solid part (arrow) with the relatively high signal on DWI and low signal on ADC map Fig. 4 A 50-year-old woman with primary adult type OGCT (IIIa). (a) The spongelike changes (arrowhead) are observed in the interior of the tumor appearing as the mostly solid mass (b). The cystic contents (arrowhead) give the low signal on DWI (c) and relatively high signal on ADC map (d), while the solid part (arrow) with the relatively high signal on DWI and low signal on ADC map Page 6 of 9 Zhang et al. Journal of Ovarian Research (2018) 11:46 Fig. 5 An 18-year-old woman with histological proven SST at the right ovary. Discussion On coronal T2WI (a) and sagittal FS-T2WI (b), the solid mass (arrow) appears as the “comb” sign with centrally hyperintense signal surrounded by peripherally isointense signal. After injection of the contrast medium, the mass shows flush-in on early stage enhancement (c) and flush-out effect on late stage postcontrast image (d) Fig. 5 An 18-year-old woman with histological proven SST at the right ovary. On coronal T2WI (a) and sagittal FS-T2WI (b), the solid mass (arrow) appears as the “comb” sign with centrally hyperintense signal surrounded by peripherally isointense signal. After injection of the contrast medium, the mass shows flush-in on early stage enhancement (c) and flush-out effect on late stage postcontrast image (d) polyps in two patients and vaginal bleeding in one patient. All other accompanied lesions were incidentally detected on MRI. In terms of tumor component, OGCTs appeared as purely solid (8/20) to entirely cystic (4/22) mass with various morphology. Our findings are in accordance with the literature that OGCTs has more heterogeneity than OSCs. It is reported that a spongelike, multilocular cystic acquisition data at our single institution within nearly 10 years. To the best of our knowledge, this is the first study to describe the detailed MRI characteristics in the largest OGCT samples. Owing to production of estrogen, OGCTs can be associ- ated with endometrial hyperplasia, polyps, and carcinoma [18]. In our studied samples, we did observe endometrial Fig. 6 Stem-and-Leaf Plots of the calculated ADC values (10−3/ mm2/s) within four groups. The mean ADC value in OGCT is lower than that in other three groups (p = 0.002) with some overlap with fibrothecoma and fibroma Fig. 7 The diagnostic performance of ADC value in discriminating OGCT from OSC Fig. 6 Stem-and-Leaf Plots of the calculated ADC values (10−3/ mm2/s) within four groups. The mean ADC value in OGCT is lower than that in other three groups (p = 0.002) with some overlap with fibrothecoma and fibroma Fig. 7 The diagnostic performance of ADC value in discriminating OGCT from OSC Fig. 7 The diagnostic performance of ADC value in discriminating Fig. 6 Stem-and-Leaf Plots of the calculated ADC values (10−3/ mm2/s) within four groups. The mean ADC value in OGCT is lower than that in other three groups (p = 0.002) with some overlap with fibrothecoma and fibroma Fig. 6 Stem-and-Leaf Plots of the calculated ADC values (10−3/ mm2/s) within four groups. Discussion The possible reason may be that cystic changes often occur in the large tumor in both OGCT and fibrothe- coma, resulting in a wide range of measurable ADC values. mass filled with blood degradation products is characteris- tic MR imaging sign for OGCT [2, 9, 19]. We observed similar MRI appearances in 6 cases (Fig. 3). In our study, over 50% of OGCTs (11/20) displayed high or mixed sig- nal intensity on T1WI images, which may be related with the blood degradative components. Kim et al. reported that hemorrhage in the tumor was a common MRI finding in their seven cases [20]. This feature may be useful in discriminating OGCT from OSC since it is not noticed in the latter group. Regarding the lesion enhancement, 22 tu- mors examined in the present study showed mild (14/22, 63.6%) and moderate (8/22, 36.4%) enhancement relative to that of the myometrium. Avid and minor enhancement were not identified in OGCTs; while 4 SSTs noticed with avid enhancement and 12 fibromas and 4 fibrothecomas with minor enhancement. Although the enhancement type between OGCT and OSC did not differ significantly, it could be useful in discriminating them from broad liga- ment fibroids because the latter always show marked en- hancement after injection of contrast medium. j Being a problem-solving modality, MRI could provide valuable information in differentiating malignant tumors from benign gynecological diseases. Numerous studies focusing on this issue have been reported with promis- ing results [15, 21, 22]. In current study, MRI could well indicate the morphology and components of the OGCTs with an accuracy of approximately 90% in distinguishing OGCT from OSC. Four lesions were preoperatively mis- diagnosed as fibroid (three lesions) and fibrothecoma (one lesion) because of either small size or homogeneous signal intensity. Pathological fluid (a large amount of ascites) was a rare condition in OGCT group (one case); however, it more often occur in OSC group (11 cases). The true mechanism is still unknown and may result from more estrogen secretion in the latter group. No lymphadenopathy was detected on MRI in all OGCT cases, which means it is a low potential malignant tumor unlike ovarian epithelial cancer. In one recent study, the authors also concluded that lymphadenectomy was not recommended in initial staging surgery of ovarian sex cord stromal tumor due to the low lymph node metasta- sis rate [23]. There are several limitations to this study. Discussion The mean ADC value in OGCT is lower than that in other three groups (p = 0.002) with some overlap with fibrothecoma and fibroma Fig. 7 The diagnostic performance of ADC value in discriminating OGCT from OSC Fig. 7 The diagnostic performance of ADC value in discriminating OGCT from OSC Zhang et al. Journal of Ovarian Research (2018) 11:46 Page 7 of 9 Table 3 Diagnostic performance of MRI in diagnosis OGCTs according to two reading protocols Protocol SEN (%) SPE (%) PPV (%) NPV (%) ACC (%) Conventional MRI 85.0(17/20) [63.9–95.0] 94.0(32/34) [81.0–98.0] 89.5(17/19) [68.6–97.1] 91.4(32/35) [77.6–97.0] 90.7(49/54) [80.1–95.9] Conventional MRI plus ADC value 95.0(19/20) [76.4–99.1] 94.1%(32/34) [80.9–98.4] 90.5(19/21) [71.1–97.4] 96.9(32/33) [84.7–99.5] 94.4(51/54) [84.9–98.1] Numbers in parentheses are the data used to calculate the percentages; Numbers in brackets are 95% confidence intervals; Conventional MRI includes T1WI, T2WI, contrast-enhanced MRI and DWI tissues with ADC value. Theoretically, as a result of high cell densities and abundant cellular membranes, the movement of water molecule in cancer tissues is re- stricted on DWI images and then, the derived ADC value should be generally lower in malignant disease than in benign or healthy tissue [8, 24]. Many studies have reported ADC value could help to distinguish ovarian malignant lesions from benign conditions on both 1.5 T [25–27] and 3.0 T MR system [28, 29]. In one study, the authors reported that the mean ADC value of OGCT was 1000 ± 120 × 10−3 s/m2 in their three cases with a 1.5 T MR machine using the same b value [30], which is higher than our results(817 ± 144 × 10−3 s/m2). As for MR imaging, sometimes, OGCT need to be differentiated with fibrothecoma and ligamental myoma. Our results show that the mean ADC value of OGCT is lower than OSC with statistically significant difference. The similar results is not reported in previ- ously published studies. However, owing to the limited reported cases, the comparative ADC value should be concluded in the large cohort data. If combining the ADC value, MRI yield a sensitivity of 95.0% and a speci- ficity of 94.1% in diagnosis of OGCT, higher than with conventional MRI reading session alone. Our findings demonstrate that there is an overlap in the ADC value between OGCT and fibroma and fibrothecoma; however, there is no overlap observed between OGCT and SST. Discussion First, we re- trieved MR reports with suggested OGCT and OSC diagnosis on PACS system (within 6 years) and then, compared the MR results with pathological reports case by case. We do believe some cases may be missed for those not mentioned on MRI reports. So, the limited study samples in both OGCT and OSC group might have influenced the final results. Second, the ADC value was manually measured on the selected area based on individual habits. Standardization in measurement may influence the final results. Third, our study is based on 1.5 T MRI system while 3.0 T MRI has been used for a decade. Owing to the variable selected b – value on DWI images and the limited case studies on 3.0 T, we By displaying water molecule mobility (Brownian mo- tion), DWI is considered as a functional imaging tech- nique, permitting the quantitative evaluation of tumor Page 8 of 9 Zhang et al. Journal of Ovarian Research (2018) 11:46 Page 8 of 9 Page 8 of 9 Page 8 of 9 Received: 18 July 2017 Accepted: 20 May 2018 Received: 18 July 2017 Accepted: 20 May 2018 References 1. Jung SE, Rha SE, Lee JM, Park SY, Oh SN, Cho KS, et al. CT and MRI findings of sex cord–stromal tumor of the ovary. Am J Roentgenol. 2005;185(1):207–15. 1. Jung SE, Rha SE, Lee JM, Park SY, Oh SN, Cho KS, et al. CT and MRI findings of se cord stromal t mor of the o ar Am J Roentgenol 2005185(1)207 15 1. Jung SE, Rha SE, Lee JM, Park SY, Oh SN, Cho KS, et al. CT and MRI findings of sex cord stromal tumor of the ovary Am J Roentgenol 2005;185(1):207 15 sex cord–stromal tumor of the ovary. Am J Roentgenol. 2005;185(1) 2. 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Long-term survival in a patient with an advanced ovarian juvenile granulosa cell tumor with Para-aortic lymph node metastasis. Taiwanese Journal of Obstetrics and Gynecology. 2016;55(6):907–9. The authors declare that all data supporting the findings of this study are available within the article. The authors declare that all data supporting the findings of this study are available within the article. Received: 18 July 2017 Accepted: 20 May 2018 cannot compare the ADC value between these two mo- dalities. However, for fibrothecoma, the mean ADC value do not differ with the results reported by our pre- vious study with 3.0 T [31]. Studies with more OGCT samples on 3.0 T unit still be needed to determine the true differences in the future. Conclusion 3. Chen VW, Ruiz B, Killeen JL, Coté TR, Wu XC, Correa CN, et al. Pathology and classification of ovarian tumors. Cancer. 2003;97(S10):2631–42. 3. Chen VW, Ruiz B, Killeen JL, Coté TR, Wu XC, Correa CN, et al. Pathology and classification of ovarian tumors. Cancer. 2003;97(S10):2631–42. In conclusion, MRI could provide useful information in accurately diagnosing OGCT. The heterogeneous signal intensity on both T1WI and T2WI and high signal in- tensity on DWI images are more suggestive of OGCT diagnosis. ADC value might be useful in differentiating OGCT from OSC. 4. Cancer O. Version 1.2016, NCCN clinical practice guidelines in oncology. J Natl Compr Cancer Netw. 2016;14:1134–63. 4. Cancer O. Version 1.2016, NCCN clinical practice guidelines in oncology. J Natl Compr Cancer Netw. 2016;14:1134–63. J Natl Compr Cancer Netw. 2016;14:1134–63. 5. Seagle B-LL, Ann P, Butler S, Shahabi S. Ovarian granulosa cell tumor: a National Cancer Database study. Gynecol Oncol. 2017;46(2):285–91. 5. Seagle B-LL, Ann P, Butler S, Shahabi S. Ovarian granulosa cell tumor: a National Cancer Database study. Gynecol Oncol. 2017;46(2):285–91. 5. Seagle B LL, Ann P, Butler S, Shahabi S. 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Ovarian tumor with functioning stroma. Comput Med Imaging Graph. 2002;26(3):193–7. Consent for publication 19. Horta M, Cunha TM. Sex cord-stromal tumors of the ovary: a comprehensive review and update for radiologists. Diagn Interv Radiol. 2015;21(4):277–86. 19. Horta M, Cunha TM. Sex cord-stromal tumors of the ovary: a comprehensive review and update for radiologists. Diagn Interv Radiol. 2015;21(4):277–86. All authors read and approved the final manuscript. All authors agreed to submit this article. All authors read and approved the final manuscript. All authors agreed to submit this article. 20. Kim SH, Kim SH. Granulosa cell tumor of the ovary: common findings and unusual appearances on CT and MR. J Comput Assist Tomogr. 2002;26(5): 756–61. Competing interests The authors declare that they have no competing interests. Zhang et al. Journal of Ovarian Research (2018) 11:46 31. Zhang H, Zhang GF, Wang TP, Zhang H. 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Br J Radiol. 2011;84(1003):600–11 31. Zhang H, Zhang GF, Wang TP, Zhang H. Value of 3.0 T diffusion-weighted imaging in discriminating thecoma and fibrothecoma from other adnexal solid masses. J Ovarian Res. 2013;6:58.
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Adaptive selection of diurnal minimum variation: a statistical strategy to obtain representative atmospheric CO&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt; data and its application to European elevated mountain stations
Atmospheric measurement techniques
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Atmos. Meas. Tech., 11, 1501–1514, 2018 https://doi.org/10.5194/amt-11-1501-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. Correspondence: Ye Yuan (yuan@wzw.tum.de) Correspondence: Ye Yuan (yuan@wzw.tum.de) Received: 29 August 2017 – Discussion started: 12 September 2017 g p Revised: 1 February 2018 – Accepted: 11 February 2018 – Published: 15 March 2018 Abstract. Critical data selection is essential for determining representative baseline levels of atmospheric trace gases even at remote measurement sites. Different data selection tech- niques have been used around the world, which could po- tentially lead to reduced compatibility when comparing data from different stations. This paper presents a novel statisti- cal data selection method named adaptive diurnal minimum variation selection (ADVS) based on CO2 diurnal patterns typically occurring at elevated mountain stations. Its capabil- ity and applicability were studied on records of atmospheric CO2 observations at six Global Atmosphere Watch sta- tions in Europe, namely, Zugspitze-Schneefernerhaus (Ger- many), Sonnblick (Austria), Jungfraujoch (Switzerland), Izaña (Spain), Schauinsland (Germany), and Hohenpeis- senberg (Germany). Three other frequently applied statisti- cal data selection methods were included for comparison. Among the studied methods, our ADVS method resulted in a lower fraction of data selected as a baseline with lower max- ima during winter and higher minima during summer in the selected data. The measured time series were analyzed for long-term trends and seasonality by a seasonal-trend decom- position technique. In contrast to unselected data, mean an- nual growth rates of all selected datasets were not signifi- cantly different among the sites, except for the data recorded at Schauinsland. However, clear differences were found in the annual amplitudes as well as the seasonal time structure. Based on a pairwise analysis of correlations between stations on the seasonal-trend decomposed components by statistical data selection, we conclude that the baseline identified by the ADVS method is a better representation of lower free tro- pospheric (LFT) conditions than baselines identified by the other methods. Adaptive selection of diurnal minimum variation: a statistical strategy to obtain representative atmospheric CO2 data and its application to European elevated mountain stations Ye Yuan1, Ludwig Ries2, Hannes Petermeier3, Martin Steinbacher4, Angel J. Gómez-Peláez5,a, Markus C. Leuenberger6, Marcus Schumacher7, Thomas Trickl8, Cedric Couret2, Frank Meinhardt9, and Annette Menzel1,10 1, Ludwig Ries2, Hannes Petermeier3, Martin Steinbacher4, Angel J. Gómez-Peláez5,a, g g Markus C. Leuenberger6, Marcus Schumacher7, Thomas Trickl8, Cedric Couret2, Frank Meinhardt9, and Annette Menzel1,10 1Department of Ecology and Ecosystem Management, Technical University of Munich (TUM), Freising, Germany 2German Environment Agency (UBA), Zugspitze, Germany g y ( ), g p , y 3Department of Mathematics, Technical University of Munich (TUM), Freising, Germany 4Empa, Laboratory for Air Pollution/Environmental Technology, Dübendorf, Switzerland p y ( ) g y 4Empa, Laboratory for Air Pollution/Environmental Technology, Dübendorf, Switzerland 5Izaña Atmospheric Research Center, Meteorological State Agency of Spain (AEMET), Santa Cruz de Tenerife, Spain 6Climate and Environmental Physics Division, Physics Institute and Oeschger Centre for Climate Change Research, University of Bern, Bern, Switzerland 7Meteorological Observatory Hohenpeissenberg, Deutscher Wetterdienst (DWD), Hohenpeissenberg, Germany 8Institute of Meteorology and Climate Research, Atmospheric Environmental Research (IMK-IFU), Karlsruhe Institute of Technology, Garmisch-Partenkirchen, Germany 9German Environment Agency (UBA) Schauinsland Germany 9German Environment Agency (UBA), Schauinsland, Germany 9German Environment Agency (UBA), Schauinsland, Germany 10 10Institute for Advanced Study, Technical University of Munich (TUM), Garching, Germany anow at: Meteorological State Agency of Spain (AEMET), Delegation in Asturias, Oviedo, Spain 10Institute for Advanced Study, Technical University of Munich (TUM), Garching, Germany anow at: Meteorological State Agency of Spain (AEMET), Delegation in Asturias, Oviedo, Spain 1 Introduction data filtering method is fixed time window selection, by se- lecting data in a certain time interval of the day based on local and mesoscale mechanisms of air mass transport. For selecting well-mixed air at elevated mountain sites, night- time is usually chosen with a special focus on the exclusion of afternoon periods due to the influence of convective up- ward transport (Bacastow et al., 1985). Brooks et al. (2012), for example, limited their mountaintop CO2 results in the Rocky Mountains (USA) by “time-of-day” from 0 a.m. till 4 a.m. local time (LT) to increase the likelihood of sam- pling the free tropospheric environment at the station. Apart from this, modeling techniques such as backward trajectories are very helpful for analyzing the origins and transport pro- cesses of air masses arriving at the station in detail (Cui et al., 2011). Uglietti et al. (2011) focused on the origins of at- mospheric CO2 at Jungfraujoch (Switzerland) by the FLEX- ible PARTicle dispersion model. Using tracers, data selec- tion can be performed by investigating the correlations be- tween the air components of interest. Many tracers have been tested and compared with CO2. Threshold limits of 300 ppb for CO and 2000 ppb for CH4 were defined by Sirignano et al. (2010) to perform a regional analysis of CO2 data at Lut- jewad (the Netherlands) and Mace Head (Ireland). Similar approaches with black carbon and CH4 were performed by Fang et al. (2015) at Lin’an (China). Moreover, Chambers et al. (2016) applied a data selection technique to identify base- line air masses using atmospheric radon measurements at the stations Cape Grim (Australia), Mauna Loa (Hawaii, USA), and Jungfraujoch (Switzerland). Continuous in situ measurements of greenhouse gases (GHGs) at remote locations have been established since 1958 (Keeling, 1960). Knowledge of background atmospheric GHG concentrations is key to understanding the global car- bon cycle and its effect on climate, as well as the GHG responses to a changing climate. A critical issue when us- ing data from remote stations remains the identification of time periods that are representative of larger spatial areas and their differentiation from periods influenced by local and regional pollution. 1 Introduction If these two regimes are well disag- gregated, the available datasets can represent more reliable information about long-term changes of undisturbed atmo- spheric GHG levels or be used to investigate local and re- gional GHG sources and sinks when specifically analyzing deviations from baseline conditions. In this study, the base- line conditions refer to a selected subset of data from the val- idated dataset, representing well-mixed air masses with min- imized short-term external influences (Elliott, 1989; Calvert, 1990; Balzani Lööv et al., 2008; Chambers et al., 2016). Measurement results depend on sampling methods, ana- lytical instrumentation, and data processing. Validated data (labeled as VAL in this study to differentiate from the se- lected data) are usually obtained after signal correction, for example due to interferences from other GHGs such as water vapor, calibration accounting for sensitivity changes of the analyzer, and validation based on plausibility checks. Base- line data selection starts with validated data and identifies in subsequent steps a final subset of the validated dataset based on predefined criteria for specific qualities such as represen- tativeness. These data will be referred to as “selected baseline data” or simply as “selected data” in the following. g j Unlike most of the methods mentioned above, which re- quire additional data or advanced transport modeling, statis- tical data selection only relies on the time series of interest and typically investigates the variability of signal. It is usu- ally assumed that the most representative CO2 data are found during well-mixed conditions revealing small variations in time (Peterson et al., 1982) and in space (Sepúlveda et al., 2014). For continuous measurements, it is possible to investi- gate within-hour and hour-to-hour variability in the datasets. The within-hour variability is often expressed as the stan- dard deviation of the measured data within 1 h. The hour-to- hour variability compares the differences between hourly av- eraged concentrations either during a certain time period, or from one hour to the next. Pales and Keeling (1965) marked ambient data as “variable” when the within-hour variability for the air sample was significantly larger than the within- hour variability for the reference gas. Consequently, they only considered CO2 data to belong to background condi- tions when the concentrations were in “steady” conditions for 6 h or more. Similarly, Peterson et al. (1982) rejected sampled CO2 data values for adjacent hours when the hour- to-hour variability exceeded 0.25 ppm. Thoning et al. Y. Yuan et al.: Adaptive selection of diurnal minimum variation 1502 data filtering method is fixed time window selection, by se- lecting data in a certain time interval of the day based on local and mesoscale mechanisms of air mass transport. For selecting well-mixed air at elevated mountain sites, night- time is usually chosen with a special focus on the exclusion of afternoon periods due to the influence of convective up- ward transport (Bacastow et al., 1985). Brooks et al. (2012), for example, limited their mountaintop CO2 results in the Rocky Mountains (USA) by “time-of-day” from 0 a.m. till 4 a.m. local time (LT) to increase the likelihood of sam- pling the free tropospheric environment at the station. Apart from this, modeling techniques such as backward trajectories are very helpful for analyzing the origins and transport pro- cesses of air masses arriving at the station in detail (Cui et al., 2011). Uglietti et al. (2011) focused on the origins of at- mospheric CO2 at Jungfraujoch (Switzerland) by the FLEX- ible PARTicle dispersion model. Using tracers, data selec- tion can be performed by investigating the correlations be- tween the air components of interest. Many tracers have been tested and compared with CO2. Threshold limits of 300 ppb for CO and 2000 ppb for CH4 were defined by Sirignano et al. (2010) to perform a regional analysis of CO2 data at Lut- jewad (the Netherlands) and Mace Head (Ireland). Similar approaches with black carbon and CH4 were performed by Fang et al. (2015) at Lin’an (China). Moreover, Chambers et al. (2016) applied a data selection technique to identify base- line air masses using atmospheric radon measurements at the stations Cape Grim (Australia), Mauna Loa (Hawaii, USA), and Jungfraujoch (Switzerland). 2.2 ADVS CO2 measurements from six European mountain stations (see Fig. 1) within the Global Atmosphere Watch (GAW) network were used. The data were taken from mountain sta- tions due to their remote locations, being subjected to lim- ited anthropogenic influence and this provided increased rep- resentativeness. Three high alpine measurement sites were included: Zugspitze-Schneefernerhaus (ZSF, DE, 47◦25′ N, 10◦59′ E, 2670 m a.s.l.), Jungfraujoch (JFJ, CH, 46◦33′ N, 7◦59′ E, 3580 m a.s.l.), and Sonnblick (SNB, AT, 47◦03′ N, 12◦57′ E, 3106 m a.s.l.). They are often above the planetary boundary layer (PBL) and thus exposed to free and presum- ably clean lower tropospheric air masses, but periodically influenced by regional emissions from lower altitudes. Ad- ditionally, to test data selection for a less remote environ- ment, CO2 measurements were investigated from Schauins- land (SSL, DE, 47◦55′ N, 7◦55′ E, 1205 m a.s.l.) at a much lower elevation, in the mid-range Black Forest. Data selec- tion was also applied to three recently started CO2 time series from different sampling heights above ground on a tall tower at the Hohenpeissenberg observatory (HPB, DE, 47◦63′ N, 11◦01′ E, 934 m a.s.l.), located in the northern foothills of the Alps. Henne et al. (2010) presented a method of categoriz- ing site representativeness based on the influence and vari- ability of population and deposition by the surface fluxes. JFJ and SNB were classified as “mostly remote,” while ZSF was considered as “weakly influenced, constant deposition,” and SSL and HPB were considered as “rural” (Henne et al., 2010). Finally, the station Izaña on Tenerife Island (IZO, ES, 28◦19′ N, 16◦30′ W, 2373 m a.s.l.) in the North Atlantic was chosen as a reference due to its location above the subtropi- cal temperature inversion layer, which means that the station ADVS is a tool for automated and systematic analysis of di- urnal CO2 cycles at elevated mountain stations in order to select consecutive time sequences with minimum variation, which can be regarded as representing well-mixed air con- ditions. Even though such measurement sites are remotely located, the CO2 levels are still influenced by local sources and sinks. For example, at ZSF, these can be characterized by episodic CO2 enhancements due to anthropogenic emissions, detectable especially in winter during the day, whereas in summer the convective upwind transport results in episodes with depleted CO2 concentrations due to photosynthetic up- take of CO2 at lower altitudes. Y. Yuan et al.: Adaptive selection of diurnal minimum variation is rarely affected by any local or regional CO2 sources and sinks (Gomez-Pelaez et al., 2013). of Baseline Signal”, to estimate the baseline curves general- ized for atmospheric compounds, which is available in the R package IDPmisc (Locher and Ruckstuhl, 2012). For this study, unless otherwise indicated, hourly data were used consistently for the purpose of evaluating the data selection method since the method should be easily ap- plicable to data obtained from standard data centers such as the World Data Centre for Greenhouse Gases (WD- CGG) where data are commonly stored with hourly resolu- tion. The validated CO2 hourly averages from all stations were downloaded from WDCGG (http://ds.data.jma.go.jp/ gmd/wdcgg/). Data with higher time resolution required for some sensitivity analysis in this study were provided directly by the station investigators. All time stamps refer to the be- ginning of the averaging interval. Descriptions of the sam- pling elevation and time period of available data are given in Table 1. Further information on each station can be found in Schmidt et al. (2003) for SSL, Gilge et al. (2010) for HPB and SNB, Gomez-Pelaez et al. (2010) for IZO, Risius et al. (2015) for ZSF, and Schibig et al. (2015) for JFJ. Practi- cal data selections and analyses in this study were performed using the R Statistical Environment (R Core Team, 2017). The present study focuses on the comparison of results from previous statistical data selection methods with the new adaptive diurnal minimum variation selection (ADVS) method proposed in this study. The ADVS is seen as a possi- ble alternative to already known data selection methods as discussed above. The results obtained with ADVS for the atmospheric CO2 records from six European mountain sta- tions are compared with those derived from three other sta- tistical data selection methods. To investigate the potential influences of trend and seasonality, further analyses focus on the decomposition of validated and selected datasets into trend and seasonal components. Finally, differences between ADVS and other data selection methods are assessed by cor- relation analysis. 1 Introduction (1989) combined these two strategies using an iterative approach by selecting data according to deviations of daily averages from a spline curve fit. Ruckstuhl et al. (2012) developed a method based on robust local regression, called “Robust Extraction p y g Data selection methods can be categorized into meteoro- logical, tracer, and statistical selection methods (Ruckstuhl et al., 2012; Fang et al., 2015). Meteorological data selec- tion makes use of the meteorological information at the mea- surement sites, which provides valuable information about the surrounding environment as well as air mass transport (Carnuth and Trickl, 2000; Carnuth et al., 2002). Forrer et al. (2000), Zellweger et al. (2003), and Kaiser et al. (2007) intensively studied the relationship between measured trace gases (such as O3, CO, and NOx) and meteorological pro- cesses at Zugspitze, Jungfraujoch, Sonnblick, and Hohen- peissenberg. For CO2, the most common parameters applied in the literature are wind speed and wind direction. They can provide information on critical variations at stations with sources and sinks in their vicinity, while these parameters are less suited at stations in largely pristine environments. For example, Lowe et al. (1979) performed a pre-selection on the CO2 record at Baring Head (New Zealand) using pe- riods with southerly winds only (clean marine air). Massen and Beck (2011) found that the CO2 versus wind speed plot can be valuable for baseline CO2 estimation without a local influence of continental measurements. Another widely used Atmos. Meas. Tech., 11, 1501–1514, 2018 www.atmos-meas-tech.net/11/1501/2018/ 1503 Y. Yuan et al.: Adaptive selection of diurnal minimum variation Atmos. Meas. Tech., 11, 1501–1514, 2018 2.2 ADVS Although high altitude moun- tain stations do not have vegetation in their surroundings, mountain stations at lower altitudes that are still in the vege- tation zone may be influenced by plant respiration, especially at night. As these effects of upward transport photosynthesis and respiration all vary diurnally, the basic strategy that we follow in this study is to identify the most stable time peri- ods of the day, i.e., periods with minimum variation, which in turn can be used for selecting representative data. How- ever, the duration of this time window during the day varies with the season and from day to day because of variations in the dynamics of transport to the site (e.g., Birmili et al., 2009; Herrmann et al., 2015). In summer, larger variabilities in the CO2 signal are observed due to more prevalent convec- tive boundary-layer air-mass injections influencing the diur- nal pattern, resulting in shorter periods of stable conditions, whereas in winter, significantly longer stable periods occur. No upwind air masses with depleted CO2 levels due to pho- tosynthesis by vegetation are recorded in winter. To preserve as much representative data as possible, it is desirable to se- Atmos. Meas. Tech., 11, 1501–1514, 2018 www.atmos-meas-tech.net/11/1501/2018/ www.atmos-meas-tech.net/11/1501/2018/ 1504 Y. Yuan et al.: Adaptive selection of diurnal minimum variation Figure 1. Locations of six European elevated mountain stations. Symbols from left to right stand for: IZO – Izaña, Spain; SSL – Schauinsland, Germany; JFJ – Jungfraujoch, Switzerland; HPB – Hohenpeissenberg, Germany; ZSF – Schneefernerhaus-Zugspitze, Germany; SNB – Sonnblick, Austria. Figure 1. Locations of six European elevated mountain stations. Symbols from left to right stand for: IZO – Izaña, Spain; SSL – Schauinsland, Germany; JFJ – Jungfraujoch, Switzerland; HPB – Hohenpeissenberg, Germany; ZSF – Schneefernerhaus-Zugspitze, Germany; SNB – Sonnblick, Austria. Table 1. Information of measured CO2 datasets at six GAW mountain stations. Table 1. Information of measured CO2 datasets at six GAW mountain stations. Station (GAW ID) Sampling elevation (a.s.l.) Time period (yyyy.mm) Data provider Hohenpeissenberg (HPB) 984/1027/1065 m 2015.09–2016.06 DWD Schauinsland (SSL) 1210 m 2010.01–2015.12 UBA-De Izaña (IZO) 2403 m 2010.01–2015.12 AEMET Zugspitze-Schneefernerhaus (ZSF) 2670 m 2010.01–2015.12 UBA-De Sonnblick (SNB) 3111 m 2010.01–2015.12 UBA-At Jungfraujoch (JFJ) 3580 m 2010.01–2015.12 Empa – Result: the start time window [istart,...,iend]. With the focus on elevated mountain stations, starting se- lection is purposely designed with the moving window 1j of 6 h, and the starting hour istart to be between 6 p.m. and 5 a.m. LT for this study. For other stations with possibly different diurnal patterns, starting selection can be adjusted accordingly. For instance, at urban stations or stations com- pletely within the continental PBL, the start time window can be chosen based on their best mixing conditions, which often occur in the afternoon with a shorter moving window, when the PBL reaches its maximum depth after “ingesting” free tropospheric air during its growth. Being aware that calculat- ing the start time window from all data could differ from the start time windows calculated by season, the overall gener- ated start time windows have been compared with seasonally generated start time windows for high altitude mountain sta- tions (see Supplement Sect. S1.1). Because these differences were mostly small to moderate and this work aims at a me- thodical comparison under identical conditions, the start time windows are always derived from overall data. – Step 5: the new absolute difference between xf and xW is calculated, as well as the new threshold criteria. If condition xf −xW ≤κ · sW holds, xf is included in W and ADVS goes back to Step 4. Otherwise, ADVS stops for this day and proceeds to the next day. When data selection for all days is finished, ADVS con- tinues with backward adaptive selection. Afterwards, it proceeds to the result. – Result: this is the final selection time window, which is a combination of Wforward and Wbackward for the day in question. – Result: this is the final selection time window, which is a combination of Wforward and Wbackward for the day in question. The following limitations of the forward and backward ex- pansions of the time window should be considered. ADVS always runs for no longer than 24 h including the start time window, i.e., f ≤24· tr, where tr is the time resolution in data points per hour of the input data. This sometimes results in an overlap of “selected” and “unselected” data for two con- secutive days. We always label the data as “selected” once it has been selected by ADVS. The threshold parameter κ is the controlling factor for the length of the selection time window. 2.2.1 Starting selection lect the time window dynamically. ADVS is constructed to select a subset from the measured data, being best represen- tative for baseline conditions with an adaptive selection time window specific for every day. For a given validated hourly dataset, ADVS starts data selec- tion by finding a start time window for all days. The stan- dardized selection procedure for the start time window re- sults from site-specific parameters. This time interval is set as the most stable period from the diurnal variation. The step is referred to as starting selection. It begins by analyzing the mean diurnal cycle of the data input. The algorithm is based on two basic assumptions. First, air masses measured at elevated stations represent well-mixed air, closest to baseline levels, within a certain time window of several hours during the day. For the elevated mountain stations discussed in this paper, this time interval is around midnight. Different diurnal patterns are apparent at each sta- tion, so the selection time window should be adjusted ac- cordingly. Second, it is assumed that real baseline conditions are not subject to local influences and thus represent unper- turbed lower free tropospheric air masses. This indicates that the variability of the measured CO2 signal should be minimal within this selection time window. The methodological steps of ADVS are introduced in detail below in the two sections “starting selection” and “adaptive selection”. – Step 1: detrending is done by subtracting a 3-day aver- age for each day, including the neighboring two days. It is the shortest possible time window to remove sudden changes in the time series related to the previous and posterior days while preserving the diurnal pattern. – Step 2: the overall mean diurnal variation, di (i = 0 to 23 h), is calculated from the complete set of detrended data. Atmos. Meas. Tech., 11, 1501–1514, 2018 www.atmos-meas-tech.net/11/1501/2018/ 1505 – Result: the start time window [istart,...,iend]. As κ increases, the length of the selection time win- dow increases. A value of 2 was chosen heuristically for this study as a compromise between selecting as many data points as possible and achieving the least data variability. Similar values of sensitivity-controlling parameters in other data se- lection methods can be found (Thoning et al., 1989; Sirig- nano et al., 2010; Uglietti et al., 2011; Satar et al., 2016). In Step 2, values of 0.3 ppm and 0.5 indicate the threshold values for si and πmissing. We denote them as si,threshold and πmissing,threshold. Less remote stations at lower altitudes may require a larger value than 0.3 ppm because of different mix- ing conditions. When performing ADVS data selection at lower sites such as HPB and SSL, we recommend a higher si,threshold, such as 1.0 ppm. However, throughout this study we used the described parameter setting (0.3 ppm) for a me- thodical inter-comparison of selection methods at all stations. Potential influences of these parameter sizes (si,threshold and tr) are discussed in Supplement Sect. S1.2 and S1.3. Y. Yuan et al.: Adaptive selection of diurnal minimum variation – Step 3: the absolute difference between xf and xi is cal- culated, and the following threshold criterion is applied: xf −xi ≤κ ·si, where κ is the threshold parameter. If this criterion holds, xf is included in W and ADVS con- tinues. Otherwise, ADVS stops for this day with only the start time window, and proceeds to the next day. – Step 3: the standard deviations s1j from the overall mean diurnal variation di are calculated on a moving window 1j (j = 6 h). To be able to place a full set of 24 moving time windows over the overall mean diurnal variation, time windows across midnight (e.g., 6 h from 11 p.m. to 4 a.m. LT) are also included, that is, its first j hours are appended to the end of the 24 h in the over- all mean diurnal variation. The time window with the smallest standard deviation is selected as the start time window. – Step 4: mean xW and standard deviation sW for the new selection time window W are calculated. If sW ≤ 0.3 ppm (CO2), ADVS continues with the next data point xf with f = f + 1. Otherwise, ADVS stops for this day with the previous selection time window and proceeds to the next day. – Result: the start time window [istart,...,iend]. www.atmos-meas-tech.net/11/1501/2018/ 2.3 Other statistical data selection methods for comparison the robustness weights to reduce the influences of extreme values for the next run of the inner loop (Cleveland et al., 1990). We compared ADVS with three statistical data selection methods. The first method named SI is based on “steady in- tervals” (Lowe et al., 1979; Stephens et al., 2013). Steady intervals, which are considered as baseline conditions, are defined by a standard deviation being lower than or equal to 0.3 ppm for six or more consecutive hours. Although this method has some similarity with ADVS, it treats all hours of the day equally without giving preference to hours where the variability is, on average, the smallest. For this study, we used the implemented function stl in R (R Core Team, 2017). Owing to functional limitation of stl, full time coverage of monthly data is needed in order to reduce the risk of large time gaps or unequal spacing (Pick- ers and Manning, 2015). All data were first aggregated to monthly averages. Then, missing data were substituted by linear interpolation, using R function na.approx (Zeileis and Grothendieck, 2005). For the application of STL, two param- eters need to be specified, which are the seasonal smooth- ing parameter n(s) (s-window in function stl) and the trend smoothing parameter n(t) (t-window in function stl). As n(s) and n(t) increase, the seasonal and trend components get smoother (Cleveland et al., 1990). For optimal compatibility in this study, the same parameters were chosen for all stations as n(s) = 7 and n(t) = 23, based on the recommendation of Cleveland et al. (1990). Another parameter combination of n(s) = 5 and n(t) = 25 was also tested according to Pickers and Manning (2015), but with no significant differences in results. Second, we adopted a method applied by NOAA ESRL, which originated from Thoning et al. (1989). This se- lection routine has been applied specifically for measure- ments of background CO2 levels at Mauna Loa. This method (referred to as THO) was applied as described on the website: http://www.esrl.noaa.gov/gmd/ccgg/about/co2_ measurements.html. The first step of THO examines the within-hour variability by selecting hours with hourly stan- dard deviation less than 0.3 ppm. For the hourly data used in this study, the within-hour variability is not applicable so that the first step is skipped. Y. Yuan et al.: Adaptive selection of diurnal minimum variation 1506 3.1 Start time window ADVS was applied to the validated hourly averages from all six stations with the parameter settings as described above. The detrended mean diurnal cycles were obtained together with the start time window for each station by starting se- lection (see Fig. 2, for conventional mean diurnal plots see Supplement Sect. S2). The observed differences in the start time windows, as well as in the widths of the confidence in- tervals (gray shades), reflect the characteristics of differently situated measurement sites and different sampling levels. The first subplot column (HPB50, HPB93, and HPB131), repre- senting the three sampling heights at HPB, shows similar de- trended diurnal patterns with similar start time windows. The slightly different start time window at HPB131 potentially indicates different dynamics of the atmospheric transport at higher elevation. The decreasing amplitude with increasing sampling height indicates that the higher the sampling inlet is above the ground, the less it is affected by the local sur- face fluxes. The three start time windows suggest that the most stable period at HPB occurs during the last few hours of a day, including midnight. However, in contrast to all other stations covering at least a full year, HPB data are only from September of 2015 to June of 2016. The results may not be fully comparable, but instead it shows that the data selection method is also applicable to data with time periods shorter than one year. The last method compared is a moving average technique (MA). A moving time window of 30 days and a threshold cri- terion of two standard deviations from the moving averages were applied to discard outliers. Afterwards, new moving av- erages and new threshold criteria were calculated for data exclusion. This step is repeated until no more outliers were found. A more detailed description can be found in Uglietti et al. (2011) and Satar et al. (2016). 2.3 Other statistical data selection methods for comparison Second, it computes hourly averages and checks the hour-to-hour variability by retaining any two consecutive hourly values where the hour-to-hour difference is less than 0.25 ppm. The last step is based on the diurnal pattern (similar to ADVS), by excluding data from 11 a.m. to 7 p.m. LT due to transported air influenced by photosynthe- sis. 2.2.2 Adaptive selection The second component, adaptive selection, is designed to de- termine the most suitable time window for each day, based on the data variability. Through this method, the length of the start time window is expanded in both directions in time. Adaptive selection is performed on a daily basis, starting with the first day of the given dataset. The following steps only describe the forward adaptive selection. ADVS also runs the backward adaptive selection in an analogous manner but backwards in time. – Step 1: the mean molar fraction xi, standard devia- tion si, and the proportion of missing values πmissing are calculated from data in the start time window [istart,...,iend]. – Step 2: if si ≤0.3 ppm (CO2) and πmissing ≤0.5, ADVS continues to advance in time, examine whether the next data point xf can be included in the selection time win- dow W with f = iend + 1. Otherwise, it is considered that the start time window does not fulfill the assump- tions. In this case, no baseline data is selected for the present day and the algorithm proceeds to the next day. Atmos. Meas. Tech., 11, 1501–1514, 2018 www.atmos-meas-tech.net/11/1501/2018/ 2.4 Seasonal-trend decomposition STL To analyze the results from different data selection methods and compare them with the original validated datasets, we applied the seasonal-trend decomposition technique based on locally weighted regression smoothing (Loess), named STL (Cleveland, 1979; Cleveland et al., 1990). STL has been widely applied to measurements of atmospheric CO2 and other trace gases (Cleveland et al., 1983; Carslaw, 2005; Brailsford et al., 2012; Hernández-Paniagua et al., 2015; Pickers and Manning, 2015). It decomposes a time series of interest into a trend component T , a seasonal component S, and a remainder component R, which allows detailed sepa- rate analyses of trend and seasonality. Two recursive proce- dures are included in the STL technique: an inner loop where seasonal and trend smoothing based on Loess are performed and updated in each pass, and an outer loop that computes Regarding the second subplot column (SSL, SNB, and IZO), the start time windows can be found from midnight Atmos. Meas. Tech., 11, 1501–1514, 2018 www.atmos-meas-tech.net/11/1501/2018/ www.atmos-meas-tech.net/11/1501/2018/ Y. Yuan et al.: Adaptive selection of diurnal minimum variation Y. Yuan et al.: Adaptive selection of diurnal minimum varia Figure 2. Detrended mean diurnal cycles of validated CO2 datasets (black) with 95 % confidence intervals (gray) from six GAW sta- tions (hours in LT). Measurements at HPB are differentiated by the sampling heights (e.g., HPB50 for 50 m a.g.l.). The covered time periods (top text), resulting start time windows (middle text, also in light blue shades), and mean diurnal amplitudes (bottom text) are shown in each subplot. 1507 Figure 3. Time series plots of validated CO2 datasets (gray), and selected datasets by ADVS (black) at six GAW stations. 3 i i l f lid d CO d ( ) Figure 2. Detrended mean diurnal cycles of validated CO2 datasets (black) with 95 % confidence intervals (gray) from six GAW sta- tions (hours in LT). Measurements at HPB are differentiated by the sampling heights (e.g., HPB50 for 50 m a.g.l.). The covered time periods (top text), resulting start time windows (middle text, also in light blue shades), and mean diurnal amplitudes (bottom text) are shown in each subplot. Figure 3. Time series plots of validated CO2 datasets (gray), and selected datasets by ADVS (black) at six GAW stations. on or later in the morning. 2.4 Seasonal-trend decomposition STL The start time window for SSL encompasses its diurnal maximum, indicating that data vari- ability is considerably smaller in the early morning than in the afternoon because of its vicinity to the Black Forest re- gion, which has strong influence due to local photosynthetic activity (Schmidt et al., 2003). A similar diurnal pattern can be found at SNB. The influence of CO2 sources is not as prominent as the effect of distant CO2 sinks, since it is sit- uated at the isolated summit peak of Hoher Sonnblick sur- rounded only by mountains and glaciers, with a negligibly small number of tourists, thus anthropogenic activities are minimal. IZO is a special case, since it is located on a re- mote mountain plateau on the Island of Tenerife above the strong subtropical temperature inversion layer. Even though the start time window is limited to 6 h, IZO presents an ideal mean diurnal cycle for data selection from a potentially much longer time window. 3.2 Percentage of selected data Starting from the initial start time windows, ADVS selected the baseline data for all stations (see Fig. 3). In addition, we calculated the percentages of the complete datasets se- lected by ADVS as baseline data, which are listed in the first column of Table 2. The higher the percentage the more well-mixed air is measured at the station, which is assumed to be a representation of lower free tropospheric conditions. This holds especially for IZO, where a larger percentage of 36.2 % was selected as baseline data. The sites with interme- diate percentages are JFJ (22.1 %), SNB (19.3 %), and ZSF (14.8 %). For the three sampling heights at HPB, only 3.2 % (50 m), 4.8 % (93 m), and 6.2 % (131 m) of the data were selected by ADVS. Finally, a similarly low percentage was found for SSL (4.0 %), probably due to its higher data vari- ability. In the right column of the figure, both ZSF and JFJ find their start time windows around midnight (including hours after midnight). ZSF shows higher diurnal CO2 amplitude than JFJ, but the two sites show similar diurnal patterns. For the choice of the start time window from the mean di- urnal variation, relatively close or even local anthropogenic sources may influence the CO2 at these two stations, possibly due to touristic influences. Table 2 clearly indicates that the percentage of baseline data increases with altitude for all methods, suggesting mea- surements at higher altitudes can capture progressively well- mixed and hence representative air. Based on this finding, a linear least squares regression was applied between the ab- Atmos. Meas. Tech., 11, 1501–1514, 2018 3.3.1 Trend component From the trend components, the mean annual growth rates were estimated by linear regression (see Table 3). Based on the 95 % confidence intervals for the slope, positive trends i.e., increasing CO2 concentrations are observed. Owing to the overlap of the confidence intervals, differences in the mean annual growth rates among VAL and selected datasets at the same station are all in good agreement. This indicates that the trend component is not significantly influenced by the statistical data selection method, which agrees well with the finding of Parrish et al. (2012) from a study of baseline ozone concentrations that there were no significant differ- ences of the long-term changes between the baseline and un- filtered datasets. Moreover, the following fact is observed for all sites except for SSL. Compared to unselected data (VAL), the mean annual growth rates based on selected datasets are systematically higher approaching the growth rates at IZO. IZO can be considered as better representing the lower free tropospheric conditions and agrees well with the mean an- nual global CO2 growth rates (2.31 ppm) during the same time period (2010–2015) based on data from https://www. esrl.noaa.gov/gmd/ccgg/trends/global.html. The exception at SSL is probably caused by stronger local influences as a re- sult of its lower elevation. In addition, the confidence inter- vals of the mean annual growth rates are always smaller after data selection, which improves the precision of trends. Since the percentages of selected data indicate not only the amount of data declared as representative but also show the characteristics of the selection methods, this criterion is used for further assessment. All other methods except for MA result in higher percentages for higher altitude stations (IZO, ZSF, SNB, and JFJ) than for those of lower altitudes (HPB and SSL). ADVS always performs the strictest filter- ing in all cases. Based on the stepwise study (see Supplement Sect. S3.2), these low percentages are primarily due to the restrictive definition of the start time window requiring data with a standard deviation of less than 0.3 ppm. With adap- tive selection, the percentages of selected data increase but remain lower than those of the other methods. SI and THO, in comparison, show differences between stations at high and low elevations. Compared with SI, THO is higher at stations at lower elevations, but lower at high ones. www.atmos-meas-tech.net/11/1501/2018/ Y. Yuan et al.: Adaptive selection of diurnal minimum variation 1508 stations at high elevations. At ZSF, SNB, and JFJ, it results in the second largest, and even the largest in the case of IZO. The highest percentages of selected data (approximately 80 %) were obtained with MA at most stations except for IZO. However, IZO obtains the largest percentages from all other selection methods. This is probably caused by the very low variability of CO2 at IZO, resulting in overly strict moving-average thresholds for the MA method. Thus, we conclude that MA does not work properly in the case of very well-mixed air (IZO). At all other stations, it is possible that MA declares too much data as representative. Therefore, MA was excluded from further analyses. Table 2. Percentage of selected data in all data by different data selection methods. The bottom shows the linear regression coeffi- cients of station (HPB is represented by HPB50; IZO is excluded) altitudes and the percentages of selected data at the significance level of 0.05 (∗∗∗). stations at high elevations. At ZSF, SNB, and JFJ, it results in the second largest, and even the largest in the case of IZO. Station ID ADVS SI THO MA HPB50 3.2 13.9 21.7 79.8 HPB93 4.8 18.5 25.0 79.4 HPB131 6.2 21.3 27.3 79.8 SSL 4.0 17.9 25.4 83.2 IZO 36.2 82.2 56.0 60.5 ZSF 14.8 47.1 40.8 79.0 SNB 19.3 58.7 44.2 76.9 JFJ 22.1 62.1 46.3 77.6 Linear regression 0.996∗∗∗ 0.992∗∗∗ 0.985∗∗∗ 0.645 coefficient (γ 2) 3.3 STL components STL was applied to the validated datasets before and after baseline selection with SI, THO, and ADVS, except for HPB due to its limited length of time (less than one year). Depend- ing on data availability, STL was performed on CO2 data from 2012 to 2015 at SNB, while data inputs at SSL, IZO, ZSF, and JFJ cover the whole period from 2010 to 2015. Fig- ure 4 gives an overview of the decomposition by STL. The following sections discuss the resulting components obtained by STL, namely the trend component, the seasonal compo- nent, and the remainder component. solute altitudes and the percentages of selected data for con- tinental stations. IZO is on a remote island and therefore not comparable. This approach reveals a significant positive lin- ear trend (see coefficient in Table 2). The related figure of linear regression can be found in Supplement Sect. S3.1. To examine the characteristic growth of the percentages of selected data by ADVS during the selection process, we ad- ditionally calculated percentages after completing both the starting selection and adaptive selection steps mentioned in Sect. 2.2 (see Supplement Sect. S3.2). All results of percent- ages show an order of stations similar to that above, and the percentages increase steadily step by step for all stations. The percentages of selected data by ADVS were then compared with those of the mentioned statistical data selection meth- ods SI, THO, and MA (see Table 2, with the corresponding figure shown in Supplement Sect. S3.3). Y. Yuan et al.: Adaptive selection of diurnal minimum variation 1509 Figure 4. STL decomposition results from VAL (black), SI-selected (brown), THO-selected (yellow), and ADVS-selected (green) datasets at five GAW stations. Figure 4. STL decomposition results from VAL (black), SI-selected (brown), THO-selected (yellow), and ADVS-selected (green) datasets at five GAW stations. esults from VAL (black), SI-selected (brown), THO-selected (yellow), and ADVS-selected (green) datasets Higher values in the summer months from May to September explain underestimation of VAL due to intensified upward transport of photosynthetic signatures resulting from vege- tation. Similar patterns can be found at stations SSL, SNB, and JFJ (see Supplement Sect. S4). IZO always shows the smallest seasonal amplitude and there is almost no difference between VAL and selected datasets. Based on this consider- ation, it is very likely that the lower free troposphere will react with a delay to CO2 concentration changes of effective sources and sinks on the ground, acting like an atmospheric memory. Table 3. Mean annual growth rates (ppm yr −1) with 95 % confi- dence intervals from linear regression, applied on the trend com- ponents by STL over 2010 to 2015, except for SNB. Data at SNB were decomposed over 2012 to 2015 due to missing data from 2010 to 2011 and thus shown in italic font. Table 3. Mean annual growth rates (ppm yr −1) with 95 % confi- dence intervals from linear regression, applied on the trend com- ponents by STL over 2010 to 2015, except for SNB. Data at SNB were decomposed over 2012 to 2015 due to missing data from 2010 to 2011 and thus shown in italic font. Station VAL SI THO ADVS ID SSL 2.04 ± 0.09 1.89 ± 0.06 2.04 ± 0.06 2.03 ± 0.09 IZO 2.24 ± 0.03 2.26 ± 0.02 2.25 ± 0.02 2.25 ± 0.02 ZSF 2.13 ± 0.08 2.16 ± 0.05 2.17 ± 0.06 2.19 ± 0.06 SNB 2.02 ± 0.07 2.06 ± 0.06 2.06 ± 0.06 2.08 ± 0.04 JFJ 2.13 ± 0.03 2.15 ± 0.02 2.14 ± 0.02 2.14 ± 0.02 y A time delay of one month in the mean seasonal maxi- mum is shown in Fig. 5a at ZSF with selected datasets by SI and ADVS (March), compared with the maximum from the validated data (February). Y. Yuan et al.: Adaptive selection of diurnal minimum variation A similar time shift can also be found by other selection methods at stations SSL (one- month delay from February to March by SI and ADVS) and JFJ (two-month delay from February to April by SI, THO, and ADVS). As for station IZO (April) in Fig. 5b and station SNB (March), the seasonal maxima stay the same. The mag- nitude of these delays may be related to mixing in the lower free troposphere. Rapid changes are usually observed close to sources and sinks, e.g., from anthropogenic and biogenic activities. Thus, the higher the station is above the bound- ary layer, the later the maxima during the winter can be ob- served because of the late response due to inhibited mixing. However, this delay does not occur for the minima during the summer because of the very effective upward transport and more favorable mixing conditions at that time of year. Consequently, no change in the seasonal minima is observed at all measurement sites, which is taken as an indicator of enhanced thickness of the mixing layer as good mixing con- ditions. Taking ZSF as an example, Birmili et al. (2009) ob- served low concentrations of particle numbers in winter and 3.3.1 Trend component A major limita- tion of SI seems to be the requirement for consecutive hours, in our case of 6 h with 0.3 ppm standard deviation threshold, which might be too restrictive for stations at lower elevations. However, this criterion results in a fairly large percentage for Atmos. Meas. Tech., 11, 1501–1514, 2018 www.atmos-meas-tech.net/11/1501/2018/ Y. Yuan et al.: Adaptive selection of diurnal minimum variation Atmos. Meas. Tech., 11, 1501–1514, 2018 www.atmos-meas-tech.net/11/1501/2018/ Y. Yuan et al.: Adaptive selection of diurnal minimum variation Y. Yuan et al.: Adaptive selection of diurnal minimum variation Y. Yuan et al.: Adaptive selection of diurnal minimum variation 1510 Figure 5. Mean monthly variation of the seasonal component decomposed by STL at (a) ZSF and (b) IZO over the whole period. For a better visualization of the results of selection methods, dots have been separated horizontally and equidistantly. The 95 % confidence intervals are shown as error bars. Figure 5. Mean monthly variation of the seasonal component decomposed by STL at (a) ZSF and (b) IZO over the whole period. For a better visualization of the results of selection methods, dots have been separated horizontally and equidistantly. The 95 % confidence intervals are shown as error bars. Table 4. Standard deviations of the remainder components by STL over 2010 to 2015, except for SNB. Data at SNB were decomposed over 2012 to 2015 due to missing data from 2010 to 2011 and thus shown in italic font. Table 4. Standard deviations of the remainder components by STL over 2010 to 2015, except for SNB. Data at SNB were decomposed over 2012 to 2015 due to missing data from 2010 to 2011 and thus shown in italic font. found it representative for the free tropospheric air by ana- lyzing the annual and diurnal cycles. From spring onwards, the PBL rises with increasing temperatures. The intense ver- tical atmospheric exchange during summer months results in a daily air mass transport from the boundary layer to reach ZSF due to thermal convection (Reiter et al., 1986; Birmili et al., 2009). Thus there are optimal transport and mixing con- ditions. Therefore after data selection, the timing of seasonal peaks corresponds better among the stations. Station ID VAL SI THO ADVS SSL 1.61 1.16 1.26 1.99 IZO 0.34 0.33 0.30 0.30 ZSF 0.89 0.75 0.72 0.73 SNB 0.66 0.56 0.55 0.70 JFJ 0.56 0.45 0.48 0.47 3.3.3 Remainder component The remainder component resembles random noise from lo- cal influences in its structure, being different from site to site and statistically uncorrelated with the general signal of CO2 concentrations in the lower free troposphere (Thoning et al., 1989). The standard deviation of the remainder component is taken here as a measure for external influences (see Fig. 4). Table 4 shows the calculated standard deviations from the re- mainder components at each station. Comparable results are derived from all selected datasets. SSL, as the lowest altitude station, exhibits the largest variation. IZO with the smallest standard deviations in the remainder component proves to be the station least influenced by its surrounding environment. The three alpine measuring stations (ZSF, SNB, and JFJ) ex- hibit intermediate variability. From this perspective, STL per- forms well in showing the site characteristics. Consequently, the noise of the remainder components, given in Table 4, de- creases with increasing altitude of the continental mountain stations, which is in inverse relation to the percentages of se- lected data (Table 2). IZO was excluded in both regressions against altitude because of its maritime character. 3.3.2 Seasonal component The resulting seasonal components show systematic differ- ences between VAL and selected datasets. The mean monthly variations were calculated on a monthly scale over the en- tire period from the analyzed data. Figure 5a and b present the results at stations ZSF and IZO. At most stations (except for IZO), the seasonal amplitudes have been substantially re- duced compared to VAL (see also Fig. 4). At ZSF, the aver- aged peak-to-peak seasonal amplitude, defined as mean sea- sonal maximum minus seasonal minimum, drops the most by 18.9 % from VAL with the ADVS selected dataset. An expla- nation of this reduction is CO2 signal exclusion from local sources and sinks by data selection. When taking a closer look at the monthly averages, lower CO2 values are found in the selected datasets in the winter months from October to April, indicating that the CO2 concentrations estimated by VAL are above the background levels because of more dominant anthropogenic activities and no active vegetation. Atmos. Meas. Tech., 11, 1501–1514, 2018 www.atmos-meas-tech.net/11/1501/2018/ www.atmos-meas-tech.net/11/1501/2018/ Y. Yuan et al.: Adaptive selection of diurnal minimum variation Figure 6. Pearson’s correlation matrices of combinations of trend and seasonal components (T +S, a), and only remainder components (R, b) at stations SSL, IZO, ZSF, SNB, and JFJ by different selection methods. Correlations with no significant coefficients at the 0.05 significance level were left blank. Figure 6. Pearson’s correlation matrices of combinations of trend and seasonal components (T +S, a), and only remainder components (R, b) at stations SSL, IZO, ZSF, SNB, and JFJ by different selection methods. Correlations with no significant coefficients at the 0.05 significance level were left blank. Figure 6. Pearson’s correlation matrices of combinations of trend and seasonal components (T +S, a), and only remainder components (R, b) at stations SSL, IZO, ZSF, SNB, and JFJ by different selection methods. Correlations with no significant coefficients at the 0.05 significance level were left blank. with selected data irrespective of the selection method, es- pecially between the three Alpine stations (ZSF, SNB, and JFJ). This evaluation shows a similar result to the method presented by Sepúlveda et al. (2014) for identifying base- line conditions based on the correlation between distant mea- suring stations. Pairs including IZO after data selection by ADVS show a notable increase in the correlation coefficients, meaning better coherence between the reference station IZO and the others. data selection procedure, we applied the method to six CO2 datasets measured at GAW mountain stations in the Euro- pean Alps. The ADVS resulted in an increasing number of percentages of selected data representing the background conditions with growing altitude of continental measurement sites, which is reasonable due to the underlying atmospheric dynamics. For comparison, three well-known statistical data selection methods were applied to the same datasets and most methods yielded similar increasing percentages with growing altitude. Among all the methods, ADVS is the most restric- tive in terms of the number of selected data in the overall datasets. Conversely, when selecting representative data more effec- tively, the results should contain less local and regional influ- ences. Therefore, we compared the remainder components derived from STL pairwise to check whether the Pearson correlation coefficients decreased after data selection (see Fig. 6b). The number of insignificant correlations between the station pairings is the greatest for ADVS. Y. Yuan et al.: Adaptive selection of diurnal minimum variation For the only two coefficients significant at the 0.05 significance level (ZSF- SNB and ZSF-JFJ), they drop largely from 0.75 to 0.48, and from 0.75 to 0.40, respectively, which cannot be observed by the other selection methods. This means that by ADVS the combination of trend and seasonal components correlate best and the remaining unselected data have the lowest correlation among the methods. If these two criteria are used to separate the representative part of the data from the unrepresentative part, the ADVS method produces the best results. In addition, we applied the time series decomposition method STL to all datasets before and after data selec- tion. All statistical data selection methods resulted in the same annual trend within the 95 % confidence interval of the datasets before selection, while the seasonal signal var- ied substantially with smaller seasonal amplitudes and de- layed occurrences of seasonal maxima. We also presented an additional assessment of ADVS compared with the other sta- tistical data selection methods based on correlation analysis. For the combination of trend and seasonal components by STL, higher correlation coefficients between stations were found with ADVS data selection than SI and THO. Inversely, ADVS resulted in lower correlation coefficients in the re- mainder components than the other methods. Both indicate a better performance of selecting baseline data by ADVS. 3.4 Correlation analysis As mentioned above, data selection is defined here as an ap- proach of extracting a group of data to be the best repre- sentative for the lower free troposphere. Consequently, the selected CO2 datasets should have properties that are well correlated between the sites. For evaluating this hypothesis, we took the combination of the trend and seasonal compo- nents from STL and examined the correlations between each pair of stations in a Pearson correlation matrix (see Fig. 6a). The trend and seasonal components of all VAL and selected datasets were first compiled, and then Pearson’s correlation coefficients were calculated assuming normal distribution of data examined by the Anderson–Darling test (P < 0.05). The correlation matrices are shown for each data selection method individually, in order to enable a comparison be- tween ADVS and other methods. Data used for correlation were chosen only when available at all stations (2012–2015). In general, most pairs show higher correlation coefficients Atmos. Meas. Tech., 11, 1501–1514, 2018 www.atmos-meas-tech.net/11/1501/2018/ 1511 Atmos. Meas. Tech., 11, 1501–1514, 2018 4 Conclusions and outlook The presented method is useful for data selection of at- mospheric CO2 data representative of the lower free tro- posphere. It requires only data from a single measurement site, is easily adjustable to the local conditions, and runs We presented the novel statistical ADVS method for se- lecting representative baseline data for CO2 measurements at elevated GAW mountain stations. For assessment of the Atmos. Meas. Tech., 11, 1501–1514, 2018 www.atmos-meas-tech.net/11/1501/2018/ The Supplement related to this article is available online at https://doi.org/10.5194/amt-11-1501-2018-supplement. Brailsford, G. W., Stephens, B. B., Gomez, A. J., Riedel, K., Mikaloff Fletcher, S. E., Nichol, S. E., and Manning, M. R.: Long-term continuous atmospheric CO2 measurements at Bar- ing Head, New Zealand, Atmos. Meas. Tech., 5, 3109–3117, https://doi.org/10.5194/amt-5-3109-2012, 2012. Brooks, B.-G. J., Desai, A. R., Stephens, B. B., Bowling, D. R., Burns, S. P., Watt, A. S., Heck, S. L., and Sweeney, C.: Assess- ing filtering of mountaintop CO2 mole fractions for application to inverse models of biosphere-atmosphere carbon exchange, At- mos. Chem. Phys., 12, 2099–2115, https://doi.org/10.5194/acp- 12-2099-2012, 2012. Competing interests. The authors declare that they have no conflict of interest. Acknowledgements. This work was supported by a scholar- ship from China Scholarship Council (CSC) under grant CSC No. 201508080110. This work was supported by a MICMoR Fellowship through KIT/IMK-IFU to Ye Yuan. This work was supported by the German Research Foundation (DFG) and the Technical University of Munich (TUM) in the framework of the Open Access Publishing Program. The CO2 measurements at Zugspitze and Schauinsland were supported by the German Environment Agency (UBA). We thank Markus Wallasch for providing CO2 data obtained at Schauinsland and Ralf Sohmer for technical support. The CO2 measurements at Hohenpeissenberg were conducted by the German Meteorological Service within the ICOS Atmospheric Station Network. The CO2 measurements at Jungfraujoch were supported by the Swiss Federal Office for the Environment, ICOS-Switzerland, and the International Foundation High Alpine Research Stations Jungfraujoch and Gornergrat. Martin Steinbacher acknowledges funding from the GAW Quality Assurance/Science Activity Centre Switzerland (QA/SAC-CH), which is supported by MeteoSwiss and Empa. The Izaña (IZO) CO2 measurements were performed within the GAW Program Calvert, J. G.: Glossary of atmospheric chem- istry terms, Pure Appl. Chem., 62, 2167–2219, https://doi.org/10.1351/pac199062112167, 1990. Carnuth, W. and Trickl, T.: Transport studies with the IFU three-wavelength aerosol lidar during the VOTALP Mesolcina experiment, Atmos. Environ., 34, 1425–1434, https://doi.org/10.1016/S1352-2310(99)00423-9, 2000. 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The results provide evidence that the proposed ADVS method confers the possibility of selecting data that are representative of CO2 concentrations of a larger area of the lower free troposphere. This is an elementary prerequi- site for application of the method to a larger number of dif- ferent stations and an essential step towards generalization. It directly supports the objective of GAW to extrapolate from a set of point measurements from single stations to a larger representative area or region in the lower free troposphere (WMO, 2017). In future, there is a need to test whether such results could be used for additional applications, such as ground calibration of satellite measurements. Finally, it would be very interesting to test as a next step whether this presented method is applicable to stations in other regions and on other continents. Moreover, the issue of whether and how to include coastal stations in a systematic and practi- cally generalizable approach for selecting representative data at GAW stations will be a particular concern. at the Izaña Atmospheric Research Center, financed by AEMET. Finally, we also thank Wolfgang Spangl from the Austrian Envi- ronment Agency (UBA-At) for providing CO2 data obtained at Sonnblick. This work was supported by the German Research This work was supported by the German Research Foundation (DFG) and the Technische Universität München within the funding programme This work was supported by the German Research Foundation (DFG) and the Technische Universität München within the funding programme München within the funding programme Open Access Publishing. Open Access Publishing. 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https://genomebiology.biomedcentral.com/track/pdf/10.1186/s13059-019-1855-4
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Common DNA sequence variation influences 3-dimensional conformation of the human genome
Genome biology
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Gorkin et al. Genome Biology (2019) 20:255 https://doi.org/10.1186/s13059-019-1855-4 RESEARCH Open Access Common DNA sequence variation influences 3-dimensional conformation of the human genome David U. Gorkin1,2†, Yunjiang Qiu1,3†, Ming Hu4*†, Kipper Fletez-Brant5,6, Tristin Liu1, Anthony D. Schmitt1,7, Amina Noor2, Joshua Chiou8,9, Kyle J. Gaulton8, Jonathan Sebat2,10, Yun Li11, Kasper D. Hansen5,6 and Bing Ren1,2,12* Abstract Background: The 3-dimensional (3D) conformation of chromatin inside the nucleus is integral to a variety of nuclear processes including transcriptional regulation, DNA replication, and DNA damage repair. Aberrations in 3D chromatin conformation have been implicated in developmental abnormalities and cancer. Despite the importance of 3D chromatin conformation to cellular function and human health, little is known about how 3D chromatin conformation varies in the human population, or whether DNA sequence variation between individuals influences 3D chromatin conformation. Results: To address these questions, we perform Hi-C on lymphoblastoid cell lines from 20 individuals. We identify thousands of regions across the genome where 3D chromatin conformation varies between individuals and find that this variation is often accompanied by variation in gene expression, histone modifications, and transcription factor binding. Moreover, we find that DNA sequence variation influences several features of 3D chromatin conformation including loop strength, contact insulation, contact directionality, and density of local cis contacts. We map hundreds of quantitative trait loci associated with 3D chromatin features and find evidence that some of these same variants are associated at modest levels with other molecular phenotypes as well as complex disease risk. Conclusion: Our results demonstrate that common DNA sequence variants can influence 3D chromatin conformation, pointing to a more pervasive role for 3D chromatin conformation in human phenotypic variation than previously recognized. Introduction Three-dimensional (3D) organization of chromatin is essential for proper regulation of gene expression [1–3] and plays an important role in other nuclear processes including DNA replication [4, 5], X chromosome inactivation [6–9], and DNA repair [10, 11]. Many recent insights about 3D chromatin conformation have been enabled by a suite of technologies based on Chromatin Conformation Capture (3C) [12]. A high-throughput * Correspondence: hum@ccf.org; biren@ucsd.edu † David U. Gorkin, Yunjiang Qiu and Ming Hu contributed equally to this work. 4 Department of Quantitative Health Sciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA 1 Ludwig Institute for Cancer Research, La Jolla, CA, USA Full list of author information is available at the end of the article version of 3C called “Hi-C” enables the mapping of 3D chromatin conformation at genome-wide scale [13] and has revealed several key features of 3D chromatin conformation including (1) compartments (often referred to as “A/B compartments”), which refer to the tendency of loci with similar transcriptional activity to physically segregate in 3D space [13–15]; (2) chromatin domains (often referred to as Topologically Associating Domains, or TADs) demarcated by boundaries across which contacts are relatively infrequent [16–18]; (3) chromatin loops, which describe point-to-point interactions that occur more frequently than expected based on the linear distance between interacting loci, and often anchored by convergent CTCF motif pairs [14]; and (4) Frequently Interacting Regions (FIREs), which are regions of © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gorkin et al. Genome Biology (2019) 20:255 increased local interaction frequency enriched for tissuespecific genes and enhancers [19, 20]. Previous studies have used Hi-C to profile 3D chromatin conformation across different cell types [14, 16, 21], different primary tissues [19], different cell states [22], and in response to different genetic and molecular perturbations [23–27], producing a wealth of knowledge about key features of 3D chromatin conformation. However, to our knowledge, no study to date has measured variation in 3D chromatin conformation across more than a handful of unrelated individuals. Several observations demonstrate that at least in some cases DNA sequence variation between individuals can alter 3D chromatin organization with pathological consequences [28]. Pioneering work by Mundlos and colleagues described several cases in which rearrangements of TAD structure lead to gene dysregulation and consequent developmental malformations [29, 30]. In cancer, somatic mutations and aberrant DNA methylation can disrupt TAD boundaries leading to dysregulation of protooncogenes [31, 32]. Moreover, many genetic variants associated with human traits by Genome-Wide Association Studies (GWAS) occur in distal regulatory elements that loop to putative target gene promoters in 3D, and in some cases, the strength of these looping interactions has been shown to vary between alleles of the associated SNP [33, 34]. Although these studies demonstrate that both large effects as well as more subtle aberrations of 3D chromatin conformation are potential mechanisms of disease, population-level variation in 3D chromatin conformation more broadly has remained unexplored. In the present study, we set out to characterize interindividual variation in 3D chromatin conformation by performing Hi-C on lymphoblastoid cell lines (LCLs) derived from individuals whose genetic variation has been cataloged by the HapMap or 1000 Genomes Consortia [35]. LCLs have been used as a model system to study variation in several other molecular phenotypes including gene expression, histone modifications, transcription factor (TF) binding, and chromatin accessibility [36–42]. These previous efforts provide a rich context to explore variation in 3D chromatin conformation identified in this model system. Through integrative analyses, we found that inter-individual variation in 3D chromatin conformation occurs on many levels including compartments, TAD boundaries, FIREs, and looping interactions. Moreover, we found that variation in 3D chromatin conformation coincides with variation in activity of the underlying genome sequence as evidenced by transcription, histone modifications, and TF binding. Although our sample size is small, we observed reproducible effects of DNA sequence variation on 3D chromatin conformation and identify hundreds of quantitative trait loci (QTLs) associated Page 2 of 25 with features of 3D chromatin conformation. Our results demonstrate that variation in 3D chromatin conformation is readily detectable from Hi-C data, often overlaps with regions of transcriptomic and epigenomic variability, and is influenced in part by genetic variation that may contribute to disease risk. Results Mapping 3D chromatin conformation across individuals To generate maps of 3D chromatin conformation suitable for comparison across individuals, we performed “dilution” Hi-C on LCLs derived from 13 Yoruban individuals (including one trio), one Puerto Rican trio, and one Han Chinese trio (19 individuals total; Additional file 2: Table S1). We also included published HiC data from one European LCL (GM12878) generated previously by our group using the same protocol [43], for a total of 20 individuals from four different populations. Many of these same LCLs have been used in previous genomic studies [38, 40, 42], allowing us to leverage multiple transcriptomic and epigenomic datasets in our analysis below (Additional file 2: Table S1). Importantly, 18 of these individuals have had their genetic variation cataloged by the 1000 Genomes Consortium [35, 44] (Additional file 2: Table S2), which allowed us to examine the influence of genetic variation on 3D chromatin conformation. Two replicates of Hi-C were performed on each LCL, with each replicate performed on cells grown independently in culture for at least two passages (Additional file 2: Table S3). All Hi-C data were processed using a uniform pipeline that incorporates the WASP approach [40, 45] to eliminate allelic mapping biases (see the “Alignment with WASP” section). For each sample, we mapped a series of well-established HiC-derived features including 40-Kb resolution contact matrices, compartmentalization [13], directionality index (DI) [16], and insulation score (INS) [7] (Fig. 1a; Additional file 1: Figure S1a-c). Compartmentalization is standardly measured by the first principal component (PC1) of Hi-C contact matrices, and thus, we use the acronym “PC1” below to refer to this measure of compartmentalization. DI and INS are both related to TAD boundaries (Additional file 3: Table S4), but they represent different quantitative phenotypes. INS measures the abundance of interactions spanning a given region of interest, whereas DI measures imbalance in the directionality of contacts from a given region of interest (i.e., upstream or downstream). TAD boundaries show a signature of strong insulation reflected by low INS values, as well as an abrupt shift from strong upstream to strong downstream DI. However, strong DI values can occur elsewhere in the genome. We also identified FIREs and their corresponding “FIRE scores” [19], which measure how frequently a given region interacts with its neighboring regions (15~200 Kb). The concept of FIRE is based on the Gorkin et al. Genome Biology (2019) 20:255 Page 3 of 25 Fig. 1 Biological variability in multiple aspects of 3D chromatin conformation. a Browser view to illustrate the Hi-C-derived molecular phenotypes examined here: contact matrices, FIRE, DI, INS, and PC1 (chr8:125,040,000-132,560,000; hg19). Only 4 individuals shown here for illustrative purposes. The full set of individuals is shown in Additional file 1: Figure S1. b Boxplots show correlation between biological replicates from the same cell line (Individuals = “within”, N = 20), and between replicates from different cell lines (Individuals = “between”, N = 760). Statistical significance calculated by two-sided Wilcoxon rank-sum test. See Additional file 1: Figure S4a for schematic of shuffling strategy. These and all boxplots in this manuscript show median as a horizontal line, interquartile range (IQR) as a box, and the most extreme value within 1.5*IQR or − 1.5*IQR as whiskers extending above or below the box, respectively. c The Pearson correlation coefficient between quantile normalized Hi-C matrix replicates from the same cell line (solid line) or different cell lines (dotted line) is plotted as a function of genomic distance between anchor bins. Distances between 0.1 and 1 Mb are highlighted in the magnified sub-panel to the right. d Significance of the difference between the “within” and “between” values in c was calculated at multiple points along the distance-correlation curve by two-sided Wilcoxon rank-sum test. Note that the scale of contact distance here is linear. Yellow bars indicate significance exceeding a Bonferroni-corrected significance threshold (dotted line) observation that the frequency of contacts in this distance range is not evenly distributed across the genome, but rather, tends to peak in regions showing epigenomic signatures of transcriptional and regulatory activity (Additional file 1: Figure S1d-e, S2). As we have shown previously [19, 46], FIRE regions often overlap putative enhancer elements (Additional file 1: Figure S1d-e). We did not call “chromatin loops” in this study because our data was not of sufficient resolution, but we used a set of loops previously reported in the LCL GM12878 [14] Gorkin et al. Genome Biology (2019) 20:255 to examine variation in loop strength among the LCLs in our study. Aggregate analysis shows that these published LCL loops are generally reproduced in our data (Additional file 1: Figure S3). 3D chromatin conformation variations between individuals After uniformly processing all Hi-C data (see the “Hi-C data processing” section), we compared chromatin conformation across LCLs at the level of contact matrices and multiple derived features (PC1, DI, INS, and FIRE). From a genome-wide perspective, each of these 3D chromatin features shows a signature consistent with reproducible inter-individual variation whereby replicates from the same individual (i.e., same LCL) are more highly correlated than datasets from different individuals (PC1 p = 3.5e−12, INS p = 5.6e−9, DI p = 2.1e−8, FIRE p = 2.6e−4 by two-sided Wilcoxon rank-sum test; Fig. 1b–d, Additional file 1: Figure S4a-e). The Hi-C data also cluster by population (Additional file 1: Figure S4f-g). We note that this inter-individual and population variation is consistent with an influence from genetic background, but can also be caused by other factors such as sample acquisition [47], batch effects, and other differences between the LCLs. Despite generally high correlations of Hi-C data across individuals, we frequently observed regions where 3D chromatin conformation varies reproducibly between individuals (example shown in Fig. 2a, Additional file 1: Figure S5a). To more systematically identify regions of variable 3D chromatin conformation, we used a linear normal model with an empirical Bayes variance estimator (as implemented in the “eBayes” function in limma [48]) to identify regions where variation between individuals was significantly higher than variation between two replicates from the same individual. We applied this approach to DI, INS, FIRE, and PC1. For each metric, we first defined a set of testable 40-Kb bins across the genome by filtering out bins with low levels of signal across all individuals or near structural variants that can appear as aberrations in Hi-C maps [49] (see the “F test for variable bins” section). We then applied a false discovery rate (FDR) threshold of 0.1 and merged neighboring variable bins, resulting in the identification of 2318 variable DI regions, 2485 variable INS regions, 1996 variable FIRE regions, and 7732 variable PC1 regions (Fig. 2b, Additional file 1: Figure S5b, Additional file 4: Table S5). We note that there is strong overlap between the variable DI, INS, FIRE, and PC1 regions detected across all 20 LCLs and those detected using only the 11 unrelated YRI LCLs, which suggests that potential confounding effects of variation between different populations are not driving the identification of these variable regions (Additional file 1: Figure S5c). Although each metric has Page 4 of 25 a unique set of testable bins, we found significant enrichment for bins that are variable in more than one metric (Fig. 2c, Additional file 1: Figure S5d), indicating that the same regions often vary across multiple features of 3D chromatin conformation. We next used fluorescent in situ hybridization (FISH) to examine whether variable regions detected by Hi-C are consistent with distance measurements from imaging data. Focusing on a variable DI region on chromosome 15 (Fig. 2a, Additional file 1: Figure S6a-b), we performed FISH in LCLs from four individuals that showed different levels of DI at the variable region being evaluated (YRI-4 and YRI-8 showing high upstream contact bias; YRI-3 and YRI-5 showing no upstream contact bias). We used three BAC probes that hybridize respectively to the variable DI region (“center”, probe covers chr15:96715965-96898793), a region approximately 668 Kb upstream (“upstream”, probe covers chr15: 95897555-96047720), or a region approximately 590 Kb downstream (“downstream”, probe covers chr15: 97488414-97648104). We found that distances between the center probe and flanking probes vary significantly between individuals with strong upstream contact bias as measured by DI and individuals without this upstream contact bias (Fig. 2d, e, center-upstream distance p = 0.017, center-downstream distance p = 1.7e−5 by two-sided Wilcoxon rank-sum test). In the two individuals with strong upstream DI signal at the central variable DI region, we found that the center probe is closer to the upstream than the downstream probe, as expected (p = 3.2e−3 for YRI-3, p = 1.5e−4 for YRI-5 by one-sided Wilcoxon rank-sum test). However, in individuals without upstream DI signal, the center probe is closer to the downstream probe (p = 0.021 for YRI-4, p = 0.1 for YRI8 by one-sided Wilcoxon rank-sum test) (Additional file 1: Figure S6c). We also sought to identify variable entries in the Hi-C contact matrix itself (“Hi-C contact matrix cells”). To facilitate this search, we used a method called Bandwise Normalization and Batch Correction (BNBC) that we recently developed to normalize Hi-C data across individuals (Fletez-Brant et al. Pre-print: https://doi.org/10. 1101/214361). BNBC takes contact distance into account as a covariate because batch effects in Hi-C data can be distance-dependent. To identify variable matrix cells, we performed a variance decomposition on Hi-C contact matrix cells, resulting in a measure of biological variability for each bin in the contact matrix (see example in Fig. 2a and Additional file 1: Figure S5a). To identify matrix cells with significant levels of biological variability, we estimated FDR using the IHW framework [50] to include the distance between anchor bins as an informative covariate. At an FDR threshold of 0.1, we identified 115, 817 matrix cells showing significant variability between Gorkin et al. Genome Biology (2019) 20:255 Page 5 of 25 Fig. 2 Variable regions of 3D chromatin conformation. a Example of a variable region (chr15:93,040,000-100,560,000; hg19). Triangular heatmaps from top to bottom: Four Hi-C contact heatmaps from individuals showing variable 3D chromatin architecture, a heatmap in blue showing the degree of variation measured across LCLs, and a second heatmap in blue showing variable cells in the matrix at IHW-FDR < 0.1 (var = variable, ns = not significant). Standard tracks from top to bottom (zoomed in on chr15:95,482,152-98,025,591; hg19): BAC probes used for FISH experiment in panels d and e, variable DI regions called using all 20 LCLs, variable DI regions called using just 11 YRI LCLs, 12 DI tracks from four different individuals. For each individual, DI tracks are shown from two biological replicates and from Hi-C data merged across both replicates. As indicated to the right of the tracks, two individuals have strong upstream contact skew in the boxed regions (YRI-4, YRI-8), while the other two individuals have weak or no upstream contact skew in that region (YRI-3, YRI-5). b The number of testable bins and significantly variable regions for each 3D chromatin phenotype examined here. c Significance of pairwise overlap between different sets of variable regions. p values calculated by chisquare test. Additional details in the “Methods” section and Additional file 1: Figure S5 and S6. d Boxplots showing the distance between indicated probe sets in four different LCLs. Probe labels same as in panel a. p values calculated by two-sided Wilcoxon rank-sum test. Number of nuclei measured for each LCL and probe pair, from left to right, are 140, 91, 111, 70, 128, 124, 219, 70. e Representative images of nuclei corresponding to panel d. f Blue line shows the fraction of variable matrix cells distributed across a range of interaction distances. Black shows the fraction of all matrix cells distributed across the same range of interaction distances. g Top panel shows the percentage of all variable matrix cell anchor bins that overlap variable INS, FIRE, INS, or PC1 regions, respectively. Middle panel is similar to top, but only including variable matrix cell anchor bins that were also tested for INS, FIRE, INS, or PC1 variability. The shade of blue is scaled with overlap percentage. Bottom panel shows the statistical significance of overlaps as calculated by chi-square test, and plotted with same color scale as (c) Gorkin et al. Genome Biology (2019) 20:255 samples (Additional file 5: Table S6). These variable bins are skewed toward shorter contact distances (Fig. 2f, Additional file 1: Figure S6d-e), likely due in part to higher read counts and thus increased power to detect biological variability at these distances. We observed that the anchor regions of variable matrix cells overlap with variable regions of DI, INS, FIRE, and PC1 more often than would be expected by chance (Fig. 2g; Additional file 1: Figure S6f). We also found that variable matrix cells tend to occur in groups (Figs. 2a and 3a; Additional file 1: Figure S6 g), suggesting that variation in 3D chromatin conformation often affects more than one adjacent genomic window. We did not find evidence that variable matrix cells are skewed toward interTAD or inter-compartment interactions (Additional file 1: Figure S6 h-i). Coordinated variation of the 3D genome, epigenome, and transcriptome To investigate the relationship between variation in 3D chromatin conformation and gene regulation, we analyzed multiple published datasets including RNA-seq, ChIP-seq, and DNase-seq data generated from some of the same LCLs in our study (Additional file 2: Table S2). Strikingly, for all external datasets examined here, we see an enrichment for regions where 3D chromatin conformation across individuals is correlated with measures of genome activity in the same 40-Kb bin (see example in Fig. 3a and Additional file 1: Figure S7a). To assign a level of statistical significance to these observations, we approximated the null distribution by randomly permuting the sample labels of external datasets, thus disrupting the link between Hi-C and ChIP/RNA/DNase-seq data from the same individual, but not changing the underlying data structure (see schematic in Additional file 1: Figure S7b). We used these permutations to calculate the bootstrap p values in Fig. 3b and Additional file 1: Figure S8. Among variable PC1 regions, we observed a significant enrichment for regions at which PC1 values across individuals are positively correlated with histone modifications indicative of transcriptional activity including H3K27ac, H3K4me1, and H3K4me3 (Bootstrap p < 0.0001; Fig. 3b). The correlations between PC1 and marks of transcriptional activity occur in the expected direction, i.e., higher PC1 values are associated with higher gene expression and more active histone modifications. Similar correlations were apparent in two distinct sets of ChIP-seq data generated by different groups [40, 42], and observed whether we use variable regions identified across all 20 LCLs or only across the 11 unrelated YRI LCLs (Additional file 1: Figure S8). At variable FIRE regions, we similarly found an abundance of regions where FIRE score is positively correlated with marks of cis-regulatory activity including H3K27ac and H3K4me1 (Bootstrap p < 0.0001; Fig. 3b, Additional file 1: Page 6 of 25 Figure S8), consistent with the previously reported relationship between FIREs and cis-regulatory activity [19, 46]. At variable DI and INS regions, these metrics tend to be correlated with histone modification levels as well as CTCF and Cohesin subunit SA1 binding (Bootstrap p < 0.0001; Fig. 3b, Additional file 1: Figure S8), which are known to influence these 3D chromatin features [16, 51, 52]. For INS, the relationship is directional as expected such that higher CTCF/ Cohesin binding corresponds to more contact insulation (i.e., lower INS score). However, at variable DI regions, the correlations are not as clearly directional, consistent with current understanding that the direction of DI (i.e., upstream vs downstream contact bias) is arbitrary relative to strength of CTCF/Cohesin binding. We performed similar analysis on variable cells in the contact matrix and found that the interaction frequency in these matrix cells tends to be correlated with epigenetic or transcriptional properties of one or both corresponding “anchor” bins (Bootstrap p < 0.0001; Fig. 3b, Additional file 1: Figure S8). Importantly, for all types of variable regions examined here, we found correlation with RNA-seq signal across individuals, indicating that at least in some regions, variation in 3D chromatin features accompanies variation in gene expression. We examined further whether 3D chromatin conformation at a given variable region tends to be correlated with a single epigenomic property, or with several properties simultaneously. We found that PC1, FIRE, INS, and DI values across individuals are often correlated with multiple features of active regions (e.g., H3K27ac, H3K4me1, RNA), and anti-correlated with the repressive H3K27me3 histone modification (Fig. 3c, d). For DI and INS, where numerical direction of the score is not as clearly linked to magnitude of gene regulatory activity, we note a larger set of regions with anti-correlation to features of active regions (e.g., H3K27ac, H3K4me1, RNA) and positive correlation with H3K27me3 (Fig. 3e, f). These results demonstrate that variation in 3D chromatin conformation is often accompanied by variation in transcriptional and regulatory activity of the same region. Genetic loci influencing 3D chromatin conformation To examine genetic influence on 3D chromatin conformation, we first considered genetic variants overlapping CTCF motifs at chromatin loop anchors [14], because disruption of these CTCF motifs by genome engineering has been shown to alter chromatin looping [23]. We focused on SNPs at key positions in anchor CTCF motifs (“motifs disrupting SNPs,” defined as SNPs in positions in the CTCF Position Weight Matrix (PWM) where a single base has a probability of > 0.75, Fig. 4a). We observed a significant linear relationship between SNP genotype and the strength of corresponding loops (p = 7.6e−5 by linear regression; Fig. 4b, c). We Gorkin et al. Genome Biology (2019) 20:255 Page 7 of 25 Fig. 3 Coordinated variation of the 3D genome, epigenome, and transcriptome. a Example of a variable region where 3D chromatin phenotypes are correlated with epigenomic and transcriptomic phenotypes (chr6:126,280,000-131,280,000; hg19). Six triangular heatmaps from top to bottom: Hi-C contact heatmaps from four individuals, variability matrix, and variable cells in the matrix (var = variable, ns = not significant). Standard tracks below show 3D chromatin, epigenomic, and transcriptomic properties from four individuals in zoomed region (chr6:127,680,918-129,416,097; hg19). All ChIPseq and RNA-seq data in the Figure from Kasowski et al. [42]. b Density plots show the distribution of Spearman correlation coefficients at variable regions between the epigenomic or transcriptomic phenotype indicated in the top margin and the 3D chromatin phenotype indicated in the right margin of panel. Gray lines show the distributions from 100 random permutations selected at random from the 10,000 permutations performed (due to plotting limitations). ***p < 0.0001 by permutation test as described in the “Identifying biological variability in Hi-C contact matrices” section, which applies to all observations in this panel except RNA-seq at INS regions (p = 0.0018) and RNA-seq at FIRE regions (p = 0.0096). c Heatmap showing Spearman correlation coefficients between PC1 and multiple epigenomic/transcriptomic phenotypes, arranged by k-means clustering (k = 4). Tick marks to the right show boundaries between clusters. Each row (N = 518) is one variable PC1 region, limited to the subset of variable PC1 regions that contain RNA-seq signal and at least one peak in at least one individual for each ChIP-seq target included here (H3K27ac, H3K4me1, H3K27me3, CTCF, Cohesin). d Similar to c, showing correlations with FIRE at N = 132 variable FIRE regions. e Similar to c, showing correlations with DI N = 265 variable DI regions. f Similar to c, showing correlations with INS at N = 154 variable INS regions also examined whether individuals heterozygous for anchor disrupting SNPs showed allelic imbalance in loop strength. To facilitate this analysis, we used the HaploSeq [43] method to generate chromosome-span haplotype blocks for each LCL (Additional file 6: Table S7). Although few Hi-C read pairs overlap a SNP allowing haplotype assignment (mean 7.89% of usable reads per LCL), we did observe that the haplotype bearing the stronger motif allele tends to show more reads connecting the corresponding loop anchors Gorkin et al. Genome Biology (2019) 20:255 Fig. 4 (See legend on next page.) Page 8 of 25 Gorkin et al. Genome Biology (2019) 20:255 Page 9 of 25 (See figure on previous page.) Fig. 4 A genetic contribution to variations in 3D chromatin conformation. a A CTCF Position Weight Matrix (PWM) is shown (Jaspar MA0139.1). Eight positions boxed by dashed lines have probability > 0.75 for a single base. We refer to SNPs at these positions as “motif disrupting SNPs.” Alleles matching the consensus base in the motif are labeled “strong motif alleles (S),” and alleles matching any other base are labeled “weak motif alleles (W).” b Boxplot shows the distribution of interaction frequencies at loops with exactly one anchor containing a CTCF motif disrupting SNP (N = 138), separated according to genotype. For each SNP, loop strengths are normalized to the mean value of the heterozygous genotype (WS). There is significant linear relationship between normalized loop strength and genotype by linear regression (p = 7.6e-5). c Aggregate contact map shows the average difference in interaction frequency per loop between SS and SW genotypes (top; N = 117 SNPs), and between SW and WW genotypes (bottom; N = 31 SNPs). The cross point of dotted lines indicates the 40-Kb bin containing the loop being evaluated. d Histogram shows the allelic imbalance in reads connecting loop anchors on the S vs W haplotypes in WS heterozygotes (N = 135 loops). The mean percentage of reads on the S haplotypes is significantly larger than 0.5 (p = 5.9e−4 by one-sided t test). e Line plots show signal of FIRE-QTL, INS-QTL, and DI-QTL by QTL genotype using 11 independent YRI individuals. Each plot shows the indicated phenotype as lines with light color, medium color, and dark color representing average signal across LCLs with the low signal genotype, medium signal genotype, and high signal genotype, respectively. For DI-QTLs, we split all 40-Kb QTL bins into two groups, based on the presence of either upstream DI bias (upper panel) or downstream DI bias (bottom panel). f For C-QTLs, an aggregate contact plot analogous to panel c is used to show the average difference in BNBC corrected interaction frequency (“Δ log(norm contacts)”) between the high and medium contact genotypes (top; N = 138 interactions), and between the genotypes medium and low genotypes (bottom; N = 94 interactions). The cross point of dotted lines indicates the 40-Kb test bin in question. g Boxplots show signal by QTL genotype using additional 6 individuals as a validation set. In each boxplot, three boxes with light shade, medium shade, and dark shade represent the average signal in the 40-Kb QTL bin from individuals with the low signal genotype, medium signal genotype, and high signal genotype, respectively. h Results of permutation test to evaluate the statistical significance of results in g. The solid vertical lines show the linear regression slope values obtained from the validation set (N = 6 individuals). The gray curves show the distributions of slope values obtained from 1000 random permutations. Corresponding bootstrap p values indicated in the upper left corner of each subpanel. i Line plot shows the fraction of “Y” QTL SNPs with nominal significance (p < 0.5) when tested for association with phenotype “X” (“nominal fraction”). Yellow diamonds show nominal fractions for phenotype “Y” QTLs. Red dashed lines show nominal fractions for all SNPs tested for association with phenotype “X,” and open triangles show nominal fractions for all SNPs tested for association with phenotype “Y.” Black circles and lines indicate the median and middle 95% range of 10,000 permutations in which SNPs were selected at random from the phenotype “Y” test SNPs. The number to the right of each line indicates the bootstrap p value (fraction of permutations with a nominal fraction higher than observed for “Y” QTLs). j QQ plot shows FIRE-QTL search results, including all SNPs tested for FIRE association (black points, N = 128,137), and several subsets of FIRE-QTL test SNPs as follows: SNPs also tested for DI association (light green, N = 46,784), SNPs also tested for INS association (light red; N = 6238), SNPs also tested for contact frequency association (light blue; N = 69,847), DI-QTLs (dark green, N = 152), INSQTLs (dark red, N = 60), C-QTLs (dark blue, N = 53) (p = 5.9e−4 by one-sided t test of mean > 0.5; Fig. 4d). Our observation that CTCF motif SNPs can modulate 3D chromatin conformation is consistent with similar findings reported from ChIA-PET data [53] and a recent report of haplotype-associated chromatin loops published while this manuscript was in preparation [27]. Motivated by these preliminary observations of genetic effects on 3D chromatin conformation, we next searched directly for QTLs associated with Hi-C-derived features of 3D chromatin conformation. Power calculations indicated that, despite limited sample size, we were moderately powered to find QTLs with strong effect sizes using a linear mixed effects model (LMM) approach that incorporates Hi-C replicates for each LCL (Additional file 7: Table S8). We conducted a targeted search for QTLs associated with variation in FIRE, DI, INS, and contact frequency. We did not include PC1 in the QTL search because we reasoned that individual genetic variants would be more likely to have detectable effects on local chromatin conformation rather than large-scale features like compartmentalization. For this same reason, we used modified versions of DI scores and INS calculated with a window size of 200 Kb upstream and downstream of the target bin, rather than the standard 2-Mb window size for DI [16] or 480-Kb window size for INS [8]. We also limited our QTL searches to the 11 unrelated YRI individuals in our study (referred to below as the “discovery set”) to mitigate potential confounding effects of differences between populations. For each 3D genome phenotype under study, we identified a list of testable bins that showed appreciable levels of signal in at least one individual in our discovery set (see the “Identification of QTLs” section for full description of test bin and SNP selection). We also identified a unified set of test SNPs with at most one tag SNP among those in perfect LD in each 40-Kb bin. For each testable bin, we measured the association of the given 3D chromatin phenotype with all test SNPs in that bin. In cases where multiple SNPs in the same bin were associated with the phenotype, we selected only the most significantly associated SNP per bin for our final QTL list. Ultimately, at FDR < 0.2, we identified 387 FIREQTLs (i.e., testable bins in which FIRE score is associated with at least one SNPs in that bin; comprising 6.6% of tested bins), 545 DI-QTLs (4.2% of tested bins), and 911 INS-QTLs (12.0% of tested bins) (Fig. 4e, Additional file 1: Figure S9a, Additional file 8: Table S9). For analysis of DI-QTLs, we separated the testable bins into those with upstream bias and those with downstream bias (see the “FIRE, DI, and INS QTL searches” section), because we observed a “Simpson’s paradox”, which occurs when a trend present in different groups of data Gorkin et al. Genome Biology (2019) 20:255 disappears when all data is combined (Additional file 1: Figure S9b). We also searched for QTLs associated directly with interaction frequency in individual contact matrix cells using an LMM approach like that described above for FIRE, DI, and INS. The large number of cells in a Hi-C contact matrix, together with limited sample size, made a true genome-wide QTL search impractical. Thus, we limited our QTL search for contact matrix QTLs (“CQTLs”) to matrix cells that showed significant biological variability in our samples, as described above. We tested for association in our discovery set between the BNBCnormalized interaction frequency in these variable matrix cells and the genotype of test SNPs in either of the two anchor bins. We selected at most one QTL SNP per matrix cell, using association p value to prioritize, finally yielding 345 C-QTL SNPs associated with 463 matrix cells at an IHW-FDR threshold of 0.2 (Fig. 4f, Additional file 1: Figure S9d, Additional file 9: Table S9). To evaluate the reproducibility of each of these QTLs sets (FIRE-QTLs, DI-QTLs, INS-QTLs, and C-QTLs), we examined Hi-C data from six individuals who were not included in our discovery set (we refer to these six individuals as our “validation set”; Additional file 2: Table S2). These individuals come from four different populations (CEU, PUR, CHS, YRI) and include a child of two individuals in the discovery set (YRI-13/NA19240 is a child of YRI-11/NA19238 and YRI-12/NA19239). In each case, we found a significant linear relationship in the validation set between QTL genotype and the corresponding 3D chromatin phenotype (p = 1.8e−14 for FIRE-QTLs, p = 2.5e−7 for DI-QTLs at positive DI bins, p = 0.008 for DI-QTLs at negative DI bins, p = 3e−4 for INS-QTLs, p = 4.1e−9 for C-QTLs; Fig. 4g). To provide an additional and more stringent estimate of the significance of these observations, we performed permutations by randomly selecting sets of test SNPs and measuring the linear relationship between genotype and phenotype in the validation set. In all cases, the observed relationship was also significant by this more conservative bootstrap approach (p < 0.001 for FIRE-QTLs, p < 0.001 for DI-QTLs at positive DI bins, p = 0.041 for DI-QTLs at negative DI bins, p = 0.005 for INS-QTLs, p = 0.006 for C-QTLs; Fig. 4h). We also found that the linear relationship between genotype and phenotype at these QTLs is generally consistent with Hi-C data analyzed at a variety of resolutions (i.e., bin sizes), and when the data is normalized with Knight-Ruiz (KR) matrix balancing [14] instead of the HiCNorm method we used for the rest of our analysis (Additional file 1: Figure S9c). There is little direct overlap between our different QTL sets (Additional file 1: Figure S9e), likely due to limited power and different testable bins for each metric. However, we observed genotype-dependent INS at FIRE- Page 10 of 25 QTLs and C-QTLs and genotype-dependent FIRE score at INS-QTLs and DI-QTLs (Additional file 1: Figure S10a), suggesting that overlapping signal between different types of 3D chromatin QTLs might exist below the level of genome-wide significance. To more rigorously assess overlapping signal between our QTL sets, we examined shared association below the threshold of multiple test correction, inspired by similar approaches reported elsewhere [54]. Our underlying assumption in this analysis is that genetic association studies of two different phenotypes “X” and “Y” with overlapping (or partially overlapping) genetic architecture may have few direct QTL overlaps due to limited power or different study designs, but the shared signal should become apparent when results below the level of genome-wide significance are considered. We thus calculated the fraction of QTLs for a given phenotype X that exceed a nominal level of significance (p < 0.05) when tested for association with a different phenotype Y. We refer to this value as the “nominal fraction” below and in Fig. 4i. To assign a level of statistical significance to these nominal fractions, we approximated the null distribution by calculating nominal fractions for 10,000 sets of SNPs selected randomly from among all test SNPs. In almost all pairwise comparisons between 3D chromatin QTL types examined here, we found that the observed nominal fractions are significantly higher than fractions that would be expected in the absence of shared genetic architecture (Fig. 4i, j). Contribution of 3D chromatin QTLs to molecular phenotypes and disease risk Given the correlation observed between 3D chromatin variation and epigenome variation, we next investigated whether 3D chromatin QTLs could modulate both the epigenome and 3D genome. Here, we used published ChIP-seq data for histone modifications (H3K4me1, H3K4me3, H3K27ac) in a large set of 65 YRI LCLs [39], DNase-seq data from 59 YRI LCLs [38], and CTCF ChIP-seq data from 15 CEU LCLs [55]. In many cases, we found a significant linear relationship between 3D chromatin QTL genotypes and these different epigenetic phenotypes, even when only considering individuals in these datasets were not included in our QTL discovery or validation sets (Fig. 5a, Additional file 1: Figure S10b; 54/65 individual for histone modification ChIP-seq, 48/ 59 for DNase-seq, and all 15/15 for CTCF ChIP-seq). For example, at FIRE-QTLs, the high-FIRE allele is also associated with higher levels of active histone modifications and chromatin accessibility (Fig. 5a). We note that although these associations are all significant by linear regression, only H3K27ac and H3K4me1 passed more conservative permutation testing in which the null distribution is approximated by selecting random SNPs from Gorkin et al. Genome Biology (2019) 20:255 Page 11 of 25 Fig. 5 Contribution of 3D chromatin QTLs to other molecular and organismal phenotypes. a Boxplots show signal for epigenetic phenotypes separated by genotype at FIRE-QTLs (top row), C-QTLs (middle row), and INS-QTLs (bottom row). Epigenetic signals averaged across all peaks in 40-Kb bin. Linear regression p and beta values shown above each plot. p value < 0.05 in bold, others in italics. b Line plots shows beta values of linear relationships between QTL genotypes indicated to the left and epigenetic phenotype indicated above each subpanel. Yellow diamonds show beta values for the true QTLs sets as shown in (a) or Additional file 1: Figure S10b. Black circles and lines indicate the median and middle 95% range, respectively, of 1000 permutations in which SNPs were selected from those tested in the QTL search indicated to the left of each line. The number to the right of each line indicates the bootstrap p value (fraction of permutations with abs(beta) higher than observed for the true QTL set). Yellow boxes highlight values < 0.05. c Genome browser view (chr2:201,222,342-201,386,844; hg19) showing examples of a DI-QTL (chr2:201333312) and FIRE-QTL (chr2:201254049). All signals plotted as a function of DI-QTL genotype (L = Low DI, M = medium DI, H=High DI). Gray boxes highlight regions where epigenetic signals stratify by DI-QTL genotype. d Left subpanel shows the enrichment values for 3D QTL SNPs with nominal significance in the indicated GWAS study calculated as follows: (fraction of indicated 3D QTL SNPs with nominal significance in the indicated GWAS)/(fraction of 3D test SNPs with nominal significance in the indicated GWAS). Asterisks mark values with p < 0.05 by chi-square test (middle panel), and permutation test (right panel). Right panel shows the proportion of 1000 random SNP subsets (selected from the tested SNPs) with enrichment values higher than the indicated true QTL set. Dotted lines mark p < 0.05. e QQ plot shows the results of UC GWAS with all tested SNPs shows as black points, and two subsets as follows: SNPs also tested in our INS-QTL search (light red), and SNP called as INS-QTLs or in perfect LD with INS-QTLs in the same 40-Kb bin (dark red). f QQ plot shows the results of IBD GWAS with all tested SNPs shows as black points, and two subsets as follows: SNPs also tested in our FIRE-QTL search (light green), and SNP called as FIRE-QTLs or in perfect LD with FIRE-QTLs in the same 40-Kb bin (dark green) Gorkin et al. Genome Biology (2019) 20:255 the full set of tested SNPs (Fig. 5b). At INS-QTLs, the slope of these genotype-phenotype relationships is inverted such that higher levels of histone modifications and chromatin accessibility are associated with the low INS score allele (i.e., more contact insulation), although only the association with chromatin accessibility is significant by both linear regression and permutation test (p = 3.6e−35 by linear regression, bootstrap p = 0.018; Additional file 1: Figure S10b,d). The genotype-phenotype relationships observed at DI-QTLs are not as clear as for other metrics (Fig. 5b, Additional file 1: Figure S10a), but this may be expected because increased histone modifications or chromatin accessibility can influence DI in either direction, potentially confounding this type of aggregate analysis. Anecdotally, we did observe examples of individual DI-QTLs where genotype appears to correlate with epigenomic phenotype (Fig. 5c). At C-QTLs, the highcontact alleles show higher levels of the enhancerassociated mark H3K4me1 in the two anchor bins that connect the corresponding matrix cell. Moreover, the fraction of C-QTLs with p < 0.05 association with H3K4me1 in a published set of H3K4me1-QTLs is significantly higher than expected in the absence of shared genetic association (p = 6.9e−6 by chi-square test, bootstrap p = 0.028; Additional file 1: Figure S10c, d). Finally, we sought to examine whether 3D chromatin QTLs might contribute risk for complex diseases. There are 44 direct overlaps between our 3D chromatin QTLs (or SNPs in perfect LD in the same 40-Kb bin) and NHGRI-EBI GWAS catalog [56] (Additional file 9: Table S10). However, the significance of these direct overlaps is hard to assess given the differences between the populations and study designs. Thus, we examined overlaps below the level of genome-wide significance by looking at “nominal fractions” (described above) to assess the shared signal between association studies. We compiled full summary statistics for large GWAS (> 50,000 individuals) of immune-relevant phenotypes including Crohn’s disease (CD), ulcerative colitis (UC), and inflammatory bowel disease (IBD) [57], as well as studies of the non-immune phenotypes height [58] and body mass index (BMI) [59]. We observed striking enrichments for INS-QTLs among variants with nominal associations for UC and IBD risk (1.67- and 1.65-fold, respectively), and these enrichments are significant by both chi-square and permutation tests (INS-QTL with UC chi-square p = 2.5e−16 and bootstrap p = 0.024; INS-QTL with IBD chi-square p = 5.5e−17 and bootstrap p = 0.018; Fig. 5d, e). We also note a trend in which FIRE-QTLs show nominal association with UC and IBD (1.36- and 1.58fold enrichment, respectively), although these observations fall just below the threshold of significance by the more stringent permutation test (FIRE-QTL with UC chi-square p = 7.6e−6 and bootstrap p = 0.090; FIRE- Page 12 of 25 QTL with IBD chi-square p = 4.2e−8 and bootstrap p = 0.056; Fig. 5d, f). Discussion Our results provide the first systematic characterization of chromatin conformation variation across unrelated individuals at the population level (Fig. 6). The most important finding of our study is that genetic variation influences multiple features of 3D chromatin conformation, and does so to an extent that is detectable even with limited sample size and Hi-C resolution. To the best of our knowledge, this represents the first report of QTLs directly associated with 3D chromatin conformation. However, there are limitations to our QTL search that are important to note here. First, the small sample size means that our power to detect QTLs is limited, and thus, our QTL sets should not be considered comprehensive, even within the targeted regions over which the QTL searches were performed. Second, in order to identify QTL sets that could be analyzed in aggregate, we tolerated elevated type I error by using an FDR threshold of 0.2 (as done previously for molecular QTL studies with limited power [40]). While the QTL sets reported here likely contain false positives, the abundance of true positives is suggested by aggregate analyses showing that the genotype-phenotype relationships are reproduced in an independent set of six individuals, and consistent genotype-phenotype relationships at 3D QTLs are apparent in orthogonal epigenomic datasets generated independently by other labs on material isolated independently, and from different LCLs/individuals. Another key finding of our study is that regions which vary in 3D chromatin conformation between individuals also tend to vary in measures of transcriptional and regulatory activity. This supports the existence of shared mechanisms that underlie variation in 3D chromatin conformation, transcription, and epigenomic properties. Our data does not resolve whether variation in higherorder chromatin conformation leads to variation in gene expression and histone modification state, or vice versa. However, we suspect that no single mechanism or causal hierarchy applies to all regions of the genome with variation in one or more of these molecular phenotypes. Our QTL results suggest that, in at least some cases, genetic variation is likely the underlying mechanism that leads to the observed multi-omic variation. This raises the question of whether 3D chromatin QTLs are fundamentally the same as QTLs previously described for other molecular phenotypes (e.g., eQTLs, dsQTLs, histoneQTLs; collectively referred to below as “molQTLs”) or, rather, represent a separate set of QTLs not detectable with other methods. This question is difficult to answer in the present study for two main reasons: (1) Our power is limited, and thus, we cannot say with Gorkin et al. Genome Biology (2019) 20:255 Page 13 of 25 Fig. 6 Summary of findings related to 3D chromatin variability and genetic influence. a There are thousands of regions that vary between individuals in one or more features of 3D chromatin conformation. b These regions tend to vary across individuals in multiple 3D chromatin features as well as in histone modifications, TF binding, and gene expression. c We identify hundreds of QTLs associated with 3D chromatin variation at a subset of these variable regions. d SNPs that disrupt CTCF binding motifs modulate chromatin loop strength confidence that a given SNP is not a 3D chromatin QTL. Many molQTL studies also have limited power and are thus also prone to high type II error (i.e., false negatives). (2) Our QTL searches, like most molQTL studies, are not truly genome-wide because testable regions and testable SNPs are pre-selected to limit the search space. These selection criteria can differ widely across studies, making direct QTL-to-QTL comparisons challenging. The observation of genotype-dependent epigenetic signal at 3D chromatin QTLs suggests that at least some 3D chromatin QTLs could also be detected as other types of molQTLs if those studies had enough statistical power. However, the limited overlap between 3D chromatin QTLs and published molQTLs (even when considering SNPs with only a nominal level of significance) suggests that the QTLs with largest effects on 3D chromatin conformation are not necessarily the same as those with large effects on other molecular phenotypes, and vice versa. Therefore, it is likely that QTL studies directed toward different types of molecular phenotypes (including 3D chromatin features) are likely to be complimentary rather than redundant. We expect that future studies with higher resolution Hi-C data and larger sample size will be important to identify functional variants modulating 3D chromatin conformation, and to further dissect the mechanistic relationships between genetics, 3D chromatin conformation, and other molecular phenotypes. We anticipate that these studies will continue to reveal cases in which perturbation of 3D chromatin conformation is a molecular mechanism through which disease-associated genetic variants confer disease risk. The present study provides initial discoveries of genetic influence on 3D chromatin conformation and an analytical framework that we believe will facilitate future efforts to unravel the molecular basis of genetic disease risk. Methods Hi-C data generation Hi-C was performed as previously described [13]. We note that all Hi-C experiments were performed using a “dilution” HindIII protocol, rather than the newer “in situ” version of the protocol, for consistency because data generation began before the invention of in situ Hi-C. In addition, the resolution of 40 Kb used here for most analysis was determined primarily by sequencing depth rather than choice of a restriction enzyme. Thus, even if a 4cutter like MboI had been used, the prohibitive cost of Gorkin et al. Genome Biology (2019) 20:255 sequencing would have prevented us taking advantage of the additional possible resolution. Hi-C data processing Alignment with WASP Read ends were aligned to the hg19 reference genome using BWA-MEM [60] v0.7.8 as single-end reads with the following parameters: -L 13,13. We used the WASP pipeline [40, 45] to control for potential allelic mapping biases, with some modifications to account for unique aspects of Hi-C data: BWA-MEM can produce split alignments where different parts of a read are aligned to different parts of the genome. This is critical for Hi-C data, because a read can span a Hi-C ligation junction between two interacting fragments. In the case of a split alignment, BWA-MEM will mark the higher-scoring alignment as the primary alignment. For Hi-C data, this is not ideal—we want the five-prime-most alignment (before the ligation junction) to be the primary alignment. To account for this, we further processed the alignments from BWA-MEM to select the five-primemost alignment in cases where one read was split. Reads without an alignment to the five-prime end of the read were filtered out, as were alignments with low mapping quality (< 10). The WASP pipeline was then used to generate alternative reads by flipping the allele in reads overlapping SNPs, and these reads were then realigned using the same pipeline. As input to WASP, we included all SNPs and indels present in the PUR individuals in our set (HG00731, 732, 733), all CHS individuals in 1000 genomes (we included all CHS to account for the fact that no 1000 genomes genotype calls were available for HG00514), all YRI individuals in 1000 genomes (we included all YRI individuals to account for the fact that no 1000 genomes genotype calls were available for GM19193), and the H1 cell line [21] (to facilitate uniform processing and comparisons between LCLs and H1-derived datasets). After alignment of the alternative reads, alignment locations of the original reads and alternative reads were compared (using WASP), and only the original reads for which all alternative reads aligned at the same location with same CIGAR string were kept. Reads overlapping indels were removed. Reads were then re-paired, and only pairs in which both reads survived this filtering were kept. PCR duplicates were removed using Picard tool (http://broadinstitute.github.io/picard/) with default parameters. To ensure that our adapted WASP pipeline removed allelic mapping biases effectively, we simulated all possible 100-bp single-end reads spanning SNPs in our LCLs for chromosome 22 and aligned them back to the genome using our adapted WASP pipeline. We found that no SNPs depart from 50/50 mapping ratio between reference and alternative allele in these simulations. Page 14 of 25 We also took steps to remove any potential artifacts due to HindIII polymorphisms. Hi-C data was obtained by cutting the genome with HindIII, so we reasoned that SNPs or indels that disrupt existing HindIII sites or create novel HindIII sites could lead to differential cutting of two alleles and thus the appearance of differential contact frequency. To mitigate these potential artifacts, we identified all HindIII sites that would be disrupted or created by genetic variants present in our samples, and removed all reads within 1 Kb of these polymorphisms in all individuals. Contact matrix calculations Matrices were generated and normalized as previously described [21, 61]. Briefly, intra-chromosomal read pairs were divided into 40-Kb bin pairs based on five-prime positions. The number of read pairs connecting each pair of 40-Kb bins was tallied to produce contact matrices for each chromosome. Raw counts in the contact matrices were then normalized using HiCNorm [61] to correct for known sources of bias in Hi-C contact matrices (GC content, mappability, fragment length) [62]. Bins that are unmappable (effective fragment length, GC content, or mappability is 0) were assigned NA values. These normalized matrices were further quantile normalized across samples to account for differing read depths and mitigate potential batch effects. One such quantile normalized matrix was generated for each chromosome in each replicate, as well as in each sample (replicates pooled together). We eliminated chromosomes X and Y from all downstream analyses due to the gender differences between our samples. For KR norm matrices used in Additional file 1: Figure S9, we used the Juicer pipeline to generate matrices and performed normalization using Juicebox tools [63]. PC1 score PC1 scores were computed using methods defined previously [13]. Briefly, quantile normalized matrices for each chromosome were transformed to observed/expected (O/E) matrices by dividing each entry in the matrix by the expected contact frequency between regions in that matrix at a given genomic distance. For a given matrix, the expected contact frequencies were computed by averaging contact frequencies at the same distance in that each matrix. The O/E matrices were further transformed to Pearson correlation matrices by the “cor” function in R and eigen vectors (principal components) were computed using the “cov” function in R. Generally, the first eigenvector (“PC1”) reflects A/B compartmentalization. However, for some chromosomes, we have seen that the second or third eigenvector sometimes reflects compartmentalization, while the first eigenvector reflects other features like the two chromosome arms. To identify such cases, we examined the first three Gorkin et al. Genome Biology (2019) 20:255 eigenvectors for each chromosome in each replicate by calculating their correlation with the gene density, because the true compartmentalization pattern is correlated with gene density, while other properties like chromosome arms are not. We required that PC1 show the highest correlation with gene density among the first three eigenvectors in every replicate. If this was not the case for a given chromosome, we eliminated that chromosome from all downstream PC1 analyses in all individuals. Six chromosomes were eliminated in this way: chr1, chr9, chr14, chr19, chr21, and chr22. For the chromosomes that passed this filter, the sign of the first eigenvector, which is arbitrary, was adjusted such that positive PC1 values correspond to compartment A (higher gene density). We note that gene density was not used in the actual PCA calculations, but was only used to orient the otherwise arbitrary direction of PC1, and to systematically eliminate problematic chromosomes where we could not be sure that the first eigenvector captures compartmentalization as opposed to other chromosome features. Directionality index Directionality index was computed as previously described [16]. Briefly, upstream and downstream contacts within 2-Mb window for each 40-Kb bin were counted, and chi-square statistics were calculated under equal assumption. The sign of the chi-square statistics was adjusted such that positive values represent upstream bias. For some bins, there are more than five NA bins within 2-Mb window, and DI for those bins are not calculated. As noted in the main text, we made a slight variation of these DI scores for the QTL searches in which DI was recalculated using a window size of 200 Kb to capture more local features. Insulation score Insulation scores were computed as previously described [7] with some adjustments. Briefly, contacts linking upstream and downstream 400-Kb windows for each 40Kb bin were calculated in the O/E matrices instead of raw matrices. We further divided the contact frequency by the average of upstream and downstream 400-Kb windows, to account for differences in contact density across the chromosome. The insulation scores were then ranged from 0 to 1, representing absolute insulation and no insulation respectively. Insulation scores for bins in which more than 50% of matrix cells in the 400-Kb window have NA values were not computed. For the QTL search, we re-calculated insulation scores using 200-Kb window. TADs calling TADs were called using the same approach as described previously [16]. DI values for each 40-Kb bins were used to build a Hidden Markov Model and predict the Page 15 of 25 probability upstream bias, no bias, and downstream bias. Regions switching from upstream bias to downstream bias were called as boundaries. FIRE We first calculated FIRE score for each of 20 individuals, as described in our previous study [19]. Specifically, we mapped the raw reads to the reference genome hg19 as described above. Next, we removed all intrachromosomal reads within 15 Kb and created 40-Kb raw Hi-C contact matrix for each individual for each autosome. For each 40-Kb bin, we calculated the total number of intra-chromosomal reads in the distance range of 15–200 Kb. We then filtered bins as follows, starting from 72,036 autosomal 40-Kb bins: First, we removed 40-Kb bins with zero effective fragment size, zero GC content, or zero mappability score [61]. Next, we filtered out 40-Kb bins within 200 Kb of the bins removed in the previous step. We further filtered out 40-Kb bins overlapping with the chr6 MHC region (chr6:28,477, 797-33,448,354; hg19), which has extremely high SNP density that can make it difficult to correct for allelic mapping artifacts. This left 64,222 40-Kb bins for downstream analysis. Next, we applied HiCNormCis [19] to remove systematic biases from local genomic features, including effective fragment size, GC content, and mappability. The normalized total number of cis intrachromosomal reads is defined as FIRE score. We further performed quantile normalization across multiple individuals using R package “preprocessCore.” The final FIRE score is log transformation log2(FIRE score + 1) and converted into a Z-score to create a mean of 0 and standard deviation of one. To identify significant FIRE bins in each individual, we used one-sided p value < 0.05. Ultimately, merging across all individuals, we identified 6980 40-Kb bins which are FIRE bin in at least one of 12 YRI individuals. Consistent with our previous findings [19], we observed significant enrichment of GM12878 typical enhancers and super enhancers among these 6980 40-Kb FIRE bins (Additional file 1: Figure S1d). GREAT analysis [64] further showed immunerelated biological pathways and disease ontologies are enriched in these 6980 40-Kb FIRE bins (Additional file 1: Figure S1e). Comparison of intra-individual vs inter-individual variation To estimate variability between replicates, we computed the Pearson correlation coefficient for all pairs of replicates for each score (DI, INS, FIRE, and PC1). Replicate pairs then can be divided into two groups based on whether they are from the same individuals or different individuals, as illustrated in Additional file 1: Figure S4c. We then tested if the distribution of Pearson correlation Gorkin et al. Genome Biology (2019) 20:255 coefficients were different comparing two groups. Similar analysis was performed for contact matrices. For contact matrices, we calculated the Pearson correlation coefficient for each distance and each chromosome separately as shown in Fig. 1c. Variable regions F test for variable bins To identify regions that are variable across genomes, we used a linear normal model with an empirical Bayes variance estimator using the “eBayes” in limma [48] function with default parameters. First, values for each 40-Kb bin in hg19 reference genome were calculated for each metric tested (DI, FIRE, INS, PC1) as described above. DI, PC1, and INS scores were calculated based on contact matrices quantile normalized across 40 replicates. FIRE scores were calculated based on raw counts using HiCNormCis [19] and then quantile normalized across 40 replicates. Second, we filtered out bins that are not testable. Specifically, FIRE scores were only tested for bins that are FIRE regions (p < 0.05) in any of 40 replicates. DI scores were only tested for bins where strong biases are observed (abs(DI) > 10.82757, which correspond to chi-squared test p value 0.001) in any of 40 replicates. INS were only tested for bins where strong insulation is observed (z-score transformed INS score < − 1) in any of 40 replicates. No similar filterers were performed for PC1 scores. Third, we filtered out any bins that overlapping large SVs (> 10,000 bp) to avoid effect caused by SVs. Specifically, for FIRE, INS, and DI scores, bins that are within 200 Kb, 400 Kb, and 2 Mb respectively upstream or downstream of large SVs were removed. For PC1 scores, bins directly overlapping large SVs were removed. Lastly, we applied limma standard model with individual as a fixed factor and eBayes correction. To estimate empirical false-positive rate (FDR), we bootstrapped replicates to calculate the number of false positives in random background. Briefly, we randomly selected 40 or 22 replicates with replacement for LCL20 and YRI11 respectively and identified variable regions as mentioned above. We performed 1000 permutations and calculated empirical FDR as the average positive hits in 1000 permutations divided by number of hits in real data. Normalizing Hi-C contact matrices using BNBC normalization To directly compare individual Hi-C contact matrix cells across samples, we sought to remove unwanted per-matrixcell variation owing to date of processing or other unknown “batch” effects. To this end, we developed Bandwise Normalization and Batch effect Correction (BNBC), described and evaluated in a separate manuscript (preprint on bioRxiv https://www.biorxiv.org/content/10.1101/214361v1 Page 16 of 25 ). A brief description follows. For each chromosome and for each strata of distance between loci (a matrix “band,” hence the term “bandwise”), we correct for unwanted variation by taking the log counts-per-million-transformed values of all samples and generating a matrix whose entries are the observations for that chromosome’s matrix band across all samples (columns indexes samples and rows indexes contact matrix cells with anchor bins separated by a fixed distance). We then quantile normalize this matrix and regress out the impact of known batches (here, date of processing) using ComBat [65] (specifically we correct both mean and variance). This procedure essentially conditions on genomic distance. We correct the majority of each contact matrix for each chromosome for each sample: we correct all but the 8 most distal matrix bands, for which we set all values to 0. The choice of the last 8 bands is empirical and reflects the small number of observations in each band matrix. The procedure is implemented in the bnbc package available through Bioconductor (http://www. bioconductor.org/packages/bnbc). Correction of contact matrices was performed on replicate-level data using the following LCLs: GM18486 (YRI-1), GM18505 (YRI-2), GM18507 (YRI-3), GM18508 (YRI-4), GM18516 (YRI-5), GM18522 (YRI-6), GM19099 (YRI-7), GM19141 (YRI-8), GM19204 (YRI-10), GM19238 (YRI-11), GM19239 (YRI12), GM19240 (YRI-13), HG00731 (PUR-1), HG00732 (PUR-2), HG00512 (CHS-1), HG00513 (CHS-2). We note that NA19239 (YRI-12) replicate 1 and NA19240 (YRI-13) replicate 2 were excluded because the BNBC algorithm requires multiple samples from a given experimental batch to estimate batch effect parameters. Identifying biological variability in Hi-C contact matrices To identify contacts with significant levels of betweenindividual variability, we employed the following procedure, which mimics the analysis for INS, DI, FIRE, and PC1, on contact matrices normalized by BNBC (see the “Normalizing Hi-C contact matrices using BNBC normalization” section). For each contact matrix cell (representing loci separated by less than 28 Mb, this is a subset of the matrix cells normalized by BNBC), we used a linear model with individual modeled as a fixed factor, note we have 2 growth replicates for almost every individual. We used a parametric likelihood ratio test (equivalent to an F test) to test whether there was significant between-individual variation. We used the IHW framework [50] with the distance between anchor bins as informative covariate, to increase power and estimate false discovery rate. We used a FDR of 10% as significance threshold, resulting in 115,817 contact matrix cells with significant biological variability across the autosomes. To estimate effect size (depicted in Figs. 2a and 3a and Additional file 1: Figure S5), we used a linear mixed effects model with individual as random effect, to decompose the Gorkin et al. Genome Biology (2019) 20:255 variance into between-individual variability (biological) and within-individual variability (technical). As the measure of biological variability in these figures, we used the estimated biological variance. For this analysis, all 16 samples we normalized using BNBC were used. Correlation with other datasets To examine correlation between 3D genome organization and other genome features, we re-identified variable regions with the same pipeline mentioned above using only individuals for which data is available for other genome features, and then computed the Spearman correlation coefficient between 3D genome metrics (DI, INS, PC1, and FIRE) and other genome features (RNA-seq, ChIP-seq, and DNase-seq) for each variabel 40-Kb bin. Signals for each 40-Kb bins were calculated by averaging signals for the bin. Specifically, signals for ChIP-seq were the average signal of all peaks within the bin, signals for RNA-seq were the average FPKM of all genes in the bin, and DNase signals were average signal for each base pair in the bin. In some cases, serval consecutive bins were identified as variable. In these cases, we only kept the bin with strongest signal for other genome features among consecutive bins. To generate random backgrounds, we permutated individual labels for the same set of bins and recomputed Spearman correlation coefficient. Ten thousand such permutations were used to calculate the statistical significance of departure from the null hypothesis in which the median value of true correlation values and permutated correlation values are equal. Similar analysis was performed for variable matrix cells with the following modifications. First, we used the variable matrix cells in the preceding section “Identifying biological variability in HiC contact matrices.” Second, to correlate matrix-cell-level contacts with bin-level DNase and ChIP-seq signals, we sued the anchor bins of variable matrix cells. Since each anchor bin may belong to more than one matrix cells, we only used each bin once and selected the matrix cell value with the highest Spearman correlation coefficient. Exactly same approach was performed during permutation to ensure a fair comparison. Phasing variants Phasing of variants was performed based on HaploSeq pipeline [43]. Briefly, (1) variants were filtered to keep only bi-allelic SNPs heterozygous in a given individual; (2) aligned Hi-C bam files were realigned and recalibrated using GATK 3.4.0 [66] based on SNPs in the individual; (3) filtered SNPs and realigned bam files were then used as input to run HAPCUT [67]; (4) results from HAPCUT were further filtered to keep only the largest haplotype block and combined with homozygous alt SNPs as input for imputation using Beagle 4.0 [68] using 1000 Genome Phase 3 data excluding individual to Page 17 of 25 phase as reference panel; (5) results from Beagle were then combined with results of HAPCUT by removing conflicting phased SNPs. For all auto chromosomes except 1 and 9 in 18 out 20 individuals, we were able to obtain a single haplotype block. For chromosome 1 and 9, two arms were phased separately because of large heterochromatin region surrounding centromere. X chromosome was only phased for female individuals. We excluded NA19193 and HG00514 from phasing because of the lack of available genotypes through 1000 genomes at the time of phasing. We evaluated accuracy of phasing in three probands in trios (NA19240, NA12878, HG00733) and found phasing results are of very high accuracy (~ 97.71%). Specifically, we calculated accuracy as percentage of correctly phased variants among total phased variants. Only variants whose transmission from parents can be unambiguously identified were used in calculation of accuracy where at least one parent is homozygous. Detailed statistics for phasing are listed in Additional file 5: Table S6. CTCF motif variation and looping strength GM12878 loops and motif positions were obtained from Rao et al. [14] (GSE63525_GM12878_primary+replicate_ HiCCUPS_looplist_with_motifs.txt.gz; N = 9448 HiCCUPS loops). We limited our analysis to autosomal cis loops in which a CTCF motif in one of the anchor regions overlaps a SNP (N = 572). To evaluate the impact of motif disruption, we first identified eight “key” positions in the CTCF PWM (Jaspar MA0139.1) [69] in which a single base has higher than 0.75 probability. We refer to SNPs at these positions in motif occurrences with one allele matching the high-probability base as “motif disrupting SNPs.” We refer to alleles matching the consensus base in the motif as strong motif alleles (S), and alleles matching any other base as weak motif alleles (W). There are N = 142 loops with a motif disrupting SNP in a convergently oriented CTCF motif, which we refer to below as testable loops. For each testable loop, we extracted the Hi-C interaction frequency in the loop bin from each LCL and classified as either “WW,” “SW,” or “SS” depending on the individual’s genotype at the corresponding motif disrupting SNP. To enable aggregation of data across different SNPs, we set the mean “SW” interaction frequency for each SNP to 1 and normalized all values for that SNP accordingly. These values are plotted in Fig. 4b. In addition, for each testable loop we extracted a submatrix including the loop bin as well as 15 bins upstream and 15 bins downstream. Submatrices with missing values were discarded. For each SNP, we calculated the mean submatrix for each genotype and then subtracted submatrices to calculate the difference in each matrix cell per “W” allele (i.e., SS-SW and SW-WW). These differences were then Gorkin et al. Genome Biology (2019) 20:255 averaged across all SNPs and plotted in Fig. 4c. Submatrices with missing values were discarded. For the allelic analysis in S/W heterozygous individuals, we used chromosome-span phasing results (see the “Variable regions” section) to split the Hi-C reads from each chromosome in each LCL into two separate haplotypes. Specifically, we required at least one base pair overlap with phased heterozygous SNPs with high base calling score (> 13) and high mapping quality (> 20). Reads overlapping indels or containing SNPs from both haplotypes were not used. Approximately 7.89% of Hi-C reads covered a heterozygous variant and could thus be assigned to one of the two haplotypes. The accuracy of haplotype assignment was evaluated by fraction of homologous-trans (h-trans) read, which contain SNPs from both haplotypes. On average, ~ 1% reads were htrans, suggesting high quality of the assignment. For each testable loop, we defined 40-Kb windows around the center of each loop anchor region and calculated the number of reads connecting these two anchor windows (“loop reads”) on each haplotype. For each heterozygous LCL, we then calculated the percentage of loop reads that occur on the haplotype containing the S allele at the motif disrupting SNP anchor. We required that at least 10 total loop reads were present for a given loop in a given heterozygous LCL, leading to a total of 218 data points from 105 different loops for inclusion in Fig. 4d. Identification of QTLs Testable bins To identify testable bins for FIRE-QTL, DI-QTL, INSQTL, and C-QTL searches, we began with 72,036 autosomal 40-Kb bins based on reference genome hg19. We eliminated “unreliable” bins with effective length, GC content, or mappability equal to zero [62], resulting in 66,597 bins remaining. We further removed any 40-Kb bins within 200 Kb of an unreliable bin, resulting in 64, 337 40-Kb bins. We also removed bins covering the chr6 MHC locus (hg19: chr6:28,477,797-33,448,354), which is extremely polymorphic and may lead to complex mapping artifacts that are difficult to correct for. To eliminate false signals in Hi-C data that could arise from large structural variations (SVs), we obtained SVs from the 1000 Genomes consortium [35] (ftp://ftp-trace.ncbi.nih. gov/1000genomes/ftp/phase3/integrated_sv_map/ALL. wgs.integrated_sv_map_v2.20130502.svs.genotypes.vcf. gz) and removed bins which overlap one or more structural variants of any size previously annotated in these individuals (N = 123,015 SVs), or within 200 Kb of large structural variations (> 10 Kb, N = 1253 SVs). These filtering steps yielded a set of 51,511 testable bins, which represent a common starting point for FIRE-QTL, DIQTL, INS-QTL, and C-QTL searches as described below. Page 18 of 25 Testable SNPs We began with a list of 15,765,667 variants among all 20 LCL individuals (Additional file 2: Table S3). We kept 14,177,284 variants among 11 unrelated YRI individuals and removed all indels, HindIII site polymorphisms, multi-allelic SNPs, and SNPs with minor allele frequency (MAF) < 5%. We also required that remaining SNPs were within the 51,511 testable bins described above and that both alleles were present in at least 2 individuals in the discovery set individuals. (N = 4,132,791 SNPs remaining). Finally, where multiple SNPs in the same bin were in perfect LD among 11 unrelated YRI individuals, we selected one with the smallest genomic position (to avoid the introduction of a random selection that would not be perfectly reproducible), ultimately yielding 1,304,404 potentially testable SNPs that served as a common input set to all QTL searches. Power calculations To explore the power of our approach and data, we performed a Monte Carlo-based power calculation. Specifically, we varied four variables: (1) the minor allele frequency of a variant, (2) the effect size of genotype (a fixed effect), (3) the variability between subjects (a random effect), and (4) the variability of the residuals. For contact QTLs, we also varied the mean of the Hi-C contact frequency in question. For analyses reported, we fixed the number of replicates per subject to be 2 (consistent with our study design). We explored a variety of settings for these parameters to assess power as each variable changes (see Additional file 6: Table S7). Each setting tested was chosen to reflect the distribution of observed values in our real Hi-C data. For each configuration of parameters, we performed the following simulation: We simulated genotypes by randomly sampling a set of alleles (one allele per subject) from a binomial distribution parameterized by the number of subjects and the MAF; we repeated this process twice and create per-subject genotypes by adding the results of the sampling of alleles. We simulated per-subject random effects, and per-sample residuals. To obtain a given sample’s simulated Hi-C contact matrix value, we added the mean Hi-C contact matrix value to that sample’s simulated genotype (multiplied by the pre-specified effect size), the specific subject’s random intercept, and the sample’s random residual. After performing this for all samples, we then fitted the same LMM model used in our QTL search. We repeated this simulation and model fitting process 1000 times and computed power as the fraction of times the null hypothesis that the effect of genotype is equal to 0 is rejected at a nominal p value of 0.05. Gorkin et al. Genome Biology (2019) 20:255 FIRE, DI, and INS QTL searches FIRE tested bins and SNPs We limited our FIRE QTL search to the subset of testable bins that were called as FIRE in at least one YRI LCL (N = 5822 FIRE test bins), and the subset of testable SNPs therein (N = 128,137 FIRE test SNPs). INS tested bins and SNPs For the INS-QTL search, we examined 328,530 test SNPs with 12,976 variable INS bins (see the “F test for variable bins” section). Page 19 of 25 them to be included in the final QTL sets. After this filtering, we ended up with 387 candidate FIRE-QTLs, 268 candidate upstream-biased DI-QTLs, 277 downstreambiased DI-QTLs, and 911 candidate INS-QTLs. As a control for each of these QTL searches, we randomly shuffled the score in question (i.e., FIRE, DI, or INS) among all 11 YRI individuals and performed QTL searches on this permuted data. In each of these tests, we found no SNPs associated with the permuted scores at FDR < 0.20. C-QTL search DI tested bins and SNPs For the DI-QTL search, we examined 181,950 test SNPs with 7590 variable DI bins (see the “F test for variable bins” section). For the DIQTL search, we further classified each DI bin based on which whether it showed stronger upstream or downstream bias, because we saw a Simpson’s paradox when we considered them together (see Additional file 1: Figure S10b). This was done as follows: for each bin, we evaluated the DI score in each of 11 unrelated YRIs and identified the DI score among these individuals with the largest absolute value. We defined a bin as “upstream DI bias” if the DI score with the highest absolute value was positive, or “downstream DI bias,” if the DI score with the highest absolute value was negative. Only 37/7590 bins (0.4%) had individuals with both positive and negative DI values. LMM QTL searches For each test SNP, we identified the 40-Kb bin it belongs to, and fitted a linear mixed effects model, using FIRE, DI (200-Kb window; see the “Directionality index” section), or INS score (200-Kb window; see the “Insulation score” section) in each biological replicate as the response variable and genotype of that testable SNP as the explanatory variable. Since two biological replicates from the same individual are dependent, we used an individual-specific random effect to specifically characterize such within-individual dependence. We used the R package “nlme” and R function “gls” to fit the linear mixed effects model. Code is available here: http://renlab.sdsc.edu/renlab_website// download/iqtl/. The quantile-quantile plots (QQ plot) showed only minor genomic inflation (median p value = 0.4821, lambda = 1.0864 for FIRE-QTLs; median p value = 0.4864, lambda = 1.0649 for upstream-biased DIQTLs; median p value = 0.4828, lambda = 1.0826 for downstream-biased DI-QTLs; median p value = 0.4865, lambda = 1.0646 for INS-QTLs). The linear mixed effects model identified 476, 315, 315, and 1092 SNPs with false discovery rate (FDR) less than 0.20 for FIRE, upstreambiased DI, downstream-biased DI, and INS, respectively. When more than one SNP in the same bin was identified, we selected the SNP with lowest p value among To find QTLs affecting Hi-C contact strength, we first identified 115,187 Hi-C contact matrix cells exhibiting substantial biological variability as described in the “Normalizing Hi-C contact matrices using BNBC normalization” section, and constrained our QTL search to these cells. We then intersected these contact cells with 1,304,404 testable SNPs by requiring a SNP to sit in one anchor bin of one of these variable matrix cells. We also filtered out matrix cells to ensure both anchor bins of the matric cell are among 51,511 testable bins. In total, we obtained 3,109,039 tests involving 687,655 SNPs and 54,880 matrix cells on all 22 autosomes. For each test, we used the BNBC normalized data described in the “Variable regions” section, but used only the 11 unrelated YRI individuals with genotypes available and fit a linear mixed effects model in which genotype is a fixed effect and subject is a random intercept. We then used “lmerTest” package in R to estimate p values for the fixed effect of genotype [70]. We used the IHW framework [50] to estimate FDR, with the distance between anchor bins as an informative covariate, and call any matrix cell with FDR < 0.2 as significant. We further filtered significant tests by selecting the most significant SNPs per matrix cell and kept the leftmost SNPs among SNPs in perfect LD in two anchor bins of the matrix cell. After filtering, we ended up with 463 tests involving 345 SNPs and 463 matrix cells. To make the aggregate contact plots in Fig. 4g, we recoded the genotypes based on the direction of effect such that 0, 1, 2 refer to the genotypes containing 0, 1, or 2 alleles associated with the increased phenotype, respectively. Next, to avoid aggregating the same submatrix multiple times, we filtered by (1) selecting only the most significant matrix cell associated with each QTL and (2) selecting only the most significant QTL associated with each anchor bin (in some cases the same bin anchors multiple matrix cells associated with different QTL SNPs). This filtering left 165 unique matrix cell QTL interactions for plotting. For each matrix cell, we then extracted a submatrix including 25 bins upstream and 25 bins downstream. Submatrices with missing values were discarded. For each QTL, we then calculated the mean submatrix values for Gorkin et al. Genome Biology (2019) 20:255 each genotype and then subtracted submatrices to calculate the difference in interaction frequency between the 1 and 0 genotypes, and between the 1 and 2 genotypes. These differences were then averaged across QTLs and plotted in Fig. 4g. Validation of QTLs in additional individuals Our validation set included six unrelated individuals not included in the discovery set: NA12878, NA19240, HG00512, HG00513, HG00731, and HG00732. For each QTL, we collected the genotype among six additional individuals, and the corresponding FIRE, DI, or INS scores. Note that a small fraction of QTLs have missing genotypes in these six individuals (coded as “-1”), and these missing data points were eliminated from validation analysis. We examined the distributions of scores for each genotype. For each QTL type (i.e., FIRE, DI, or INS), we found that the same direction of effect observed in the discovery set is observed on average in the validation set. To assess the significance of this observation, we approximated the null expectation as follows. For FIRE-QTLs, for example, we started from all 128, 137 FIRE test SNPs and 5822 FIRE test bins. Note that in our discovery set, we identified 387 FIRE-QTLs, each in a different 40-Kb bin. To create a random control SNP group, we first randomly selected 387 40-Kb bins from all 5822 FIRE test bins. Next, within each select bin, we randomly selected one SNP and combined all these 387 selected SNPs into a control SNP group. We then tested their SNP effect on the six additional individuals. We repeated such sampling with replacement 1000 times, to create a null distribution of positive and negative SNP effect, respectively. We performed the same type of permutations for DI and INS. Similar analysis was performed for C-QTLs with a few modifications. First, we only used replicates from NA19420, HG00512, HG00513, HG00731, and HG00732 as explained in the “Variable regions” section. Second, 1000 random permutations were performed by sampling matrix cells instead of bins. Third, we used values of biological replicates separately instead of as merged data because the BNBC normalization is performed at the level of replicates. Examining epigenetic variation at FIRE, DI, INS, and C-QTLs To examine epigenetic variation at 3D genome QTLs, we re-analyzed DNase-seq data from 59 LCLs [38], histone modification ChIP-Seq data (H3K27ac, H3K4me1 and H3K4me3) for 65 LCLs [39], and CTCF ChIP-seq data from 11 LCLs [55]. These data were re-mapped using the WASP pipeline to control for allelic mapping artifacts and calculating the signal in 40-Kb bins as described above in the "Alignment with WASP" section. We examined the effect of genotype at FIRE, DI, INS, or Page 20 of 25 C-QTLs on DNase-seq and ChIP-seq signal by linear regression. As a control, we randomly selected the matched number of SNPs with the same approach described in the “Validation of QTLs in additional individuals” section and re-did such validation analysis. We repeated such random sampling 1000 times to create the empirical null distribution of no genetic effect. For CQTLs, we used the sum of epigenetic features in two anchor bins to calculate correlation with contact frequency. Nominal fraction analyses Comparing between 3D chromatin QTL types To compare between different 3D chromatin QTLs, we took the raw test results for each QTL set and projected other 3D QTLs into the test results. For example, in Fig. 4j, we selected subset of SNPs that are DI-QTLs and plotted them (dark green dots) using p values from FIRE-QTLs along with all SNPs tested in the FIRE-QTL search (black dots). We also used all tested SNPs in the DI-QTL search (light green dots) as a control set. To assign significance to the overlap, we compared the fraction of SNPs with nominal significance (p < 0.05) in each set: (1) DI-QTL tested SNPs that were not significant QTLs and (2) DI-QTLs. We calculated p values for this comparison by chi-square test. To rule out the effect of sampling bias when selecting a small number of SNPs, we also performed permutation. In each permutation, we randomly selected the same number of SNPs as the real QTL set (from the full set of tested SNPs) and calculated the fraction with nominal significance. We then computed bootstrap p values using 10,000 such permutations under the null hypothesis that the fraction of nominal significance is the same between QTLs and random selected SNPs. For C-QTLs, one SNP may be tested against multiple matrix cells, so we only kept the most significant p value for each SNP to avoid biases toward SNPs with multiple tests. Comparing 3D chromatin QTLs to other molQTLs Similar approaches were used to assess overlap between 3D chromatin QTLs and other molQTLs. We obtained full test results (all tested SNPs with the p values) from previous molQTL studies and projected 3D chromatin QTLs into those test results. We calculated the fraction of nominal significance and used chi-square test to evaluate significance between 3D-QTLs and non-3DQTLs. Similarly, we performed bootstrap to estimate significance empirically. One modification is that we extended our QTL sets by incorporating all SNPs in perfect LD with the same 40-Kb bin because we may not use the same tagging SNP in our study as used in other studies. To ensure a fair comparison, we performed the same extension for the control sets of all tested SNPs. Gorkin et al. Genome Biology (2019) 20:255 Page 21 of 25 Comparing 3D chromatin QTLs to GWAS in PBS for 30 min at room temperature. Lastly, they were rinsed with 1× PBS and washed with 2× SSC for 5 min. For hybridization of probes, the prepared probes were denatured at 73 °C for 5 min in water bath. Cells were denatured in a two-step process in a 73 °C water bath: 2.5 min in 70% formamide in 2× SSC and 1 min in 50% formamide in 2× SSC. Denatured probes were transferred onto microscope slides, and coverslips were placed on top with cell-side facing down. The coverslips were sealed with rubber cement and incubated overnight at 37 °C in a dark, humid chamber. Next day, coverslips were carefully removed and transferred onto a 6-well plate. Cells were washed at 37 °C with gentle agitation, twice with 50% formamide in 2× SSC for 15 min and three times with 2× SSC for 5 min. The cells were then stained with DAPI (Invitrogen, D1306), mounted on microscope slides with ProLong Gold Antifade Mountant (Invitrogen, P36930), sealed with nail polish, and imaged. Comparison with the GWAS results was performed in the same manner as described in the “Comparing 3D chromatin QTLs to other molQTLs” section for other molQTLs. Instead of test results for other molQTLs, we used summary statistics from previous GWAS. FISH Cell preparation for FISH Approximately 100,000 cells were adhered to center of PDL-coated coverslips (Neuvitro, GG-22-15-PDL) by placing 100 uL of cells at 1 × 10 [6] cells/mL. Cells on coverslips were incubated for an hour at 37 °C, carefully washed with PBS, and fixed with 4% paraformaldehyde in 1× PBS for 10 min. PFA was quenched with 0.1 M Tris-Cl, pH 7.4 for 10 min, washed with PBS, and stored in 1× PBS at 4 °C for up to 1 month. BAC probe labeling and preparation All BAC clones were ordered from the BACPAC Resource Center at the Children’s Hospital Oakland Research Institute: “U” probe is RP11-74P5, “C” probe is RP11-337 N12, and “D” probe is RP11-248 M23. BAC DNAs were labeled with either Chromatide Alexa Fluor 488-5 dUTP (Invitrogen, C-11397) or Alexa Fluor 647aha-dUTP (Invitrogen, A32764) using nick-translation kit (Roche, 10976776001), and incubated in 15 °C for 4 h. The nick-translation reaction was deactivated using 1 μL of 0.5 M EDTA, pH 8.0, and heated for 10 min at 65 °C. The probes were then purified using illustra ProbeQuant G-50 Micro Columns (GE Healthcare, 28903408) and eluted to a concentration of 20 ng/μL. Probes were mixed with Human Cot-1 DNA (Invitrogen, 15279011) and salmon sperm (Invitrogen, 15632011), and precipitated with 1/10th volume of 3 M sodium acetate, pH 5.2, and 2.5 volume of absolute ethanol for at least 2 h at − 20 °C. Probes were then spun down, washed with cold 70% ethanol, resuspended in formamide and 40% dextran sulfate in 8X SSC, and incubated at 55 °C. Hybridization Cells on coverslips were blocked with 5% BSA and 0.1% Triton X-100 in PBS for 30 min at 37 °C and washed twice with 0.1% Triton X-100 in PBS for 10 min each with gentle agitation at room temperature. Cells were permeabilized with 0.1% saponin and 0.1% Triton X-100 in PBS for 10 min at room temperature. Next, they were incubated in 20% glycerol in PBS for 20 min, freezethawed three times with liquid nitrogen, and incubated in 0.1 M hydrogen chloride at room temperature for 30 min. Cells were further blocked for 1 h at 37 °C in 3% BSA and 100 μg/mL RNase A in PBS. Cells were permeabilized again with 0.5% saponin and 0.5% Triton X-100 Microscope and analysis Images were acquired with DeltaVision RT Deconvolution Microscope at UC San Diego’s department of neuroscience (acquired with award NS047101). Captured images were processed using the TANGO [71] plugin in ImageJ for quantitative analysis. Each FISH experiment contained two probes labeled with different color dyes (either U-C or C-D). We limited our analysis to nuclei containing 2 labeled foci for each color (4 total foci), allowing us to more confidently distinguish foci in cis from those in trans. Distances were measured from the center of one color focus to the center of the closest focus of the other color. Re-analysis of public datasets Analysis of ChIP-seq data from Kasowski et al. and McVicker et al. Raw fastq files were downloaded from SRA database for each experiment (SRP030041 and SRP026077, respectively). Reads were aligned to hg19 reference genome using BWA MEM (Kasowski) or BWA ALN [60] v0.7.8 (McVicker) with WASP pipeline [40, 45] to eliminate allelic mapping bias. Only reads with high mapping quality (> 10) were kept. PCR duplicates were removed using Picard tools v1.131 (http://broadinstitute.github.io/picard). MACS2 [72] v2.2.1 was then used to call peaks using corresponding input files. For CTCF and SA1, default parameters were used for MACS2. For H3K27ac, H3K4me1, and H3K4me3, peak calling was done using “--nomodel” parameter because we do not expect sharp peaks for histone modifications. For H3K27me3 and H3K36me3, peak calling was done using “--nomodel --broad” parameter. Bigwig files were generated by MACS2 using fold enrichment for viewing in genome Gorkin et al. Genome Biology (2019) 20:255 Page 22 of 25 browser. All Kasowski data were processed in pair-end mode, and both replicates were merged for analysis. All McVicker data were processed in single-end mode, and the pooled input data were used for all samples because there are no individual input files. To compute signals in peaks, we used a set of merged peaks across all individuals for each mark. interactions. Figure S3. Aggregate looping interactions in each sample. Figure S4. 3D chromatin variation among 20 LCLs and H1-derived lineages. Figure S5. Characterization of variable regions of 3D chromatin conformation. Figure S6. Additional characterization of variable regions of 3D chromatin conformation. FigureS7. Coordinated variation between 3D chromatin conformation and multiple molecular phenotypes. Figure S8. Correlations between 3D genome phenotypes and other molecular phenotypes. Figure S9. 3D chromatin QTLs. Figure S10. Influence of 3D chromatin QTLs on epigenomic and disease phenotypes. Analysis of RNA-seq data from Kasowski et al. Additional file 2: Tables S1-S3. Public datasets re-analyzed in this study, LCLs included in the study, and Hi-C mapping statistics. Raw fastq files were downloaded from SRA database (SRP030041). Reads were aligned to hg19 reference genome using STAR [73] v2.4.2a with the WASP pipeline in pair-end mode to eliminate allelic mapping bias. Gencode [74] v24 annotation was used to construct STAR index and computing FPKM. Only uniquely mapped reads were kept. Cufflinks [75] v2.2.1 was applied to compute FPKM values. Both replicates were merged for analysis. Additional file 3: Table S4. TAD and boundary calls for each sample. Additional file 4: Table S5. Regions showing evidence of biological variability in 3D chromatin conformation. Additional file 5: Table S6. Matrix cells showing evidence of biological variability. Additional file 6: Table S7. Summary of phasing results. Additional file 7: Table S8. Power calculations at standard p < 0.05 and at p-values corresponding to FDR < 0.2 (FIRE-QTLs p < 7e-4, INS-QTL p < 7e-4, DI-QTL p < 7e-4, C-QTL p < 3e-5). Additional file 8: Table S9. 3D chromatin conformation QTLs. Analysis of DNase-seq data from Degner et al. Raw fastq files were downloaded from SRA database for each experiment (SRP007821). Reads were aligned to hg19 reference genome using BWA ALN with the WASP pipeline in single-end mode to eliminate allelic mapping bias. Only reads with high mapping quality (> 10) were kept. PCR duplicates were removed using Picard tools. Bigwig files were generated using makeUCSCfile commands in homer tools [76] v4.9.1. Analysis of ChIP-seq data from Ding et al. Raw fastq files were downloaded from SRA database for each experiment (SRP004714). Reads were aligned to hg19 reference genome using BWA MEM v0.7.8 with the WASP pipeline to eliminate allelic mapping bias. Only reads with high mapping quality (> 10) were kept. PCR duplicates were removed using Picard tools. We performed quality control for CTCF ChIP-seq data by FRIP (Fraction of Reads In Peaks) and used datasets with FRIP > 10. Bigwig files were generated using bamCoverage commands in deepTools [77] v2.3.3. To compute signals in peaks, we used the merged CTCF peaks from Kasowski data. Analysis of ChIP-seq data from Grubert et al. Bigwig files and peaks for H3K27ac, H3K4me1, and H3K4me3 were downloaded from GEO database (GSE62742). Peaks for each mark were merged and then used to compute the averaged signal. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s13059-019-1855-4. Additional file 1: Figure S1. Hi-C derived molecular phenotypes measured across 20 LCLs. FigureS2. FIRE measures density of local Additional file 9: Table S10. Overlaps between 3D chromatin QTL and GWAS catalog. Additional file 10. Review history. Acknowledgements The authors would like to acknowledge members of the Ren lab and Dr. Graham McVicker for important discussions and feedback during preparation of this manuscript. Review history The review history is available as Additional file 10. Peer review information Yixin Yao was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Authors’ contributions Study was conceived and overseen by BR, MH, KH, JS, KG, and DUG. Hi-C experiments were performed by DUG and AS. Data analysis was performed by YQ, MH, KF-B (variable matrix cells, C-QTLs, related contact matrix analyses, power calculations), AN, YL, JC, and DUG. FISH experiments were performed by TL. The manuscript was written by DUG, YQ, MH, and KF-B with input from all authors. All authors read and approved the final manuscript. Funding This work was supported by NIH grant U54DK107977 to B.R. and M.H., and NIH grant U41HG007497 to J.S. D.U.G. was supported by fellowships from the A.P. Giannini Foundation and the NIH Institutional Research and Academic Career Development Awards (IRACDA) program. KH was supported by R01GM121459. KF-B was supported by a Maryland Genetics, Epidemiology and Medicine (MD-GEM) fellowship. Availability of data and materials All raw sequencing data and many processed data files are available through NCBI’s Gene Expression Omnibus (GEO) accession GSE128678 [78], as well as through the 4D Nucleome data portal (https://data.4dnucleome.org). Additional processed data are available on the Ren lab’s website at http:// renlab.sdsc.edu/renlab_website/download/iqtl/, or by request. The following third-party datasets were also used in this study (also listed in Additional file 2: Table S2): GSE48592 [79], GSE52457 [80], GSE63525 [81], GSE50893 [82], GSE47991 [83], GSE62742 [84], E-ERAD-141 [85], and GSE31388 [86]. Code is available at Zenodo accession 3475719 [87], and released under a Common Development and Distribution License compliant with OSI (http://opensource.org/licenses). Gorkin et al. Genome Biology (2019) 20:255 Ethics approval and consent to participate This study is exempt from IRB Review according to 45 CFR 46.101(b), criteria 4. All human samples analyzed are publicly available as part of the NIGMS Human Genetic Cell Repository, distributed through Coriell Institute for Medical Research. Competing interests B.R. is co-founder and shareholder of Arima Genomics. A.D.S. is employee and shareholder of Arima Genomics. The other authors declare that they have no competing interests. Author details 1 Ludwig Institute for Cancer Research, La Jolla, CA, USA. 2Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA. 3Bioinformatics and Systems Biology Graduate Program, University of California San Diego, La Jolla, CA, USA. 4Department of Quantitative Health Sciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA. 5McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins School of Medicine, Baltimore, MD, USA. 6Department of Biostatistics, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA. 7Current address: Arima Genomics, San Diego, CA 92121, USA. 8 Department of Pediatrics, University of California San Diego, La Jolla, CA, USA. 9Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, CA, USA. 10Department of Psychiatry, University of California, San Diego, La Jolla, CA 92093, USA. 11Department of Genetics, Department of Biostatistics, and Department of Computer Science, University of North Carolina, Chapel Hill, Chapel Hill, NC, USA. 12Institute of Genomic Medicine and Moores Cancer Center, University of California San Diego, La Jolla, CA, USA. 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Pezotettix giornae (Rossi, 1794) (Orthoptera, Acrididae, Pezotettiginae) en Galicia (NO península ibérica).
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Pezotettix giornae (Rossi, 1794) (Orthoptera, Acrididae, Pezotettiginae) en Galicia (NO península ibérica). Rubén Pino Pérez1, David Llucià-Pomares2 & Juan José Pino Pérez3 Rubén Pino Pérez1, David Llucià-Pomares2 & Juan José Pino Pérez3 1 Departamento de Biología Vegetal y Ciencias del Suelo. Universidad de Vigo. Lagoas - Marcosende 363 artamento de Biología Vegetal y Ciencias del Suelo. Universidad de Vigo. Lagoas - Marcosende 36310 o (Pontevedra España) Pontevedra ruben pino perez@gmail com 1 Departamento de Biología Vegetal y Ciencias del Suelo. Universidad de Vigo. Lagoas - Marcosende 36310 - Vigo (Pontevedra, España) Pontevedra. ruben.pino.perez@gmail.com ORCID:https://orcid.org/0000-0001-9665-3900 - Vigo (Pontevedra, España) Pontevedra. ruben.pino.perez@gmail.com ORCID:https://orcid.org/0000-0001-9665-3900 Vigo (Pontevedra, España) Pontevedra. ruben.pino.perez@gmail.com ORCID:https://orcid.org/0000-0001-9665-3900 2 2) Museu de Ciències Naturals de Barcelona. Laboratori de Natura, Departament d’Invertebrats. Picasso s/n, 08003, Barcelona (Barcelona, España) dllucia1219@hotmail.com ORCID:https://orcid.org/0000-0002-0307-5855 2 2) Museu de Ciències Naturals de Barcelona. Laboratori de Natura, Departament d’Invertebrats. Picasso s/n, 08003, Barcelona (Barcelona, España) dllucia1219@hotmail.com ORCID:https://orcid.org/0000-0002-0307-5855 3 Departamento de Ecología y Biología Animal. Facultad de Biología. Universidad de Vigo. Lagoas - Marcosende 36310 - Vigo (Pontevedra, España). jj.pino.perez@gmail.com ORCID: https://orcid.org/0000-0001-5609-9458 3 Departamento de Ecología y Biología Animal. Facultad de Biología. Universidad de Vigo. Lagoas - Marcosende 36310 - Vigo (Pontevedra, España). jj.pino.perez@gmail.com ORCID: https://orcid.org/0000-0001-5609-9458 Cómo citar este artículo: Pino Pérez, R.; Llucià-Pomares, D. & Pino Pérez, J. J. 2021. Pezotettix giornae (Rossi, 1794) (Orthoptera, Acrididae, Pezotettiginae) en Galicia (NO península ibérica). Boletín Biga, 19: 79-110. https://doi.org/10.5281/zenodo.5526973. Fecha de publicación: 24 de octubre de 2021. Cómo citar este artículo: Pino Pérez, R.; Llucià-Pomares, D. & Pino Pérez, J. J. 2021. Pezotettix giornae (Rossi, 1794) (Orthoptera, Acrididae, Pezotettiginae) en Galicia (NO península ibérica). Boletín Biga, 19: 79-110. https://doi.org/10.5281/zenodo.5526973. Fecha de publicación: 24 de octubre de 2021. Resumen El conocimiento de la ortopterofauna gallega es todavía muy incompleto. Se han realizado pocos estudios sobre ese grupo de especies, el catálogo de sus especies dista de estar completo y se des- conocen los ciclos biológicos de la mayoría de ellas. En este trabajo damos cuenta de la presencia de Pezotettix giornae (Rossi, 1794) (Acrididae) en Galicia tras las exploraciones de campo y el estudio de distintas colecciones, ofreciendo datos sobre su ciclo biológico, ecología y rango altitu- dinal. También se recoge la literatura científica de carácter faunístico conocida para el contexto ibérico (Anexo). Consideramos que se trata de una especie mesoxerófila, abundante en las zonas de bioclima mediterráneo y escaso en la franja litoral atlántica. Se aporta abundante información corológica ibérica inédita, a partir del estudio de la colección del MNCN de Madrid. Palabras clave: Pezotettix giornae, Pezotettiginae, Acrididae, Caelifera, Orthoptera, morfología, corología, fenología, ecología, distribución potencial, Galicia, NO península ibérica. Key words: Pezotettix giornae, Pezotettiginae, Acrididae, Caelifera, Orthoptera,morphology, cho- rology, phenology, ecology, potential distribution, Galicia, NW Iberian Peninsula. Boletín BIGA 19 (2021) http://www.biga.org Vol. 19: 79-110 Boletín BIGA 19 (2021) http://www.biga.org Vol. 19: 79-110 ISSN: 1886-5453 Introducción La subfamilia Pezotettiginae Brunner von Wattenwyl, 1893, encuadrada en la familia Acrididae MacLeay, 1821 (Orthoptera, Caelifera), cuenta actualmente con dos géneros, Pezotettix Burmeis- ter, 1840 y Sphenophyma Uvarov, 1934 y 10 especies (Cigliano, 2021). Sin embargo, Chapco (2013: 17) encuentra ambos géneros muy separados filogenéticamente, y recoge al género Eypre- pocnemis Fieber, 1853, que cuenta con 31 especies repartidas ampliamente por Europa, Asia y África, como el grupo más cercano a Pezotettix (Chapco, 2013: 18). En todo caso, Pezotetti- ginae y Catantopinae (s.l.) son grupos no monofiléticos, probablemente parafiléticos, puesto que Sphenophyma, restringido actualmente a Asia Menor, fue el primer género en divergir del ancestro común de todo el cuerpo de taxones de Catantopinae. Esa región pudo haber sido el lugar de origen de estas subfamilias, bien a partir de movimientos geográficos del ancestro, bien que aquel estuviera muy extendido y algunos elementos pudieran evolucionar hasta convertirse en los táxones de rango limitado que conocemos hoy en día (Chapco, 2013: l.c.). Figura 1: Distribución de Pezotettix giornae en el Mundo. Georreferencias de GBIF.org [10/07/2021]: en amarillo los países donde ha sido indicado; en rojo, especímenes preservados; en azul, observaciones. Figura 1: Distribución de Pezotettix giornae en el Mundo. Georreferencias de GBIF.org [10/07/2021]: en amarillo los países donde ha sido indicado; en rojo, especímenes preservados; en azul, observaciones. Abstract The knowledge of the Galician orthopterofauna is still very incomplete. Few studies have been carried out on this group of species, the catalog of their species is far from complete and the biolo- gical cycles of most of them are unknown. In this work we report the presence of Pezotettix giornae (Rossi, 1794) (Acrididae) in Galicia after field explorations and the study of different collections, offering data on its biological cycle, ecology and altitudinal range. The faunistic scientific literature known in for the Iberian Peninsula is also collected (Annex). We consider that it is a mesoxerop- hilic species, abundant in the Mediterranean bioclimate areas and scarce in the Atlantic coastline. Unpublished Iberian chorological information is provided, based on the study of the collection of the MNCN of Madrid. Key words: Pezotettix giornae, Pezotettiginae, Acrididae, Caelifera, Orthoptera,morphology, cho- rology, phenology, ecology, potential distribution, Galicia, NW Iberian Peninsula. 79 ISSN 1886-5453 (Edición en línea) Pino Pérez et al. Introducción Efectivamente, casi todas las especies del género Pezotettix presentan una distribución más o menos restringida a los países del entorno de la península de Asia Menor, como Turquía, Siria, Líbano, Chipre, Jordania o Israel, excepto Pezotettix giornae (Rossi, 1794) que presenta una distri- bución más amplia, por casi toda la cuenca mediterránea, en todo el S de Europa, Norte de África y Asia Menor (Albania, Alemania, Argelia, Austria, Bosnia-Herzegovina, Bulgaria, Croacia, Egipto, Eslovaquia, Eslovenia, España, Francia, Gibraltar, Grecia, Hungría, Italia, Macedonia, Marrue- cos, Moldavia, Montenegro, Portugal, Rumanía, Rusia, Serbia, Suiza, Túnez, Turquía y Ucrania) (Bolívar, 1876: 301; Brunner von Wattenwyl, 1882: 231; Bolívar, 1898: 31; Navàs, 1909: 200; Bolívar, 1915: 66; Hebard, 1925: 48; Uvarov, 1927: 213; Johnston, 1956: 2621; Bey- Bienko & Mistshenko, 1963: 188; Chopard, 1951: 359; Čejchan, 1963: 765; Gausz, 1970: 70; Harz, 1975: 330; Llorente, 1980: 143; Presa et al., 1996: 16; Kocárek, 1999: 150; Kollaros & Legakis, 1999: 285; Nagy & Nagy, 2000: 151; Braud et al., 2002: 15; Krištín, 2004: 47; Bounechada et al., 2006: 250; Sombke & Schlegel, 2007: 134; Nagy et al., 2010: 229; Baden- hausser, 2012: 399; Chobanov, 2013: 21; Moyano, 2014: 110; Šerić Jelaska & Skejo, 2014: 64; Skejo & Stanković, 2014: 17; Pina et al., 2017: 32; Sackl, 2018: 146; Cigliano, 2020) (fig. 1). Pino Pérez et al. Pezotettix giornae en Galicia 80 Boletín Biga 19 (2021): 79-110. Pino Pérez et al.: Pezotettix giornae en Galicia ISSN 1886-5453 (Edición en línea) Las citas más septentrionales las encontramos en Hungría (Nagy et al., 2010: 235), Suiza (Krištín, 2004: 49), Rumanía (Moyano, 2014: 110) y Austria (Adlbauer & Sackl, 1993: 60), si descartamos el registro de Nordrhein-Westfalenen (Alemania) en GBIF tomado de observation.org. Por el sur, las más meridionales corresponden a los registros del Alto Atlas de Marruecos (Badih, 1997: 112), la provincia marroquí del Ifni (Chopard, 1943), Argelia y Túnez (Defaut & Mo- richon, 2015). Respecto a la presencia de P. giornae en Egipto, apoyada en un único registro recogido en GBIF, y las dudas que pudiera plantear, hemos podido verificar la identidad del único ejemplar en que se basa, depositado en el Lund Museum of Zoology (MZLU), con referencia “MZ- LU:ENT: 101488”, a partir de la revisión de distintas fotografías de dicho ejemplar realizadas por Christoffer Fägerström y remitidas amablemente por Ellen Sandström. Pino Pérez et al. Introducción A pesar de ello, teniendo en cuenta que es el único registro conocido del país, y lo disyunto de dicha localización, su presencia debería ser confirmada con nuevas observaciones. Figura 2: Distribución de Pezotettix giornae en la península ibérica tomando como base las referencias bibliográ- ficas, los especímenes preservados en GBIF, los ejemplares conservados en el Museo Nacional de Ciencias Naturales (MNCN) y nuestros propios datos para Galicia. Figura 2: Distribución de Pezotettix giornae en la península ibérica tomando como base las referencias bibliográ- ficas, los especímenes preservados en GBIF, los ejemplares conservados en el Museo Nacional de Ciencias Naturales (MNCN) y nuestros propios datos para Galicia. En GBIF hay 5341 registros de P. giornae de los que 683 son de la península ibérica y uno de Tomiño (Pontevedra, Spain), 2012-08-08, 41.9951, -8.7616, Francisco Barros leg.; de ellos, 73 son especímenes preservados, pero ninguno gallego. En el portal de Bold Systems, hay 20 registros [2021/09/27] pero solo 5 públicos, todos del sur de Europa. Se considera una especie de amplia distribución circummediterránea (Galvagni, 2010: 174) y abundante en muchas zonas, e incluso se ha señalado que en ocasiones aparece como plaga (Reinhardt et al., 2003: 12). En la península ibérica se encuentra en prácticamente todo el territorio a excepción de la vertiente atlántica y NO ibérico (Navás, 1910: 242; Gangwere & Morales Agacino, 1970: 18; Pulido, 1990: 140; Barranco & Pascual, 1992: 616; Pardo et al., 1993: 77; Pardo & Gómez, 1995: 17; Vidal, 2000: 83; Llucià-Pomares, 2002: 83; Pina et al., 2017: 32) y también en Baleares (Navàs, 1909: 200); tampoco ha sido indicada del principado de Andorra. Para una relación más completa de las referencias, véase el anexo de bibliografía ibérica de este trabajo. Pezotettix giornae en Galicia 81 81 ISSN 1886-5453 (Edición en línea) En la figura 2 puede verse una distribución de P. giornae en la península ibérica solo teniendo en cuenta los datos extraídos de las fuentes bibliográficas, los especímenes preservados en GBIF, los ejemplares conservados en el Museo Nacional de Ciencias Naturales (MNCN) y nuestros propios datos para Galicia. Mientras que en la figura 3 puede verse la distribución en base a las observaciones recogidas en GBIF. Mientras que en la figura 3 puede verse la distribución en base a las observaciones recogidas e GBIF. Figura 3: Distribución de Pezotettix giornae en la península ibérica en base a las observaciones de GBIF. Pino Pérez et al. Introducción Figura 3: Distribución de Pezotettix giornae en la península ibérica en base a las observaciones de GBIF. P. giornae es un acrídido escuamíptero de alas vestigiales y hábitos eminentemente epígeos; es una especie de carácter termófilo y heliófilo, que se comporta como euritópica en su área de distri- bución (Chobanov, 2013: 13). Se trata de una especie muy común con una gran valencia ecológica (Massa et al., 2012: 408) lo que le permite colonizar biotopos muy variados como sotobosques, bosques de ribera, dehesas, alcornocales, encinares, prados naturales y de siega, pinares, o zonas con vegetación higrófila (Llorente, 1980: 143; Presa et al., 1983: 260; Llucià-Pomares, 2002: 83; Moyano, 2014: 249, Skejo & Stanković, 2014: 17; Llucià-Pomares & Fernández-Ortín, 2016: 286; Parejo-Pulido & Rodríguez, 2018: 307). No obstante, Gangwere & Llorente (1992: 67) señalan que se trata de un elemento fundamentalmente xerófilo, aunque otros autores como Massa et al. (2012: 408) o Krištín (2004: 47) lo definen como mesoxerófilo; finalmente, Defaut & Morichon (2015: 350) indican una ecología estacional que, según la bioclimatología de cada región y las particularidades microclimáticas locales, puede extenderse desde biotopos hi- perxéricos, (ej. cantera del Montmaurin, Haute-Garonne, Francia), hasta muy húmedos (ej. mont Ventoux, Vauclause, Francia). En Cataluña, es abundante sobre suelos calizos, en matorrales de jarales y brezales, garrigas, zarzales, juncales, prados sabanoides y herbazales de ribera (Olmo Vidal, 2006: 316). Por otra parte, Gauffre et al. (2015: 1725) sostienen que P. giornae tiene un patrón genético complejo como resultado de variaciones espaciales estructurales en el paisaje; las barreras lineales reducen el flujo de genes entre las poblaciones. Chopard (1951: 359) o Massa et al. (2012: 408) señalan como periodo fenológico de los adultos de junio a noviembre, pero en realidad se pueden encontrar ejemplares maduros en casi cualquier Pezotettix giornae en Galicia 82 ISSN 1886-5453 (Edición en línea) época del año dependiendo del área geográfica (Fig. 4). época del año dependiendo del área geográfica (Fig. 4). Figura 4: Fenología de Pezotettix giornae en la península ibérica Número de registros de adultos y ninfas por mes. Fuente GBIF.org (especímenes preservados) [10/07/2021], MNCN, referencias bibliográficas y datos propios. 1668 registros. Figura 4: Fenología de Pezotettix giornae en la península ibérica Número de registros de adultos y ninfas por mes. Fuente GBIF.org (especímenes preservados) [10/07/2021], MNCN, referencias bibliográficas y datos propios. 1668 registros. Pino Pérez et al. Introducción El adulto presenta diapausa invernal, muy bien documentada en algunas zonas (Brunner von Wattenwyl, 1882: 82; Chopard, 1951: 359; Harz, 1975: 330; Llorente, 1978: 143; Reinhardt et al., 2003: 12; Bounechada et al., 2006: 250; Kollaros & Legakis, 1999: 285; Moyano, 2014: 110). Según Bounechada et al. (2006: 251) desarrollan sus estadios ninfales entre finales de primavera y principios del verano. Las cópulas aparecen con mayor frecuencia entre septiembre y noviembre. Algunos adultos pasarían el invierno en diapausa para realizar las puestas hasta finales de abril, lo que sugiere a su vez, una supuesta diapausa embrionaria durante el verano. Sin embargo, el ciclo puede variar fundamentalmente en función de la latitud (Harz, 1975: 330) y la altitud. Por ejemplo, Kollaros & Legakis (1999: l.c.) descubrieron diferencias importantes en los ciclos, entre las poblaciones del N y del S de Creta. Los adultos pueden confundirse con las ninfas pero se distinguen por la presencia del tubérculo cuadrangular desarrollado en el prosterno, las carenas de las tibias posteriores (Chopard, 1951: 359) y, muy especialmente, por la presencia o no de tegminas. También es posible confundirlos con las ninfas de otros acrídidos, especialmente Calliptamus spp. P. giornae es una especie polífaga que se comporta como eurífaga en cautividad (Moyano, 2014: 271). Presenta los molares contiguos a la línea de corte con los incisivos (forvíboros sensu Gang- were, 1965), estructura adecuada para una dieta herbácea o arbustiva tierna (Moyano, 2014: 18). Se alimenta de monocotiledóneas según Gangwere & Llorente (1992: 80) pero Gausz (1970: 70) identifica a Salvia pratensis L. (Lamiaceae) como su planta nutricia en Hungría y liga su abundancia a la de esta labiada. Asimismo, según Defaut & Morichon (2015), en Francia su presencia parece estar condicionada en gran medida a la de gramíneas, siendo Brachypodium pinnatum una de sus predilectas en Chasteaux (Corrèze), aunque también puede ser abundante en zonas donde el recubrimiento de dichas plantas apenas alcanza el 10 %; estos mismos autores también indican la ingesta de la leguminosa Ononis repens y accesoriamente de Stachys recta y Anthyllis vulneraria. Por su parte, Moyano (2014: 249) registra la preferencia de las ninfas por Cistus crispus L. (Cistaceae) frente a la ingesta de gramíneas en cautividad, pero constata su capacidad adaptativa ante los diferentes recursos tróficos. Ya Brunner von Wattenwyl (1882: Pezotettix giornae en Galicia 83 ISSN 1886-5453 (Edición en línea) 231) apuntaba que eran muy abundantes en Rubus sp. (Rosaceae). Pino Pérez et al. Introducción En este sentido, recientemen- te Martín-Vega et al. (2013), incluyen a Pezotettix giornae entre las especies estudiadas que, facultativamente, pueden presentar comportamiento necrófago. En el contexto europeo, Hochkirch et al. (2016: 62) consideran a P. giornae como un taxon con preocupación menor (LC), es decir, que tras su evaluación no cumple ninguno de los criterios que definen las otras categorías de vulnerabilidad (UICN, 2012: 15). No obstante, a nivel local este estatus puede variar; así, mientras Defaut (2003: 31) lo incluye en la lista roja de especies del Midi-Pyrénées, con una regresión de hasta el 25 % en algunas zonas, otros autores como Sombke & Schlegel (2007: 136) lo consideran muy abundante en algunas islas de Croacia aunque sin alcanzar la consideración de plaga. En lo que concierne a Portugal, Pina et al. (2017: 32) lo consideran dentro de la categoría LC. Las amenazas más importantes para la viabilidad de las poblaciones son las alteraciones del pai- saje; el aumento de la superficie cultivada implica una reducción de la superficie de los pastizales y su fragmentación y ya hemos visto que Gauffre et al. (l.c.) consideran a cierta heterogenei- dad espacial como una barreta discreta para el flujo génico. También se ha constatado que sufre predación por algunos vertebrados como Falco vespertinus Linnaeus, 1766 (Szövényi, 2015: 53). Material y Métodos Los especímenes de Pezotettix giornae (Rossi, 1794), etiquetados con el sigloide LOU-Arthr están depositados en la colección de Arthropoda del Centro de Investigación Forestal (CIF) de Lourizán (Pontevedra); con el sigloide MNCN están depositados en el Museo Nacional de Ciencias Naturales (Madrid) y los etiquetados con las siglas DLP, en la colección particular de uno de los autores (David Llucià Pomares). Los ejemplares fueron capturados con los permisos preceptivos de la Xunta de Galicia. En el apartado de resultados los datos se exponen por orden alfabético de provincias, seguido del término municipal y localidad, georreferencia en el formato Military Grid Reference System (MGRS) con una precisión de 1 × 1 m, altitud en metros sobre el nivel del mar, la fecha, ecología de la zona de captura, observaciones sintaxonómicas en su caso, una referencia a su abundancia relativa, los colectores y el número de colección cuando lo tienen adjudicado, seguido finalmente del sexo y el estado, bien adulto, ninfa o ambos. Para la nomenclatura, seguimos a Cigliano et al. (2020). Para la nomenclatura, seguimos a Cigliano et al. (2020). La confección del mapa de distribución mundial (Fig. 1) se ha basado en el dataset de GBIF sobre la especie. Para el mapa de distribución de la península ibérica (Fig. 2) y gráficos fenológico y altitudinal (Figs., 4 y 9) hemos utilizado los datos de los repositorios de GBIF basados únicamente en ejemplares preservados, junto con los procedentes de referencias bibliográficas y los datos de los ejemplares del Museo Nacional de Ciencias Naturales (MNCN), además de los datos del material gallego inédito aquí presentado. Hemos representado en mapa aparte aquellas georreferencias de GBIF basadas en observaciones y cuya verificación no es por tanto posible (Fig. 3). En el anexo de este trabajo se recogen las 102 referencias bibliográficas consultadas para la confección de las figuras 2 y 3. En todos los casos, el concepto de registro utilizado se corresponde con la captura u observación de ejemplar o ejemplares, independientemente de su número y sexo, siempre y cuando pertenezcan a la misma especie o subespecie, hayan sido colectados en el mismo lugar (en sentido más o menos estricto) y fecha (día, mes y año), es decir, una unidad de información en el ámbito de la faunística. En el caso concreto del gráfico fenológico se ha tenido también en cuenta el estadio (adulto/ninfa). En el apartado de ‘Material comunicado’ hemos detallado por país y provincia, los datos de los ejemplares existentes en el MNCN y que nos fueron amablemente comunicados por Mercedes Paris, indicando en cada caso, localidad, georreferencia, altitud, ecología, sexo, estadio, número de colección, colector o colectores, determinadores y observaciones. Dado que en muchos de los regis- tros del MNCN no constan georreferencias, se asignaron coordenadas geográficas MGRS a partir de las localidades mencionadas en las etiquetas, siempre que fuera posible, con una precisión de Pezotettix giornae en Galicia 84 ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) 10 x 10 km, indicándose, en estos casos, entre corchetes. En principio, se siguió un criterio similar para la adjudicación de la altitud de las distintas localidades indicadas en los registros; cuando la localización de la cita permitía una precisión de 1 x 1 km en las coordenadas UTM, se adju- dicaba la altitud media de ese kilómetro cuadrado. Sin embargo, se mantenía cierta ambigüedad, principalmente en aquellos territorios de relieve montañoso, con grandes diferencias de altitud. Para la nomenclatura, seguimos a Cigliano et al. (2020). Utilizar la altitud máxima y mínima de cada cuadrícula tampoco fue posible porque al representar el gráfico altitudinal por rangos de 100 m, muchas de las altitudes de las cuadrículas pertenecerían a dos o más rangos, invalidando la correspondencia. Por todo ello, no se han inferido altitudes de los registros del MNCM. También se incluyen entre paréntesis los nombres de los determinadores cuando éstos están anotados en las etiquetas. Cuando no se indica como estado ninfa, se entiende que se trata de imagos. Para el gráfico de distribución de Galicia hemos utilizado nuestros propios datos (Fig. 6). No obstante, dado que los datos de presencia disponibles no cubren todo el rango de distribución natural de una especie, se ha utilizado un programa de modelación de la distribución que identifica el área en que es probable que se presente (Scheldeman & Zonneveld, 2011: 147) mediante un mapa de distribución potencial (Fig. 18). En nuestro caso, se basa en el contraste de los puntos de presencia conocidos con un conjunto de variables climáticas sobre un ráster. Las variables climáticas utilizadas se han tomado de la base de datos Wordclim [http://www.worldclim.org] con una resolución de 30’’ que equivalen a celdas de aproximadamente 1 km2 para el territorio de Galicia, N de Portugal y O de las regiones de Asturias y Castilla y León y que han sido generadas mediante la interpolación de datos climáticos mensuales. Para la nomenclatura, seguimos a Cigliano et al. (2020). Las variables utilizadas son las siguientes: BIO1 = Temperatura Media Anual BIO2 = Intervalo Diurno Medio (Media mensual de (temp max - temp m BIO3 = Isotermalidad (BIO2/BIO7) (x 100) BIO4 = Temperatura Estacional (desviación estándar x 100) BIO5 = Temperatura Máxima del Mes más Cálido BIO6 = Temperatura Mínima del Mes más Frío BIO7 = Intervalo Anual de Temperatura (BIO5-BIO6) BIO8 = Temperatura Media del Trimestre más Húmedo BIO9 = Temperatura Media del Trimestre más Seco BIO10 = Temperatura Media del Trimestre más Cálido BIO11 = Temperatura Media del Trimestre más Frío BIO12 = Precipitación Anual BIO13 = Precipitación del Mes más Húmedo BIO14 = Precipitación del Mes más Seco BIO15 = Estacionalidad de la Precipitación (Coeficiente de Variación) BIO16 = Precipitación del Trimestre más Húmedo BIO17 = Precipitación del Trimestre más Seco BIO18 = Precipitación del Trimestre más Cálido BIO19 = Precipitación del Trimestre más Frío BIO19 = Precipitación del Trimestre más Frío Hemos utilizado el programa MaxEnt, que calcula el nicho de la especie para determinadas varia- bles climáticas y la probabilidad de presencia usando un algoritmo de máxima entropía (Philips et al., 2006; Rodrigues et al., 2010), por su capacidad predictiva y la versatilidad en su configuración de partida (Merow et al., 2013: 1058). El modelo ofrece una distribución en la que cada celda de la cuadrícula tiene una probabilidad de ocurrencia o abundancia, según el ráster examinado. También se han analizado las variables climáticas de mayor relevancia en la distribución real de la especie y se presenta un nicho climático bidimensional. Pino Pérez et al. Resultados Pino Pérez, LOU-Arthr 51547-51551 5 ♀♀; Rubiá, Biobra, 29TPH7504006692, 830 m, 4/05/2018, en prados clareados de bosque de Quercus ilex, R. Pino Pérez & J. J. Pino Pérez, LOU-Arthr 51385 ♂; Rubiá, Vilardesilva, mirador, 29TPH7888702894, 607 m, 30/09/2020, en las laderas de orientación este sobre el embalse de Penarrubia, en claros de encinar sobre suelos calizos, abundante, R. Pino Pérez & J. J. Pino Pérez, LOU-Arthr 52866-52870 ♀♀, LOU-Arthr 52871 ♂; Rubiá, entre Pardollán y Sobredo, laderas del Carabouxal, 29TPH7922200805, 372 m, 30/09/2020, en terrazas sedimentarias del río Sil con Hypericum perforatum L., Foeniculum vulgare, Quercus ilex y Pteridium aquilinum en suelos calizos con cuarcitas, abundante, R. Pino Pérez & J. J. Pino Pérez, ♂, LOU-Arthr 52887-52891 5 ♂♂; LOU-Arthr 52892-52895 4 ♀♀; Orense, Orense, /08/1908, Taboada leg., MNCN 278983, 279009, 279011 y 279012 4 ♀♀, 279010 1 ♂. Pontevedra, Cangas, Sierra Poniente, Darbo, 29TNG1719378736, 80 m, 03/11/2008, en prados de siega, escasa, R. Pino & J. J. Pino, LOU-Arthr 1097 ♀; Salvaterra de Miño, Corzáns, A Fraga, 29TNG4399262739, 50 m, 19/11/2014, en la hojarasca bajo un castaño, frecuente, R. Pino Pérez, LOU-Arthr 50923 ♂. Pontevedra, Cangas, Sierra Poniente, Darbo, 29TNG1719378736, 80 m, 03/11/2008, en prados de siega, escasa, R. Pino & J. J. Pino, LOU-Arthr 1097 ♀; Salvaterra de Miño, Corzáns, A Fraga, 29TNG4399262739, 50 m, 19/11/2014, en la hojarasca bajo un castaño, frecuente, R. Pino Pérez, LOU-Arthr 50923 ♂. Todas las poblaciones que hemos encontrado en Galicia (fig. 5) se sitúan en el Bioclima Medi- terráneo Pluviestacional Oceánico definido por López Fernández et al. (2008: 230s, mapa 2), es decir, al menos dos meses consecutivos con aridez durante el período más cálido del año, en los que el valor en milímetros de la precipitación media del bimestre más cálido del trimestre estival es menor del doble de la temperatura media del bimestre más cálido del trimestre estival expresada en grados centígrados en función de la continentalidad y del índice ombrotérmico (Rivas-Martínez, 2008). En la misma línea, Rodríguez Guitián & Ramil-Rego (2007: 41) señalan como ma- crobioclima de las zonas de hallazgo de las poblaciones de Navia de Suarna, Cartelle, Cangas y Salvaterra de Miño, el templado submediterráneo, mientras que las poblaciones del oriente gallego (Quiroga y Rubiá) se adscriben claramente al tipo mediterráneo. Resultados Material estudiado. Pezotettix giornae (Rossi, 1794), Mantissa Insectorum exhibens species nuper in Etruria collectas a Petro Rossio. Adjectis faunae etruscae illustrationibus, ac emendationibus. Tom II. Pisis. ex Pino Pérez et al. Pezotettix giornae en Galicia 85 ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) Typographia polloni praesidum facultats.154 pp. + Lám VIII: 104-105 (1794) sub Gryllus giornae. España: Lugo, Navia de Suarna, Penamil, 29TPH5890256449, 436 m, pastizal en ladera rocosa (esquistos), 13/8/2007, D. Llucià, DLP 1♂y 1♀; Quiroga, Montefurado, 29TPG4700494956, 268 m, 6/10/2017, en terrazas de cuarcita sobre el río Sil, en cópula, frecuente, R. Pino Pérez & J. J. Pino Pérez, LOU-Arthr 52898 ♂, LOU-Arthr 52899 ♀; Quiroga, Os Covallos, Montefurado, 29TPG4702394888, 257 m, 30/09/2020, en terrazas sedimentarias de cuarcitas del río Sil, sobre la vegetación, formada por Phyllirea angustifolia, Centaurea langei, Asperula aristata, Foeniculum vulgare, Andryala ragusina, Setaria parviflora, Setaria faberi y Erica australis, abundante, R. Pino Pérez & J. J. Pino Pérez, LOU-Arthr 52849-52853 4 ♀♀, LOU-Arthr 52854, 52855 2 ♂♂; Quiroga, Os Covallos, Montefurado, 29TPG4702394888, 257 m, 30/09/2020, en terrazas sedimentarias de cuarcitas del río Sil, sobre la vegetación, formada por Phyllirea angustifolia, Centaurea langei, Asperula aristata, Foeniculum vulgare, Andryala ragusina, Setaria parviflora, Setaria faberi y Erica australis, en cópula, abundante, R. Pino Pérez & J. J. Pino Pérez, LOU-Arthr 52856 ♀, LOU-Arthr 52857 ♂. Ourense, A Arnoia, Valdemeixeira, A Lomba, presa sobre el río Arnoia, 29TNG7302775776, 144 m, 29/03/2019, en prados húmedos cerca del río Arnoia, en Quercetum suberis, R. Pino Pérez, J. J. Pino Pérez & J. L. Camaño Portela, LOU-Arthr 51396-51397 2 ♀♀; A Arnoia, Valdemeixeira, A Lomba, presa sobre el río Arnoia, 29TNG7302775776, 144 m, 29/03/2019, en prados húmedos cerca del río Arnoia, en Quercetum suberis, en cópula, R. Pino Pérez, J. J. Pino Pérez & J. L. Camaño Portela, LOU-Arthr 51398 ♀, LOU-Arthr 51399 ♂; Cartelle, Valdemeixeira, A Lomba, presa sobre el río Arnoia, 29TNG7380875631, 178 m, 22/03/2019, en prados húmedos cerca del río Arnoia, en Quercetum suberis, R. Pino Pérez, J. J. Pino Pérez & J. L. Camaño Portela, LOU-Arthr 51390-51393 4 ♀♀; Rubiá, Covas, 29TPH7854204540, 500 m, 12/10/2007, en encinar sobre calizas, R. Pino Pérez, LOU-Arthr 52054 ♂, LOU-Arthr 52055 ♀; Rubiá, Biobra, 29TPH7504506698, 830 m, 23/09/2017, en claros de bosque de encinas (Quercus ilex), R. Pino Pérez & J. J. Pino Pérez et al. Pino Pérez et al. Resultados Para evaluar su adaptación a los diferentes ambientes, hemos extraído los datos climáticos de los sitios de presencia en Galicia de la base de datos de BioClim y hemos obtenido, por un lado, el nicho bidimensional con base a dos variables climáticas, la temperatura media anual y la precipitación Pezotettix giornae en Galicia 86 Boletín Biga 19 (2021): 79-110. Pino Pérez et al : Pezotettix gi ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) Figura 5: Distribución conocida de P. giornae en Galicia. En gris, las provincias donde consta su presencia. En amarillo, los municipios donde se han realizado capturas y los puntos rojos representan los lugares correspondientes a las georreferencias. Figura 5: Distribución conocida de P. giornae en Galicia. En gris, las provincias donde consta su presencia. En amarillo, los municipios donde se han realizado capturas y los puntos rojos representan los lugares correspondientes a las georreferencias. anual, y por otro, una de las variables climáticas más significativa en la distribución conocida de P. giornae (BIO10). Los resultados pueden verse en las figuras 7 y 8. Estos resultados habrán de tomarse con cautela porque se trata de un número limitado de datos; según estos, P. giornae se desarrolla en Galicia en un rango climático estrecho, lo que puede hacerle vulnerable a los cambios climáticos. Se restringe a zonas con temperaturas medias anuales elevadas para el territorio (11-15º C), pero con una pluviosidad anual moderadamente elevada (700-1400 mm) (fig. 6). De hecho, la medida de la temperatura media del trimestre más cálido, se muestra como una variable climática influyente en su distribución (Fig. 7) al igual que las variables de temperatura estacional o la temperatura máxima del mes más cálido. Pezotettix giornae en Galicia 87 Boletín Biga 19 (2021): 79-110. ISSN 1886-5453 (Edición en línea) Pino Pérez et al.: Pezotettix giornae en Galicia Figura 6: Nicho climático de P. giornae en Galicia. Figura 7: Temperatura Media del Trimestre más cálido. Boletín Biga 19 (2021): 79-110. ISSN 1886-5453 (Edición en línea) Pino Pérez et al.: Pezotettix giornae en Galicia ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) Figura 7: Temperatura Media del Trimestre más cálido. Figura 6: Nicho climático de P. giornae en Galicia. Figura 7: Temperatura Media del Trimestre más cálido. Desde un punto de vista sintaxonómico, las poblaciones se localizan en comunidades de Rusco aculeati-Quercetum roboris Br.-Bl., P. Pino Pérez et al. Pezotettix giornae en Galicia 88 Pezotettix giornae en Galicia Resultados Silva et Rozeira 1956, correspondientes a los hábitats se- mihúmedos de los suelos de soutos (Castanea sativa Mill.), o en prados húmedos sobre roquedos de solana en los bosques en galería de la subass. Quercetosum suberis Amigo et al. 1998, abun- dantes en los ríos del sur de Galicia (Izco et al., 1999: 42). Las poblaciones del oriente gallego se localizan sobre las clases Quercetea ilicis Br-Bl. ex A. & O. Bolòs 1950 y Rhamno-Prunetea Rivas Goday & Borja ex Tüxen 1962, bien sobre terrazas sedimentarias del río Sil y sus tributarios en las comunidades de Genisto hystricis-Quercetum rotundifoliae P. Silva 1970, bien sobre comunidades arbustivas de Rubo ulmifolii-Rosetum corymbiferae Rivas-Martínez et Arnaiz in Arnaiz 1979, con especies espinosas y lacerantes sobre suelos calizos y de gran insolación (Amigo Vázquez et al., 2005: 14) (Fig. 8). Figura 8: Vista parcial del encinar de Biobra (Rubiá, Ourense). En primer plano, claro del encinar donde hemos observado P. giornae. Figura 8: Vista parcial del encinar de Biobra (Rubiá, Ourense). En primer plano, claro del encinar donde hemos observado P. giornae. Pino Pérez et al. Pezotettix giornae en Galicia 88 ISSN 1886-5453 (Edición en línea) En Galicia, presenta un rango altitudinal moderado, entre los 50 y los 830 m. A partir de 28 registros extraídos del material depositado en el MNCN y cuyos datos fueron comunicados por Mercedes Paris, de la bibliografía consultada (1522 registros) y nuestros propios datos (12 registros), se ha obtenido una distribución altitudinal en la península ibérica, que permite concluir una clina bimodal de las poblaciones, por debajo de los 300 m y entre los 1000 y 1600, mientras que a partir de 1700 disminuyen rápidamente las observaciones (Fig. 9). Sin embargo, estos resultados deben considerarse con cautela ya que los datos recopilados han sido obtenidos a partir de estudios faunísticos muy heterogéneos a lo amplio del territorio peninsu- lar, e irregulares en el esfuerzo de muestreo. Por otra parte, la distribución de las especies a lo largo del gradiente altitudinal no ha sido estudiada en general en los ortópteros de la península ibérica y se desconocen, en gran medida, los factores ecológicos responsables de la misma. Lomolino (2001: 10) señala que es importante conocer los sesgos de muestreo, ya que, en algunos casos, pueden crear patrones espurios o dificultar la detección de los patrones auténticos de diversidad a lo largo de gradientes de elevación. Pino Pérez et al. Resultados La disminución de registros en el rango situado entre los 400 y los 900 m, puede deberse, precisamente a esos sesgos. Por una parte, los trabajos de muestreo tienden a priorizar las cotas medio-altas y altas donde se concentran el mayor número de endemismos y las especies corológicamente de mayor interés, y por otra, las localidades situadas a cotas más bajas suelen estar sobremuestreadas por su mayor accesibilidad y proximidad a las grandes ciudades del litoral mediterráneo, ubicación de un gran número de centros de investigación. Figura 9: Gráfico altitudinal de Pezotettix giornae en la península ibérica. Fuente MNCN, referencias bibliográficas y datos propios. 1562 registros. Figura 9: Gráfico altitudinal de Pezotettix giornae en la península ibérica. Fuente MNCN, referencias bibliográfic y datos propios. 1562 registros. Desde el punto de vista fenológico, hemos encontrado adultos en marzo y mayo y tras el verano, en septiembre, octubre y noviembre. En los meses de primavera lo hemos visto en cópula repeti- damente, al igual que en el otoño, por lo que presentan diapausa invernal en estado adulto, tanto machos como hembras. Hemos comprobado, al igual que lo señalado por Reinhardt et al. (2003: 12), que las cópulas duran muchas horas por lo que es frecuente observarlos así y se muestran renuentes a separarse. Se muestran más activos en las horas centrales del día, cuando las tempe- raturas son más altas, pero hemos observado ejemplares con mayor o menor grado de actividad desde el amanecer (6:00 h) hasta el anochecer. Desarrollan su actividad preferentemente en el suelo pero también los hemos localizado sobre las plantas (gramíneas), aunque nunca a más de 50 cm del suelo, dato coincidente con lo indicado por Defaut & Morichon (2015) que sitúan el límite en la altura del recubrimiento vegetal donde puede prosperar la especie entre los 40 y los 60 cm. Pezotettix giornae en Galicia 89 ISSN 1886-5453 (Edición en línea) Nuestros ejemplares muestran cierto tamaño dimórfico, con una longitud hasta el extremo de las rodillas posteriores entre 11,85 - 13,65 mm (¯x = 12,58, = 0,51) ♂♂y entre 14,40 -17,70 mm (¯x = 15,43, = 0,72) ♀♀. Casi todas las especies de Caelifera muestran un dimorfismo del tamaño sexual (SSD) sesgado hacia las hembras (Hochkirch & Gröning, 2008: 189). Pezotettix giornae en Galicia Pezotettix giornae en Galicia Pino Pérez et al. Resultados Según Rensch (1950: 69) algunos grupos de animales muestran mayores diferencias en el tamaño corporal de ambos sexos a medida que las especies son más grandes, aunque se mantenía cauto en la aplicación de esta regla para los grupos no estudiados por él, como los ortópteros. Tanto García‐Navas et al. (2017: 11) como Hochkirch & Gröning (l.c.), concluyen que las especies de Caelifera, no cumplen la regla de Rensch, sino más bien su inverso, las hembras son proporcionalmente más grandes que los machos en las especies grandes, lo que refuerza la opinión de que dicha regla es poco frecuente en grupos taxonómicos que exhiben SSD sesgado hacia las hembras como es el caso de P. giornae. García‐Navas et al. (2017: 8) hallaron para esta especie, una variación en el grado de dimorfismo de tamaño sexual de 0,83 y una duración de la temporada de reproducción de casi 6 meses, y sostienen que ese grado de SSD sesgado para las hembras está correlacionado con la duración de la temporada de reproducción, de tal forma que aquellas especies con una fenología más amplia tienen un tamaño más dimórfico que otras con ventanas reproductivas más reducidas. (Figs. 10, 11, 12, 13, 14 y 15 para material galaico y 16, 17 a 18 para material ibérico extragalaico). Figura 11: Pezotettix giornae ♀. Cartelle (Orense). Figura 10: Pezotettix giornae ♂. Quiroga (Lugo). Figura 10: Pezotettix giornae ♂. Quiroga (Lugo). Figura 11: Pezotettix giornae ♀. Cartelle (Orense). Figura 12: Andropigio. Figura 13: Ginopigio. Figura 13: Ginopigio. Figura 12: Andropigio. Pino Pérez et al. Pezotettix giornae en Galicia Pezotettix giornae en Galicia 90 Boletín Biga 19 (2021): 79-110. ISSN 1886-5453 (Edición en línea) Pino Pérez et al.: Pezotettix giornae en Galicia ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) Figura 14: Pezotettix giornae en cópula. Rubiá (Orense). Figura 14: Pezotettix giornae en cópula. Rubiá (Orense). Figura 15: Ejemplares de Pezotettix giornae en la colección LOU-Arthr. Figura 15: Ejemplares de Pezotettix giornae en la colección LOU-Arthr. Pezotettix giornae en Galicia Pino Pérez et al. 91 Boletín Biga 19 (2021): 79-110. ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) Figura 16: Habitus dorsal del macho y hembra de Pezotettix giornae. Material ibérico extragalaico. Figura 16: Habitus dorsal del macho y hembra de Pezotettix giornae. Material ibérico extragalaico. Pezotettix giornae en Galicia Pino Pérez et al. Pino Pérez et al. Pino Pérez et al. Resultados 92 Boletín Biga 19 (2021): 79-110. ISSN 1886-5453 (Edición en línea) Pino Pérez et al.: Pezotettix giornae en Galicia Figura 17: Detalles morfológicos de P. giornae. a) Cabeza y pronoto en visión dorsal; b) placas esternales y prosterno; c) Pata posterior (cara externa e interna, respectivamente); d) Cabeza, pronoto y tegmina en visión lateral; e) detalle de tegmina. Material ibérico extragalaico. Figura 17: Detalles morfológicos de P. giornae. a) Cabeza y pronoto en visión dorsal; b) placas esternales y prosterno; c) Pata posterior (cara externa e interna, respectivamente); d) Cabeza, pronoto y tegmina en visión lateral; e) detalle de tegmina. Material ibérico extragalaico. Pino Pérez et al. 93 Pezotettix giornae en Galicia ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) 8: Terminalia abdominal (últimos terguitos, esternitos, placa subgenital, epiprocto, cercos y ov la hembra) de P. giornae. Macho: a, b, c, d. Hembra: e, f. a) en visión lateral; b) detalle de sal; d) visión ventral; e) visión dorsal y f) visión ventral. z et al. 94 Pezotettix giornae Figura 18: Terminalia abdominal (últimos terguitos, esternitos, placa subgenital, epiprocto, cercos y oviscapto en el caso de la hembra) de P. giornae. Macho: a, b, c, d. Hembra: e, f. a) en visión lateral; b) detalle del cerco; c) visión dorsal; d) visión ventral; e) visión dorsal y f) visión ventral. Figura 18: Terminalia abdominal (últimos terguitos, esternitos, placa subgenital, epiprocto, cercos y oviscapto en el caso de la hembra) de P. giornae. Macho: a, b, c, d. Hembra: e, f. a) en visión lateral; b) detalle del cerco; c) visión dorsal; d) visión ventral; e) visión dorsal y f) visión ventral. Figura 18: Terminalia abdominal (últimos terguitos, esternitos, placa subgenital, epiprocto, cercos y oviscapto en el caso de la hembra) de P. giornae. Macho: a, b, c, d. Hembra: e, f. a) en visión lateral; b) detalle del cerco; c) visión dorsal; d) visión ventral; e) visión dorsal y f) visión ventral. Pino Pérez et al. Pezotettix giornae en Galicia 94 Boletín Biga 19 (2021): 79-110. Pino Pérez et al.: Pezotettix giornae en Galicia ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) Distribución potencial. Con los datos disponibles, hemos intentado establecer un modelo teórico de la superficie ocupada por P. giornae en Galicia, mediante el programa MaxEnt. Este software asume que la muestra de puntos de presencia se ha obtenido de manera aleatoria en el territorio estudiado y que se desconoce el tamaño de la población. Con estos supuestos de partida, para probabilidades superiores al 70 %, se observa que la distribución de P. giornae se extiende sobre las cuencas fluviales meridionales de los ríos Miño y Cabe, y sobre todo del río Sil, que resulta ser la vía de acceso y contacto con las poblaciones del centro de la península ibérica, a través de la provincia de León. Por el sur, las cuencas de los valles de los ríos Limia, Támega, Mente o Pentes, entre otros, permiten la conexión de las poblaciones portuguesas con las gallegas (fig. 19), mientras que por el norte, es posible su presencia en las terrazas sedimentarias de solana de los ríos Navia y Eo y sus tributarios. Las costas del golfo ártabro y las zonas con mayor influencia atlántica parecen las menos adecuadas para sus poblaciones. En la Galicia rural, principalmente aquella sin concentración parcelaria, encontramos un aban- dono progresivo de los cultivos tradicionales y una transformación de los diferentes ecosistemas hacia su etapa climácica, favoreciendo el desarrollo de las poblaciones y apoyando su consideración como LC. Por el contrario, en la franja atlántica sur, con una densidad de población humana ele- vada y un paisaje extremadamente fragmentado y uniforme, las poblaciones se rarifican y podrían llegar a catalogarse como vulnerables al enfrentarse a un riesgo de extinción medio a alto. Figura 19. Distribución potencial de P. giornae en Galicia (MaXent). En azul, los puntos de presencia. Figura 19. Distribución potencial de P. giornae en Galicia (MaXent). En azul, los puntos de presencia. Pino Pérez et al. Material ibérico comunicado. Llorente 2006, 292289, 292302, 292301, 1 ♂, MNCN 292269. Chiclana, [29SQA53], 4 ♀♀, MNCN 292266, 292267, 292278, 292297, 2 ♂♂, MNCN 292281, 292282, 4 ♀♀, MNCN 279025, L. Cepero, 279021, 279022, Etiqueta del primer ejemplar de la serie: Pl. G. var. rufipes Br. Pezotettix giornae (Rossi, 1794) [=Platyphyma giornae var. rufipes Brunner von Wattenwyl, 1882] según OSF (acceso 09/02/2021), 292360, 3 ♂♂, MNCN 279023, 279024, 279030. El Parralejo, [30STF44], XII-2004, 4 ♀♀, MNCN 292291, M. Gª París, det. V. Llorente 2006. Estella del Marqués, [29SQA66], 292293, 292294, 292292, 2 ♂♂, MNCN 292296, 292295. La Blanca, [29SQA56], 2 ♀♀, MNCN 292290, 292303, 2 ♂♂, MNCN 292262, 292280. Medina Sidonia, [30STF33], 3 ♀♀, MNCN 292286, 292285, 292284, 2 ♂♂, MNCN 292283, 292279. S. José del Va- lle, [30STF45], 4 ♀♀, MNCN 292288, 292287, 292259, 292258, 2 ♂♂, MNCN 292260, 292264. San Roque, [30STF81], 19-IX-1996, 3 ♀♀, MNCN 292309, J. Ramírez, det. V. Llorente 2006, 292308, 292307, 4 ♂♂, MNCN 292311, 292316, 292312, 292317, 23-X-1996, 1 ♂, MNCN 292310, det. V. Llorente 2006, 27-VIII-1996, 3 ♂♂, MNCN 292313, det. V. Llorente 2006, 292315, 292314. CASTELLÓN. Benicassim: Desierto de las Palmas, [31TBE44], 05-IV-1992, 1 ♀, MNCN 292385, J Íñiguez det J Íñiguez Morella [30TYL40] 1 ♂MNCN 279016 J Royo CIUDAD REAL. Cortijos de Malagón, [30SVJ23], 6-X, 2 ♂♂, MNCN 279033, (sin datos), 279034. Guadalmez, 30SUH3285, 18-II-2019, 1 ♂, MNCN 261626, M. Domenech, det. M. Dome- nech 2019. Piedrabuena: Tabla de la Hiedra, 30SUJ9223, 550 m, 24-X-2004, prado alto vega (suelo pedregoso), 1 ♀, MNCN 292376, J. Íñiguez, det. J. Íñiguez., p g ), ♀, , g , g , CÓRDOBA. Iznájar, [30SUG82], IV-1909, 1 ♀, MNCN 278994, Exp. del Museo. Srra Córdoba, 21-III-1982, 1 ♀, MNCN 292221, A. Compte, det. V. Llorente. CUENCA. Belinchón: ctra. a Zarza de Tajo, al SW del Cerro de Santiago, 30TVK9230, 768 m 26-IX-2008 tomillar ladera yesífera 1 ♀MNCN 292379 J Íñiguez det J Íñiguez Fuente g ) ♀ g g CÓRDOBA. Iznájar, [30SUG82], IV-1909, 1 ♀, MNCN 278994, Exp. del Museo. Srra Córdoba, 21-III-1982, 1 ♀, MNCN 292221, A. Compte, det. V. Llorente. CÓR O ája , [30SUG8 ], V 909, ♀, C 7899 , xp del useo S a Có doba, 21-III-1982, 1 ♀, MNCN 292221, A. Compte, det. V. Llorente. CUENCA. Belinchón: ctra. a Zarza de Tajo, al SW del Cerro de Santiago, 30TVK9230, 768 m, 26-IX-2008, tomillar ladera yesífera, 1 ♀, MNCN 292379, J. Agradecimientos. Dejamos constancia de nuestro testimonio de gratitud con Mercedes París García, conservadora de las colecciones de Arthropoda del Museo Nacional de Ciencias Naturales por su amabilidad y eficacia; por su constante y desinteresado apoyo a nuestras peticiones, pero remarcando la calidad de sus colaboraciones, que además, siempre van más allá de lo solicitado. Gracias a la información suministrada por ella, este trabajo ha mejorado notablemente a nivel corológico en el ámbito de la península ibérica. Agradecemos a Ellen Sandström, responsable de Entomological section del Biological Museum, Lund University, por aportarnos la información y fotografías del ejemplar conservado en MZLU:ENT. Pezotettix giornae en Galicia 95 ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) Boletín Biga 19 (2021): 79-110. ISSN 1886-5453 (Edición en línea) Material ibérico comunicado. rufipes Br. Pezotettix giornae (Rossi, 1794) [=Platyphyma giornae var. rufipes Brunner von Wattenwyl, 1882] según OSF (acceso 09/02/2021), MNCN 292355, Mz. Escalera, MNCN 279029, 292356, 292352, Escalera, MNCN 292354, Mz. Es- calera, MNCN 292353, 3 ♂♂, MNCN 292357, 292358, 292359. Cabo Roche, [29SQA52], XII-2004, 4 ♀♀, MNCN 292298, M. Gª París, det. V. Llorente 2006, 292289, 292302, 292301, 1 ♂, MNCN 292269. Chiclana, [29SQA53], 4 ♀♀, MNCN 292266, 292267, 292278, 292297, 2 ♂♂, MNCN 292281, 292282, 4 ♀♀, MNCN 279025, L. Cepero, 279021, 279022, Etiqueta del primer ejemplar de la serie: Pl. G. var. rufipes Br. Pezotettix giornae (Rossi, 1794) [=Platyphyma giornae var. rufipes Brunner von Wattenwyl, 1882] según OSF (acceso 09/02/2021), 292360, 3 ♂♂, MNCN 279023, 279024, 279030. El Parralejo, [30STF44], XII-2004, 4 ♀♀, MNCN 292291, M. Gª París, det. V. Llorente 2006. Estella del Marqués, [29SQA66], 292293, 292294, 292292, 2 ♂♂, MNCN 292296, 292295. La Blanca, [29SQA56], 2 ♀♀, MNCN 292290, 292303, 2 ♂♂, MNCN 292262, 292280. Medina Sidonia, [30STF33], 3 ♀♀, MNCN 292286, 292285, 292284, 2 ♂♂, MNCN 292283, 292279. S. José del Va- lle, [30STF45], 4 ♀♀, MNCN 292288, 292287, 292259, 292258, 2 ♂♂, MNCN 292260, 292264. San Roque, [30STF81], 19-IX-1996, 3 ♀♀, MNCN 292309, J. Ramírez, det. V. Llorente 2006, 292308, 292307, 4 ♂♂, MNCN 292311, 292316, 292312, 292317, 23-X-1996, 1 ♂, MNCN 292310, det. V. Llorente 2006, 27-VIII-1996, 3 ♂♂, MNCN 292313, det. V. Llorente 2006, 292315, 292314. CASTELLÓN. Benicassim: Desierto de las Palmas, [31TBE44], 05-IV-1992, 1 ♀, MNCN 292385, J Íñiguez det J Íñiguez Morella [30TYL40] 1 ♂MNCN 279016 J Royo CÁDIZ. Algeciras, [30STF70], 1-VIII-905, Etiqueta del primer ejemplar de la serie: Pl. g. var. rufipes Br. Pezotettix giornae (Rossi, 1794) [=Platyphyma giornae var. rufipes Brunner von Wat- tenwyl, 1882] según OSF (acceso 09/02/2021), 3 ♀♀, MNCN 292343, Escalera, 292344, 292345, 7 ♂♂, MNCN 227, 292346, 292351, 292350, 292349, 292348, 292347, 3-IX-1996, 1 ♀, MNCN 292318, J. Ramírez, det. V. Llorente 2006, 5-IX-1996, 7 ♀♀, MNCN 292319, P. Coello, det. V. Llorente 2006, Etiqueta del primer ejemplar de la serie: Pl. G. var. rufipes Br. Pezotettix giornae (Rossi, 1794) [=Platyphyma giornae var. rufipes Brunner von Wattenwyl, 1882] según OSF (acceso 09/02/2021), MNCN 292355, Mz. Escalera, MNCN 279029, 292356, 292352, Escalera, MNCN 292354, Mz. Es- calera, MNCN 292353, 3 ♂♂, MNCN 292357, 292358, 292359. Cabo Roche, [29SQA52], XII-2004, 4 ♀♀, MNCN 292298, M. Gª París, det. V. Material ibérico comunicado. ESPAÑA. ALBACETE. El Bonillo, [30SWJ41], 990 m, 13-IX-1987, Etiqueta: La Donal, 08089101 Prionix, C. García, 1 ninfa, MNCN 292340, A. Sánchez, det. V. Llorente 1997, 3 ♀♀, MNCN 292325, 292326, 292327, 1 ♂, MNCN 292328, det. V. Llorente 2006. [Albacete] La Donal, 08-VIII-1991, Etiqueta: La Donal, 08089101 Prionix, C. García, 2 ♂♂, MNCN 279204, C. García, det. (V. Llo- rente), 279178. ALICANTE. Concentaina, S. de Mariola, [30SYH19], 800 m, 5 ♀♀, MNCN 279000, (sin datos), 279001, 279003, 278997, 278980, 4 ♂♂, MNCN 278998, 278999, 279002, 278981. Í LMERÍA. Berja, [30SWF07], 300 m, V-1945, 1 ♀, MNCN 278992, M. M. Cabo de Gata, 0SWF76], 200 m, 13-VI-1991, 1 ninfa, MNCN 279177, C. García, det. (V. Llorente). ALMERÍA. Berja, [30SWF07], 300 m, V-1945, 1 ♀, MNCN 278992, M. M. Cabo de Gata, [30SWF76], 200 m, 13-VI-1991, 1 ninfa, MNCN 279177, C. García, det. (V. Llorente). ÁVILA. Cebreros, [30TUK78], 800 m, 30-X-88, 1 ♀, MNCN 292219, C. Martín, det. V. Llorente 1988. Gredos, [30TUK26], 3-IX-[18]97, 1 ♀, MNCN 279026, (sin datos). Piedralaves, [30TUK56], 700 m, 8-VIII-82, 1 ♂, MNCN 292333, V. Llorente, det. V. Llorente. BARCELONA B l VII MNCN A MNCN [ ], , , , , , ( ) ÁVILA. Cebreros, [30TUK78], 800 m, 30-X-88, 1 ♀, MNCN 292219, C. Martín, det. V. Llorente 1988. Gredos, [30TUK26], 3-IX-[18]97, 1 ♀, MNCN 279026, (sin datos). Piedralaves, [30TUK56], 700 m, 8-VIII-82, 1 ♂, MNCN 292333, V. Llorente, det. V. Llorente. BARCELONA. Barcelona, 30-VII-905, 2 ♂♂, MNCN 279017, Arias, 6 ♀♀, MNCN 278989, Llenas, 278990, 278987, 278984, 279031, Antiga, 278988, Llenas, 279032, Antiga. Las Planas, [31TDF28], 15-IX-40, 1 ♀, MNCN 279050, F. Esp. S. Segimnont, Montseny, [31TDG42], 14-X- 41, 1 ♀, MNCN 228, J. Mateu. Vallvridriera, [31TDF28], 29-X-40, 1 ♂, MNCN 279053, F. Esp. CÁCERES. El Jerte, Glorieta de las Tapuas, [30TTK65], 5-IV-2007, 2 ♀♀, MNCN 292330, I. Izquierdo, det. V. Llorente, MNCN 292342, det. V. Llorente 2008. q , , , CÁDIZ. Algeciras, [30STF70], 1-VIII-905, Etiqueta del primer ejemplar de la serie: Pl. g. var. rufipes Br. Pezotettix giornae (Rossi, 1794) [=Platyphyma giornae var. rufipes Brunner von Wat- tenwyl, 1882] según OSF (acceso 09/02/2021), 3 ♀♀, MNCN 292343, Escalera, 292344, 292345, 7 ♂♂, MNCN 227, 292346, 292351, 292350, 292349, 292348, 292347, 3-IX-1996, 1 ♀, MNCN 292318, J. Ramírez, det. V. Llorente 2006, 5-IX-1996, 7 ♀♀, MNCN 292319, P. Coello, det. V. Llorente 2006, Etiqueta del primer ejemplar de la serie: Pl. G. var. Material ibérico comunicado. Llorente 1979, 292238, 292246, 292245, 292243, 2 ♂♂, MNCN 279104, V. Llorente, 279105, 23-VI- 67, 1 ninfa, MNCN 279096, det. V. Llorente 1979, 3-X-1967, 1 ♀, MNCN 292247, det. V. Llorente 1979. Coto Doñana, Alr. de Palacio, Martinazo, 10-VI-66, 1 ♀, MNCN 292234, det. V. Llorente 1980. Doñana, Alr. de Palacio, 2-V-1969, bajo Eucalipt., 3 ♀♀, MNCN 292251, det. V. Llorente 1979, 292252, 292250. Doñana, La Baqueta, [29SQB20], 2-X-1968, en juncos, 2 ♀♀, MNCN 292249, det. V. Llorente 1979, 292248, Etiqueta del primer ejemplar de la serie: Pezotettix giornae (Ros.) (♂) V. Llorente de. 1979, 3 ♂♂, MNCN 279106, 279109, 279107. Ermita de S. Isidro, Cta. Gi- braleón, S. Bartolomé, [29SPB73], XI-1988, 1 ♀, MNCN 279180, C. Martín, det. (V. Llorente). Los Marines, [29SQB09], 4-X-68, Castañar; Etiqueta del primer ejemplar de la serie: Pezotettix giornae (Rossi) [manuscrita de V. Llorente, 14 ♀♀, MNCN 279151, V. Llorente, det. (V. Llorente), 279145, 279156, 279148, 279144, 279142, 279121, 279120, 279119, 279118, 279117, 279116, 279155, 279127, 11 ♂♂, MNCN 279149, 279150, 279147, 279146, 279157, 279141, 279140, 279152, 279143, 279154, 279153. Lucena del Puerto: pr. cementerio, 29SQB0231, 120 m, 04-VIII-2006, Pastizal agostado, pastoreado (ladera arenosa), 1 ♀, MNCN 292389, J. Íñiguez, det. J. Íñiguez., Mazagón, [29SPB91], XII-2004, 5 ♀♀, MNCN 292299, M. Gª París, det. V. Llorente 2006, 292270, 292300, 292272, 292271, 4 ♂♂, MNCN 292275, 292273, 292261, 292274. Palacio Doñana, [29SQA2796], 8-VIII-1969, 1 ♀, MNCN 292361, A. Compte, det. (V. Llorente). Palos de la Frontera, [29SPB82], 5-XI-1988, 2 ♀♀, MNCN 279182, C. Martín, det. (V. Llorente), 279179, 1 ♂, MNCN 279181. Parque Nacional Doñana, [29SQB20], [20]-VII-1978, Carretera de El Rocío a Matalascañas, poste con indi- cación de las características del tendido eléctrico, 20 jul 78 ADC, 1 ♀, MNCN 292362, V. Llorente, det. (V. Llorente), 1 ♂, MNCN 292363, X-XI-1982, 1 ♂ninfa, MNCN 279201, det. (V. Llorente), 279198. San Bartolome, [29SPB84], 23-XII-90, en cupula (sic!), 1 ♀, MNCN 279081, Izquierdo, 1 ♂, MNCN 279082. HUELVA. Aljaraque, [29SPB72], 13-II-84, 2 ♂♂, MNCN 279115, M. Huertas, det. V. Llorente 2004, 279114. Almonte, [29SQB22], XII-2004, 5 ♀♀, MNCN 292324, M. Gª París, det. V. Llorente 2006, 292323, 292256, 292254, 292255, 1 ♂, MNCN 292257. Corrales, [29SPB72], 4-XI-1988, 4 ♀♀, HUELVA. Aljaraque, [29SPB72], 13-II-84, 2 ♂♂, MNCN 279115, M. Huertas, det. V. Llorente 2004, 279114. Almonte, [29SQB22], XII-2004, 5 ♀♀, MNCN 292324, M. Gª París, det. V. Llorente 2006, 292323, 292256, 292254, 292255, 1 ♂, MNCN 292257. Material ibérico comunicado. Corrales, [29SPB72], 4-XI-1988, 4 ♀♀, MNCN 279080, (sin datos). Coto Doñana, Alr. de Palacio, [29SQA2796], 10-VI-66, Etiqueta del primer ejemplar de la serie: Pezotettix giornae (Ros.) ninfas, V. Llorente, 1979, 5 ninfas, MNCN 279094, V. Llorente, det. V. Llorente, 1979, 279095, 279093, E. Mingo, 279090, 279091, 292233, 292235, V. Llorente, 292236, E. Mingo, 18-VI-67, 1 ♀ninfa, MNCN 279092, V. Llorente, det. V. Llorente 1979, 292237, 1-X1967, 1 ♀, MNCN 292244, det. V. Llorente 1979, 1-X-1967, 3 ♀♀, MNCN 292241, det. V. Llorente 1979, 292240, 292239, 1-X-67, 5 ♀♀, MNCN 292242, E. Mingo, det. V. Llorente 1979, 292238, 292246, 292245, 292243, 2 ♂♂, MNCN 279104, V. Llorente, 279105, 23-VI- 67, 1 ninfa, MNCN 279096, det. V. Llorente 1979, 3-X-1967, 1 ♀, MNCN 292247, det. V. Llorente 1979. Coto Doñana, Alr. de Palacio, Martinazo, 10-VI-66, 1 ♀, MNCN 292234, det. V. Llorente 1980. Doñana, Alr. de Palacio, 2-V-1969, bajo Eucalipt., 3 ♀♀, MNCN 292251, det. V. Llorente 1979, 292252, 292250. Doñana, La Baqueta, [29SQB20], 2-X-1968, en juncos, 2 ♀♀, MNCN 292249, det. V. Llorente 1979, 292248, Etiqueta del primer ejemplar de la serie: Pezotettix giornae (Ros.) (♂) V. Llorente de. 1979, 3 ♂♂, MNCN 279106, 279109, 279107. Ermita de S. Isidro, Cta. Gi- braleón, S. Bartolomé, [29SPB73], XI-1988, 1 ♀, MNCN 279180, C. Martín, det. (V. Llorente). Los Marines, [29SQB09], 4-X-68, Castañar; Etiqueta del primer ejemplar de la serie: Pezotettix giornae (Rossi) [manuscrita de V. Llorente, 14 ♀♀, MNCN 279151, V. Llorente, det. (V. Llorente), 279145, 279156, 279148, 279144, 279142, 279121, 279120, 279119, 279118, 279117, 279116, 279155, 279127, 11 ♂♂, MNCN 279149, 279150, 279147, 279146, 279157, 279141, 279140, 279152, 279143, 279154, 279153. Lucena del Puerto: pr. cementerio, 29SQB0231, 120 m, 04-VIII-2006, Pastizal agostado, pastoreado (ladera arenosa), 1 ♀, MNCN 292389, J. Íñiguez, det. J. Íñiguez., Mazagón, [29SPB91], XII-2004, 5 ♀♀, MNCN 292299, M. Gª París, det. V. Llorente 2006, 292270, 292300, 292272, 292271, 4 ♂♂, MNCN 292275, 292273, 292261, 292274. Palacio Doñana, [29SQA2796], 8-VIII-1969, 1 ♀, MNCN 292361, A. Compte, det. (V. Llorente). Palos de la Frontera, [29SPB82], 5-XI-1988, 2 ♀♀, MNCN 279182, C. Martín, det. (V. Llorente), 279179, 1 ♂, MNCN 279181. Parque Nacional Doñana, [29SQB20], [20]-VII-1978, Carretera de El Rocío a Matalascañas, poste con indi- cación de las características del tendido eléctrico, 20 jul 78 ADC, 1 ♀, MNCN 292362, V. Llorente, det. (V. Llorente), 1 ♂, MNCN 292363, X-XI-1982, 1 ♂ninfa, MNCN 279201, det. (V. Llorente), 279198. Material ibérico comunicado. San Bartolome, [29SPB84], 23-XII-90, en cupula (sic!), 1 ♀, MNCN 279081, Izquierdo, 1 ♂, MNCN 279082. É [ ] HUELVA. Aljaraque, [29SPB72], 13-II-84, 2 ♂♂, MNCN 279115, M. Huertas, det. V. Llorente 2004, 279114. Almonte, [29SQB22], XII-2004, 5 ♀♀, MNCN 292324, M. Gª París, det. V. Llorente 2006, 292323, 292256, 292254, 292255, 1 ♂, MNCN 292257. Corrales, [29SPB72], 4-XI-1988, 4 ♀♀, MNCN 279080, (sin datos). Coto Doñana, Alr. de Palacio, [29SQA2796], 10-VI-66, Etiqueta del primer ejemplar de la serie: Pezotettix giornae (Ros.) ninfas, V. Llorente, 1979, 5 ninfas, MNCN 279094, V. Llorente, det. V. Llorente, 1979, 279095, 279093, E. Mingo, 279090, 279091, 292233, 2006, 292323, 292256, 292254, 292255, 1 ♂, MNCN 292257. Corrales, [29SPB72], 4-XI-1988, 4 ♀♀, MNCN 279080, (sin datos). Coto Doñana, Alr. de Palacio, [29SQA2796], 10-VI-66, Etiqueta del primer ejemplar de la serie: Pezotettix giornae (Ros.) ninfas, V. Llorente, 1979, 5 ninfas, MNCN 279094, V. Llorente, det. V. Llorente, 1979, 279095, 279093, E. Mingo, 279090, 279091, 292233, 1980. Doñana, Alr. de Palacio, 2-V-1969, bajo Eucalipt. JAÉN. Cerro de La Muela, [30SWH42], 3 ♀♀, MNCN 278995, G. Llueca, 278996, 278993. Jaen, 2 ♀♀, MNCN 279006, Lopez Cano, 279008, 3 ♂♂, MNCN 279004, 279005, 279007. Jandula, [30SVH01], VI-32, 1 ninfa, MNCN 279054, E. Mor., 1 ♀, MNCN 278982, E. M. (E. Morales), X-1932, 1 ♂, MNCN 278991, F. Escalera, X-32, 1 ♀, MNCN 278986, E. Mor. N. Foncuberta, Ca- zorla, [30SVG99], VII-56, 1 ♂, MNCN 279071, E. Morales. Nava de San Pedro, [30SWG19], 1400 m, 1-IX-62, Etiqueta del primer ejemplar de la serie: Pezotettix giornae (Ros.) (♂) V. Llorente de. 1964, 4 ♀♀, MNCN 292226, V. Llorente, det. V. Llorente 1964, 292223, 292224, 292225, 3 ♂♂, MNCN 279098, 279097, 279099, 30-VIII-62, 279100, det. V. Llorente 1964. , 1400 m, 31-VIII-62, 1 ♀, MNCN 292227, det. V. Llorente 1964, 5 ♀♀, MNCN 292228, 292229, 292231, 292232, 292230, 3 ♂♂, MNCN 279103, 279102, 279101, VII-56, 279068, E. Morales, 279066, 279067, 279065. Rio Despeñaperros, [30SVH54], 4-XI-1988, 4 ♀♀, MNCN 279076, C. Martín, 279077, 279078, 279075, 3 ♂♂, MNCN 279074, 279073, 279072. Sierra de Cazorla, Valdecuevas, 11-IV-91, comiendo corola JAÉN. Cerro de La Muela, [30SWH42], 3 ♀♀, MNCN 278995, G. Llueca, 278996, 278993. Jaen, 2 ♀♀, MNCN 279006, Lopez Cano, 279008, 3 ♂♂, MNCN 279004, 279005, 279007. Jandula, [30SVH01], VI-32, 1 ninfa, MNCN 279054, E. Mor., 1 ♀, MNCN 278982, E. M. (E. Morales), X-1932, 1 ♂, MNCN 278991, F. Material ibérico comunicado. Íñiguez, det. J. Íñiguez., Fuente de la tia Perra, [30TWK86], 12-VIII-79, 1 ♀, MNCN 279079, V. Llorente. Saelices: Pinguruzo, 30SWK1621, 921 m, 04-VIII-2007, matorral calcícola-tomillar ladera rocosa caliza, 1 ♀, MNCN 292378, J. Íñiguez, det. J. Íñiguez., Saelices: r. Cigüela, pr. ruinas Castillejo, 30SWK1815, 807 m, 1 ♂, MNCN 292377. Tejadillos, [30TXK14], 11-X-81, 2 ♀♀, MNCN 279085, Caminero. Uclés, [30SWK12], 279028, Pantel!. Pino Pérez et al. Pezotettix giornae en Galicia 96 Boletín Biga 19 (2021): 79-110. Pino Pérez et al.: Pezotettix giornae en Galicia ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) GRANADA. Bubión: pr. Bco. del Tejar (Srra. Nevada), 30SVF6991, 1924 m, 28-VII-2005, To- millar + Erynacea anthyllis esquistos, prado-juncal húmedo, 1 ♂, MNCN 292386, J. Íñiguez, det. J. Íñiguez., Capileira: pr. Bco. del Tejar, 1942 m, 2 ♀♀, MNCN 292387. Granada, 279020, Cáma- ra. Prado Grande Sierra Nevada, 17-IX-1970, 2 ♀♀, MNCN 292336, A. Compte, det. V. Llorente, 292337, 2 ♂♂, MNCN 292335, 292334. Trévelez, Sierra Nevada, [30SVF79], 18-X-1970, 1 ♀, MNCN 292338, det. V. Llorente. GUADALAJARA. Azañon, [30TWL30], 1000 m, VIII-56, 1 ♀, MNCN 279060, J. Abajo. Ma- ranchón: Sabinar de Maranchón, 30TWL6447, 1318 m, 13-VIII-2005, Tomillar calcícola en sabinar (primario), 1 ♂, MNCN 292380, J. Íñiguez, det. J. Íñiguez., Riba de Saelices, [30TWL52], 24-III- 35, 1 ♂, MNCN 279037, E. Mor. Robleluengo, [30TVL75], 11-III-2000, 2 ♀♀, MNCN 292320, López Colón, det. V. Llorente 2006. Sigüenza, [30TWL24], 279055, M. Escalera, 2 ♂♂, MNCN 279056, 279057. Sn. Andres del Congosto, [30TVL93], 29-XII-34, 230, E. Mor. 279057. Sn. Andres del Congosto, [30TVL93], 29-XII-34, 230, E. Mor. HUELVA. Aljaraque, [29SPB72], 13-II-84, 2 ♂♂, MNCN 279115, M. Huertas, det. V. Llorente 2004, 279114. Almonte, [29SQB22], XII-2004, 5 ♀♀, MNCN 292324, M. Gª París, det. V. Llorente 2006, 292323, 292256, 292254, 292255, 1 ♂, MNCN 292257. Corrales, [29SPB72], 4-XI-1988, 4 ♀♀, MNCN 279080, (sin datos). Coto Doñana, Alr. de Palacio, [29SQA2796], 10-VI-66, Etiqueta del primer ejemplar de la serie: Pezotettix giornae (Ros.) ninfas, V. Llorente, 1979, 5 ninfas, MNCN 279094, V. Llorente, det. V. Llorente, 1979, 279095, 279093, E. Mingo, 279090, 279091, 292233, 292235, V. Llorente, 292236, E. Mingo, 18-VI-67, 1 ♀ninfa, MNCN 279092, V. Llorente, det. V. Llorente 1979, 292237, 1-X1967, 1 ♀, MNCN 292244, det. V. Llorente 1979, 1-X-1967, 3 ♀♀, MNCN 292241, det. V. Llorente 1979, 292240, 292239, 1-X-67, 5 ♀♀, MNCN 292242, E. Mingo, det. V. Material ibérico comunicado. Escalera, X-32, 1 ♀, MNCN 278986, E. Mor. N. Foncuberta, Ca- zorla, [30SVG99], VII-56, 1 ♂, MNCN 279071, E. Morales. Nava de San Pedro, [30SWG19], 1400 m, 1-IX-62, Etiqueta del primer ejemplar de la serie: Pezotettix giornae (Ros.) (♂) V. Llorente de. 1964, 4 ♀♀, MNCN 292226, V. Llorente, det. V. Llorente 1964, 292223, 292224, 292225, 3 ♂♂, MNCN 279098, 279097, 279099, 30-VIII-62, 279100, det. V. Llorente 1964. , 1400 m, 31-VIII-62, 1 ♀, MNCN 292227, det. V. Llorente 1964, 5 ♀♀, MNCN 292228, 292229, 292231, 292232, 292230, 3 ♂♂, MNCN 279103, 279102, 279101, VII-56, 279068, E. Morales, 279066, 279067, 279065. Rio Despeñaperros, [30SVH54], 4-XI-1988, 4 ♀♀, MNCN 279076, C. Martín, 279077, 279078, 279075, 3 ♂♂, MNCN 279074, 279073, 279072. Sierra de Cazorla, Valdecuevas, 11-IV-91, comiendo corola Pino Pérez et al. Pezotettix giornae en Galicia 97 ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) Boletín Biga 19 (2021): 79-110. ISSN 1886-5453 (Edición en línea) Narcissus longispathus, 1 ♀, MNCN 292220, C.M. Herrera, det. V. Llorente 1993. LÉRIDA. L: Àger: Srra. Montsec d’Ares, 31TCG1156, 1085 m, 05-XI-2005, Tomillar-lastonar (encinar-quejigar en calizas), 1 ♀, MNCN 292388, J. Íñiguez, det. J. Íñiguez., MADRID. Alameda del Valle, [30TVL23], 10-IX-86, 1 ♀, MNCN 279132, V. Llorente, det. V. Llorente 1988, 3 ♂♂, MNCN 279129, 279128, 279131. Brunete, [30TVK17], VII-1938, 5 ♀♀, MNCN 279043, E. Morales, 279044, 279045, 279046, 279047, 2 ♂♂, MNCN 279048, 279049. C. Univer- sitaria, [30TVK37], 2-VII-44, 279176, Junco, det. (V. Llorente), XI-61, 5 ♀♀, MNCN 292217, J. Alvarez, det. V. Llorente 1988, 292218, 292215, 292214, 292216. Canencia. Robledal. Miraflores de la Sierra, [30TVL31], 1150 m, 5-IX-76, 1 ♂, MNCN 292188, V. Llorente, det. V. Llorente. Cercedilla, [30TVL11], 21-II-1992, borde camino pinar-melojar, 3 ♀♀, MNCN 292383, J. Íñiguez, det. J. Íñiguez, 292381, 292384, 1 ♂, MNCN 292382, 1460 m, IX-1951, 2 ♀♀, MNCN 279059, J. Abajo, 279058, 2 ♂♂, MNCN 279062, 279061. Corpa, [30TVK77], XI-32, 1 ♀, MNCN 279035, D. P. (Dionisio Peláez), 1 ♂, MNCN 279036. Crta. Torrelaguna La Cabrera, [30TVL42], 7-XI-1986, 2 ♀♀, MNCN 279125, V. Llorente, det. V. Llorente 1988, 279124, 2 ♂♂, MNCN 279122, 279123. El Escorial, [30TVK09], 6-IV-35, 1 ♀, MNCN 278985, E. Mor. El Pardo, [30TVK38], 9-VIII-77, 1 ninfa, MNCN 279126, C. Morillo, III-33, 2 ♂♂, MNCN 279038, E. Mor., 279039. El Pardo (El Goloso), [30TVK48], (1-8)-VIII-91, Trampa Malaise, 1 ♀, MNCN 292253, Nieves & Rey, det. (V. Llorente). Material ibérico comunicado. El Ventorrillo, 28-IX-2001, 1 ♀, MNCN 292331, Curso FOCCITCAM, det. V. Lloren- te 2001, X-1991, 1 ♀, MNCN 292329, (sin datos), det. V. Llorente 2001, 1 ♂, MNCN 292332. Escorial, [30TVK09], IX, 1 ♀, MNCN 279015, (sin datos), Etiqueta: Platyphyma Giornae Rossi Escorial / Mazar., 1 ♂, MNCN 279019, Mazar. Est. Alpina, Cercedilla, [30TVL11], 1460 m, 1 ♀, MNCN 279063, J. Abajo. La Cabrera, [30TVL42], 24-X-85, 3 ♀♀, MNCN 292194, V. Llorente, det. V. Llorente 1988, 292190, 292192, 3 ♂♂, MNCN 292193, 292195, 292191. Loeches, [30TVK67], 20-IX-72, 1 ♀, MNCN 279170, E. Mingo, det. (V. Llorente), 20-IX-77, 1 ♀, MNCN 279083, A. Com- pte. Madarcos, [30TVL54], 1062 m, 18-XI-86, 1 ♀, MNCN 279135, E. Plaza, det. V. Llorente 1988. Miraflores de la Sierra, [30TVL31], 11-IX-71, 2 ♀♀, MNCN 279209, (sin datos), det. V. Llorente, 279210, 2 ♂♂, MNCN 279208, 279211. Mirasierra, [30TVK48], 23-IX-71, 2 ♀♀, MNCN 279203, V. Llorente, det. (V. Llorente), 279202. Montejo de la Sierra, [30TVL54], 18-XI-86, 2 ♀♀, MNCN 279136, E. Plaza, det. (V. Llorente) 1988, 279137, 2 ♂♂, MNCN 279139, 279138, 22-XII-71, 5 ♀♀, MNCN 279161, S.V. Peris, det. (V. Llorente), 279162, 279163, 279166, 279168, 4 ♂♂, MNCN 279167, 279164, 279165, 279169. Novales, [30TVK37], XI-1938, 2 ♀♀, MNCN 279041, E. Morales, 279042, 1 ♂, MNCN 279040. Patones de Arriba, [30TVL52], 19-II-1995, 1 ♀, MNCN 279087, C. Martin. Pelayos de la Presa, [30TUK86], 12-X-2000, 2 ♀♀, MNCN 292305, J.I. López Colón, det. V. Llorente 2006, 292304, 1 ♂, MNCN 292306, 5-X-2001, 1 ♀, MNCN 292321, Jesú MBA, det. V. Llorente 2006. Peña Real, C. Viejo, [30TVL30], 15-VI-84, 1 ♀, MNCN 292205, V. Llorente, det. V. Llorente, 16-IX-84, 2 ♀♀, MNCN 292209, det. V. Llorente 1988, 292208, 1 ♂, MNCN 292210, 26-IX-1980, 1 ♂, MNCN 292204, det. V. Llorente, 5-VIII-84, 2 ninfas, MNCN 292196, det. V. Llorente, 292206, 1 ♀, MNCN 292322, det. M. Coca 2010, 7-VII-85, MNCN 292198, det. V. Llorente 1988. Peña Real, Colmenar Viejo, 2-VII-78, 1 ♀, MNCN 279207, det. V. Llorente 1988, VII-83, 2 ♂♂, MNCN 292189, det. V. Llorente 1988, 292207, VIII-83, 2 ♀♀, MNCN 279205, det. V. Llorente 1988, 279206. Peña Real, Soto del Real, [30TVL31], 19-X-86, 1 ♀, MNCN 279188, det. (V. Llorente). Peña Real, Soto Real, 1 ♂, MNCN 279189, VIII-86, 3 ninfas, MNCN 279190, det. (V. Llorente), 279193, 279192, 4 ♀♀, MNCN 279183, 279184, 279191, 279089, 2 ♂♂, MNCN 279194, 279088. Pino Pérez et al. Narcissus longispathus, 1 ♀, MNCN 292220, C.M. Herrera, det. V. Llorente 1993. LÉRIDA. L: Àger: Srra. Montsec d’Ares, 31TCG1156, 1085 m, 05-XI-2005, Tomillar-lastonar (encinar-quejigar en calizas), 1 ♀, MNCN 292388, J. Íñiguez, det. J. Íñiguez., MADRID. Alameda del Valle, [30TVL23], 10-IX-86, 1 ♀, MNCN 279132, V. Llorente, det. V. Llorente 1988, 3 ♂♂, MNCN 279129, 279128, 279131. Brunete, [30TVK17], VII-1938, 5 ♀♀, MNCN 279043, E. Morales, 279044, 279045, 279046, 279047, 2 ♂♂, MNCN 279048, 279049. C. Univer- sitaria, [30TVK37], 2-VII-44, 279176, Junco, det. (V. Llorente), XI-61, 5 ♀♀, MNCN 292217, J. Alvarez, det. V. Llorente 1988, 292218, 292215, 292214, 292216. Canencia. Robledal. Miraflores de la Sierra, [30TVL31], 1150 m, 5-IX-76, 1 ♂, MNCN 292188, V. Llorente, det. V. Llorente. Cercedilla, [30TVL11], 21-II-1992, borde camino pinar-melojar, 3 ♀♀, MNCN 292383, J. Íñiguez, det. J. Íñiguez, 292381, 292384, 1 ♂, MNCN 292382, 1460 m, IX-1951, 2 ♀♀, MNCN 279059, J. Abajo, 279058, 2 ♂♂, MNCN 279062, 279061. Corpa, [30TVK77], XI-32, 1 ♀, MNCN 279035, D. P. (Dionisio Peláez), 1 ♂, MNCN 279036. Crta. Torrelaguna La Cabrera, [30TVL42], 7-XI-1986, 2 ♀♀, MNCN 279125, V. Llorente, det. V. Llorente 1988, 279124, 2 ♂♂, MNCN 279122, 279123. El Escorial, [30TVK09], 6-IV-35, 1 ♀, MNCN 278985, E. Mor. El Pardo, [30TVK38], 9-VIII-77, 1 ninfa, MNCN 279126, C. Morillo, III-33, 2 ♂♂, MNCN 279038, E. Mor., 279039. El Pardo (El Goloso), [30TVK48], (1-8)-VIII-91, Trampa Malaise, 1 ♀, MNCN 292253, Nieves & Rey, det. (V. Llorente). El Ventorrillo, 28-IX-2001, 1 ♀, MNCN 292331, Curso FOCCITCAM, det. V. Lloren- te 2001, X-1991, 1 ♀, MNCN 292329, (sin datos), det. V. Llorente 2001, 1 ♂, MNCN 292332. Escorial, [30TVK09], IX, 1 ♀, MNCN 279015, (sin datos), Etiqueta: Platyphyma Giornae Rossi Escorial / Mazar., 1 ♂, MNCN 279019, Mazar. Est. Alpina, Cercedilla, [30TVL11], 1460 m, 1 ♀, MNCN 279063, J. Abajo. La Cabrera, [30TVL42], 24-X-85, 3 ♀♀, MNCN 292194, V. Llorente, det. V. Llorente 1988, 292190, 292192, 3 ♂♂, MNCN 292193, 292195, 292191. Loeches, [30TVK67], 20-IX-72, 1 ♀, MNCN 279170, E. Mingo, det. (V. Llorente), 20-IX-77, 1 ♀, MNCN 279083, A. Com- pte. Madarcos, [30TVL54], 1062 m, 18-XI-86, 1 ♀, MNCN 279135, E. Plaza, det. V. Llorente 1988. Miraflores de la Sierra, [30TVL31], 11-IX-71, 2 ♀♀, MNCN 279209, (sin datos), det. V. Llorente, 279210, 2 ♂♂, MNCN 279208, 279211. Mirasierra, [30TVK48], 23-IX-71, 2 ♀♀, MNCN 279203, V. Llorente, det. (V. Llorente), 279202. Montejo de la Sierra, [30TVL54], 18-XI-86, 2 ♀♀, MNCN 279136, E. Plaza, det. (V. Llorente) 1988, 279137, 2 ♂♂, MNCN 279139, 279138, 22-XII-71, 5 ♀♀, MNCN 279161, S.V. Peris, det. (V. Llorente), 279162, 279163, 279166, 279168, 4 ♂♂, MNCN 279167, 279164, 279165, 279169. Novales, [30TVK37], XI-1938, 2 ♀♀, MNCN 279041, E. Morales, 279042, 1 ♂, MNCN 279040. Patones de Arriba, [30TVL52], 19-II-1995, 1 ♀, MNCN 279087, C. Martin. Pelayos de la Presa, [30TUK86], 12-X-2000, 2 ♀♀, MNCN 292305, J.I. López Colón, det. V. Llorente 2006, 292304, 1 ♂, MNCN 292306, 5-X-2001, 1 ♀, MNCN 292321, Jesú MBA, det. V. Llorente 2006. Peña Real, C. Viejo, [30TVL30], 15-VI-84, 1 ♀, MNCN 292205, V. Llorente, det. V. Llorente, 16-IX-84, 2 ♀♀, MNCN 292209, det. V. Llorente 1988, 292208, 1 ♂, MNCN 292210, 26-IX-1980, 1 ♂, MNCN 292204, det. V. Llorente, 5-VIII-84, 2 ninfas, MNCN 292196, det. V. Llorente, 292206, 1 ♀, MNCN 292322, det. M. Coca 2010, 7-VII-85, MNCN 292198, det. V. Llorente 1988. Peña Real, Colmenar Viejo, 2-VII-78, 1 ♀, MNCN 279207, det. V. Llorente 1988, VII-83, 2 ♂♂, MNCN 292189, det. V. Llorente 1988, 292207, VIII-83, 2 ♀♀, MNCN 279205, det. V. Llorente 1988, 279206. Peña Real, Soto del Real, [30TVL31], 19-X-86, 1 ♀, MNCN 279188, det. (V. Llorente). Peña Real, Soto Real, 1 ♂, MNCN 279189, VIII-86, 3 ninfas, MNCN 279190, det. (V. Llorente), 279193, 279192, 4 ♀♀, MNCN 279183, 279184, 279191, 279089, 2 ♂♂, MNCN 279194, 279088. Pinilla del Valle, [30TVL33], 10-IX-86, 2 ♀♀, MNCN 279134, det. (V. Llorente) 1988, 279133, 1 ♂, MNCN 279130. Ribas, [30TVK56], 16-IX-94, 1 ♀, MNCN 279014, Bolivar. San Fernando de Henares, [30TVK57], 15-XI-2008, 1 ♂, MNCN 292339, J.L. Colón, det. V. Llorente. Soto del Real, [30TVL31], 11-XI-79, Etiqueta del primer ejemplar de la serie: Pezotettix giornae (Ros.) (♂) V. Llorente de. 1979, 1 ♀, MNCN 292213, V. Llorente, det. V. Llorente 1988, 2 ♂♂, MNCN 279110, 279111, 11-XI-81, 2 ♀♀, MNCN 292201, det. V. Llorente 1988, 292202, 16-IX-79, 2 ♀♀, MNCN 292212, det. V. Llorente 1988, 292211, Etiqueta del primer ejemplar de la serie: Pezotettix giornae (Ros.) (♂) V. Llorente de. 1979, 1 ♂, MNCN 279108. Soto del Real, C. Viejo, 11-XI-84, 2 ♀♀, MNCN 292200, det. V. Llorente 1988, 292199, 13-X-85, 1 ♂, MNCN 292197, det. V. Llorente 1988, 21-VII-85, 1 ♀, MNCN 69912, 6-X-85, 1 ♂, MNCN 279212, det. V. Llorente. Urb. Peña Real, Colmenar Viejo, [30TVL30], 26-IX-1980, 1 ♂, MNCN 292203, det. V. Llorente 1988. Valdemorillo, [30TVK08], X-XI-1982, 3 ♂, MNCN 279197, (sin datos), det. (V. Llorente), 279195, 279196. Material ibérico comunicado. Pinilla del Valle, [30TVL33], 10-IX-86, 2 ♀♀, MNCN 279134, det. (V. Llorente) 1988, 279133, 1 ♂, MNCN 279130. Ribas, [30TVK56], 16-IX-94, 1 ♀, MNCN 279014, Bolivar. San Fernando de Henares, [30TVK57], 15-XI-2008, 1 ♂, MNCN 292339, J.L. Colón, det. V. Llorente. Soto del Real, [30TVL31], 11-XI-79, Etiqueta del primer ejemplar de la serie: Pezotettix giornae (Ros.) (♂) V. Llorente de. 1979, 1 ♀, MNCN 292213, V. Llorente, det. V. Llorente 1988, 2 ♂♂, MNCN 279110, 279111, 11-XI-81, 2 ♀♀, MNCN 292201, det. V. Llorente 1988, 292202, 16-IX-79, 2 ♀♀, MNCN 292212, det. V. Llorente 1988, 292211, Etiqueta del primer ejemplar de la serie: Pezotettix giornae (Ros.) (♂) V. Llorente de. 1979, 1 ♂, MNCN 279108. Soto del Real, C. Viejo, 11-XI-84, 2 ♀♀, MNCN 292200, det. V. Llorente 1988, 292199, 13-X-85, 1 ♂, MNCN 292197, det. V. Llorente 1988, 21-VII-85, 1 ♀, MNCN 69912, 6-X-85, 1 ♂, MNCN 279212, det. V. Llorente. Urb. Peña Real, Colmenar Viejo, [30TVL30], 26-IX-1980, 1 ♂, MNCN 292203, det. V. Llorente 1988. Valdemorillo, [30TVK08], X-XI-1982, 3 ♂, MNCN 279197, (sin datos), det. (V. Llorente), 279195, 279196. Pino Pérez et al. Pezotettix giornae en Galicia 98 Boletín Biga 19 (2021): 79-110. Pino Pérez et al.: Pezotettix giornae en Galicia ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) MÁLAGA. Estepona: Reales de Sierra Bermeja, 30SUF0240, 1422 m, 02-VIII-2006, Pinar (P. pinaster) con pinsapo, Cistus populifolius…, 1 ♂, MNCN 292390, J. Íñiguez, det. J. Íñiguez., Be- nalmadena, [30SUF65], 20-VIII-82, 1 ♂, MNCN 279084, V. Llorente, 22-VIII-82, 2 ♀♀, MNCN 279185, det. (V. Llorente), 279187, 1 ♂, MNCN 279186. Fuengirola, [30SUF54], VIII-82, 279159, det. (V. Llorente), 279160. Málaga, 1 ♀, MNCN 279013, E. Gros. ORENSE. Orense, VIII-1908, 4 ♀♀, MNCN 278983, Taboada, 279009, 279011, 279012, 1 ♂, MNCN 279010. SEGOVIA. Espinar, VIII-1894, 1 ♀, MNCN 279027, Bolivar. SEVILLA. Arroyo del Rey, [29SQB67], 4-X-68, entre adelfas, 1 ♀, MNCN 279158, V. Llorente, det. (V. Llorente). Carmona, [30STG65], 7-XI-71, 1 ♂, MNCN 279113, det. V. Llorente 2004. Rio Rivera de Huelva, [29SQB66], 4-XI-88, 2 ♀♀, MNCN 279172, C. M. Albadalejo (Albaladejo), det. (V. Llorente), 279171, 3 ♂♂, MNCN 279175, 279174, 279173. Sevilla, XII-1961, 2 ♀♀, MNCN 279069, (sin datos), 279070. Villamanrique, [29SQB32], XII-2004, 2 ♀♀, MNCN 292263, M. Gª París, det. V. Llorente 2006, 292277, 3 ♂♂, MNCN 292268, 292276, 292265. , , , , , , TARRAGONA. Material ibérico comunicado. 99 Pezotettix giornae en Galicia ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) Bibliografía. Adlbauer, W. & Sackl, P. 1993. Zum Vorkommen und zur Verbreitung seltener Heuschrec- ken und Grillen in der Steiermark. Mitteilungen der Abteilung für Zoologie des Landesmuseum Joanneum, 47: 55-66. Amigo Vázquez, J., Pulgar Sañudo, Í. & Giménez de Azcárate Cornide, J. 2005. Guía da flora do Parque Natural “Serra da Enciña da Lastra”. Consellería de Medio Ambiente. Santiago de Compostela. 95 pp. Badenhausser, I. 2012. Estimation d’abondance des criquets (Orthoptera: Acrididae) dans les écosystèmes prairiaux. Annales de la Société Entomologique de France, 48: 3-4, 397-406. https://doi.org/10.1080/00379271.2012.10697787 Badih, A. 1997. Caelifera (Insecta, Orthoptera) del Norte de Marruecos (Rif y Depresión Baja del Muluya). Faunística, Ecología y Biogeografía. 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Larose, Paris. 447 pp. Chopard, L. 1951. Faune de France. Orthoptéroïdes 56. Material ibérico comunicado. Capsanes, [31TCF15], 30-IX-43, 1 ♀, MNCN 229. TARRAGONA. Capsanes, [31TCF15], 30-IX-43, 1 ♀, MNCN 229. TOLEDO. Almendral de la Cañada (Sierra S. Vicente): cº Pico Cruces, 30TUK5246, 1245 m, 29-VIII-2004, pastizal agostado borde melojar, 1 ♀, MNCN 292367, J. Íñiguez, det. J. Íñiguez, 1 ♂, MNCN 292368. Calzada de Oropesa: Embalse de Rosarito, 30TUK0340, 312 m, 16-IX-2007, Pastizal-juncal borde embalse, parcela roturada en jaral, 1 ♀, MNCN 292372, det. J. Íñiguez, 1 ♂, MNCN 292371. Campillo de La Jara: pr. vértice Reventón, 30SUJ2382, 750 m, 1 ♀, MNCN 292364. Escalona, [30TUK84], 550 m, 14-VI-1980, 1 ♂ninfa, MNCN 279199, I. Marcos, det. (V. Llorente). Hinojosa de S Vicente: Pico S Vicente, lad. S, 30TUK5243, 1245 m, 17-X-2004, pradera con Pteridium aquilinum, 1 ♂, MNCN 292365, J. Íñiguez, det. J. Íñiguez., Hontanar: Risco de las Paradas, 30SUJ6882, 4-X-2003, claros jaral, 1 ♂, MNCN 292366, det. J. Íñiguez. Layos: Em- balse de Guajaraz, 30TVK0705, 617 m, 13-IX-2007, Espartal en ladera terrosa-rocosa, solana, 1 ♀, MNCN 292373, det. J. Íñiguez. Oropesa: Cañada Leonesa Occ., pr. Pte. sobre r. Guadyerbas, 30TUK2432, 381 m, 15-X-2006, codesar (A. aureus, Cytisus …), 1 ♀, MNCN 292369, det. J. Íñi- guez. Oropesa: río Tiétar, 30TUK1140, 319 m, 16-IX-2007, pradera herbazal orilla del río, al borde del agua, 1 ♂, MNCN 292370, det. J. Íñiguez. San Pablo de los Montes: La Morra, 30SUJ8675, 1286 m, 11-III-2007, rebrotes melojo en claro pinar repoblado, 1 ♀, MNCN 292374, det. J. Íñiguez. Villarrubia de Santiago: pista rural Zarza de Tajo, 30SVK7126, 675 m, 08-VII-2007, ladera solana, yesos: tomillar, albardinar, claros…, 1 ♀, MNCN 292375, det. J. Íñiguez. VALENCIA. Godelleta, [30SXJ96], 29-IX-1938, 1 ♀, MNCN 279051, (sin datos), 2 ♂♂, MNCN 279052. 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Notes Orthopterologiques V. Les orthoptères du ”Sitio”dans la Sierra de Cuenca. Anales de la Sociedad Española de Historia Natural, 25: 59-118 Lam. I. Pardo, J.E. 1992. Estudio faunístico y ecológico del superorden Orthopteroidea de los principales sistemas montañosos de Castilla-La Mancha. Tesis Doctoral, 43. Universidad de Valencia. 658 pp. Parejo-Pulido, D. & J. Lagar-Carvajal 2018. Primeras citas de ortópteros (Insecta: Orthop- tera) de la provincia de Badajoz (Extremadura, España). Boletín de la Sociedad Entomológica Aragonesa, 63: 269-270. Pascual, F. 1978. Estudio preliminar de los Ortópteros de Sª Nevada, I: Introducción general e inventario de especies. Boletín de la Asociación española de Entomología, 1 (1977): 163-175. Peinado, M.V., J. Mateos & F.J. Santibañez 1996. Estudio de las superpoblaciones de ortópteros en zonas de las provincias de Segovia, León y Zamora. Memoria Final. Instituto Universitario de Ciencias Ambientales IUCA, Madrid.510 pp. Pérez Arcas, L. 1877. Una excursión entomológica a Navacerrada. Anales de la Sociedad Espa- ñola de Historia Natural (Actas), 6: 54-61. Pina, S., S. Vasconcelos, L. Reino, J. Santana, P. Beja, J.S. Sánchez-Oliver, I. Catry, F. Moreira & S. Ferreira 2017. Consejería de Agricultura y Ganadería. Colección Mundo Rural. 182 pp. The Orthoptera of Castro Verde Special Protection Area (Southern Portugal): new data and conservation value. Zookeys, 691: 19-48. Pinedo, M.C. & V. Llorente 1988. Orthopteroidea de la provincia de Castellón, en especial de la Marisma de Oropesa. Graellsia, 43: 93-109. Presa, J.J. 1978. Los Acridoidea (Orthoptera) de la sierra de Guadarrama. Trabajos de la Cátedra de Artrópòdos, 26. Tesis de Licenciatura. Dpto. Zoologia, Facultad de Biología. Catedra de Entomología. Universidad Complutense de Madrid. Madrid. 274 pp. Presa, J.J. & M.D. García 1982. Contribución al conocimiento de los Acridoidea de la región murciana (II): Sª Seca de Revolcadores. Boletín de la Asociación española de Entomología, 6(1): 127-132. Presa, J.J. & V.J. Montserrat 1978. Contribución al conocimiento de los Acridoidea de Cádiz. Boletín de la Asociación española de Entomología, 2: 41-48. Prunier, F. 2014. Apuntes sobre los ortópteros (Orthoptera) de la provincia de Málaga y presencia de especies oromediterráneas en Sierra Tejeda (España). Boletín de la Sociedad Entomológica Aragonesa, 54: 317-328. Prunier, F. 2017. Nemobius sylvestris sylvestris (Bosc, 1792), especie nueva en Málaga, España (Orthoptera: Trigonidiidae). Revista gaditana de Entomología, 8(1): 245-247. Pulido García, M.L. 1990. Los Orthoptera, Mantodea, Phasmoptera (Insecta) de la Sierra de Alcaraz (Albacete), S.E. España. Boletín de Sanidad Vegetal. Plagas (Fuera de serie), 20: Pino Pérez et al. Pezotettix giornae en Galicia 109 ISSN 1886-5453 (Edición en línea) ISSN 1886-5453 (Edición en línea) Pezotettix giornae en Galicia 137-144. 137-144. Pulido García, M.L. 1990. Contribución al conocimiento de los Orthopteroidea (Insecta) de la Sierra de las Almenaras y Calar del Mundo (Sierra de Alcaraz, Albacete). Al-Basit, 27: 163-174. Pulido García, M.L. 1995. Estudio faunístico y ecológico de los órdenes mantodea, phasmatodea y orthoptera (Insecta) de la Sierra de Alcaraz. Colección Tesis Doctorales, nº 12. Universidad de Castilla-La Mancha. 405 pp. Quiñones-Alarcón, J. & D. Llucià-Pomares 2018. Estudio faunístico de los ortópteros (In- secta: Orthoptera) de la provincia de Málaga (Andalucía, España). Boletín de la Sociedad En- tomológica Aragonesa, 62: 181-219. Ragge, D.R. 1965. Ortópteros y Dermápteros colectados en la Península Ibérica durante los años de 1962 y 1963 por misiones del British Museum (Natural History). Graellsia, 21: 95-119. Rambur, J.P. 1838. Faune entomologique de l’Andalousie: Dermaptères et Orthoptères, 2. Arthus Bertrand, Librairie de la Soc. Geographique, París. 336 pp. Riso, S., M.J. Arabolaza, J. Braga, N. Martins, M. Nóvoa & G. da Silva-Méndez 2016. Influência das alteraçôes agricolas em populaçôes de Orthoptera no noreste de Portugal. Boletín de la Sociedad entomológica Aragonesa, 58: 217-220. Salvador, M. de 1915. Suplemento a la narración de la excursión a Arnes en el Boletín de Octubre de 1914. Boletín de la Sociedad Aragonesa de Ciencias Naturales, 15: 117-123. Santos, J.L., P. Arana & R. Giráldez 1983. Chromosome C-banding patterns in Spanish Acridoidea. Genetica, 61: 65-74. Schmidt, G.H. 1999. Ein Beitrag zur Höhenverteilung der Orthopteroidea in der Sierra Neva- da/Spanien. Articulata, 14(1): 45-61. Schmidt, G.H., A.P. Martinho & M.R. Paiva 2009. The saltopteran fauna of Portugal: new records and biogeographical aspects (Orthopteroidea). Fragmenta entomologica, Roma, 41(1): 15-67. Seabra, A.F. de 1939. Contribuïção para a História da Entomologia em Portugal. Catálogo das Colecções Entomológicas do Laboratório de Biologia Florestal em 1937. Publicações da Direcção Geral dos Serviços Florestais e Aqüicolas, 6(2): 155-301. Seabra, A.F. de 1939. Contribuïção para a História da Entomologia em Portugal. Catálogo das Colecções Entomológicas do Laboratório de Biologia Florestal em 1937. Publicações da Direcção Geral dos Serviços Florestais e Aqüicolas, 6(1): 5-148. Pino Pérez et al. 110 Pezotettix giornae en Galicia
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Review of: "The Outcome of Emergency Admissions and Associated Factors Among Children Admitted to the Pediatric Emergency Unit at Selected Public Hospitals at Addis Ababa, Ethiopia — Retrospective Cross-Sectional Study"
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Gebrehiwot Berie Mekonnen1 1 Debre Tabor University Potential competing interests: No potential competing interests to declare. Qeios, CC-BY 4.0 · Review, April 25, 2024 Review of: "The Outcome of Emergency Admissions and Associated Factors Among Children Admitted to the Pediatric Emergency Unit at Selected Public Hospitals at Addis Ababa, Ethiopia — Retrospective Cross-Sectional Study" Review of: "The Outcome of Emergency Admissions and Associated Factors Among Children Admitted to the Pediatric Emergency Unit at Selected Public Hospitals at Addis Ababa, Ethiopia — Retrospective Cross-Sectional Study" Gebrehiwot Berie Mekonnen1 1 Debre Tabor University Gebrehiwot Berie Mekonnen1 Qeios ID: CN4HYO · https://doi.org/10.32388/CN4HYO Qeios, CC-BY 4.0 · Review, April 25, 2024 Potential competing interests: No potential competing interests to declare. Thank you for your work adding to the scientific community in the area of pediatrics. I'm especially happy with the background section, even if it's too long. Please respond to the following issues before the next steps or publication: 1. Your title is too long to convey clear and precise scientific meaning. If possible, keep it under 20 words. 1. Your title is too long to convey clear and precise scientific meaning. If possible, keep it under 20 words. 2. From the abstract section, the method subsection is too shallow. Please try to add information about the study area, sample size, your tool, data processing, and analysis. 2. From the abstract section, the method subsection is too shallow. Please try to add information about the study area, sample size, your tool, data processing, and analysis. 3. From the abstract section, the result subsection “….children who were treated with fluid during admission” was not stated. Please add it since it's a significant variable. 4. Your conclusion from the abstract section and the main body of the document is not well addressed based on your results. 5. I'm not clear on your source population. Please rewrite it again because it's paradoxical with the sampling procedures. Could you think that your study is a generalization for Addis Ababa government public hospitals or selected study hospitals? 6. You said that “…….children whose charts had incomplete information were not included in the study” - when do you say a chart has incomplete information? 7. You stated a 100% data completeness rate. Have you done missing data management? If yes, please clarify in the last part of the method section? 12. From the discussion section, significant variables were not discussed well. Please revise it again. Good luck! Qeios ID: CN4HYO · https://doi.org/10.32388/CN4HYO 1/2 Qeios, CC-BY 4.0 · Review, April 25, 2024 Qeios ID: CN4HYO · https://doi.org/10.32388/CN4HYO 2/2
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Research on Supply Chain Coordination Decision Making under the Influence of Lead Time Based on System Dynamics
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Citation: Zhang, M.; Wang, Y.; Zhang, Y. Research on Supply Chain Coordination Decision Making under the Influence of Lead Time Based on System Dynamics. Systems 2024, 12, 32. https://doi.org/10.3390/ systems12010032 systems systems systems systems systems Mingli Zhang 1,2, Yanan Wang 1 and Yijie Zhang 1,* 1 School of Economics and Management, Yanshan University, Qinhuangdao 066004, China; nanyi@stumail.ysu.edu.cn (Y.W.) 1 School of Economics and Management, Yanshan University, Qinhuangdao 066004, China; nanyi@stumail.ysu.edu.cn (Y.W.) 1 School of Economics and Management, Yanshan University, Qinhuangdao 066004, China; nanyi@stumail.ysu.edu.cn (Y.W.) 2 Hebei Construction Material Vocational and Technical College, Qinhuangdao 066004, China * Correspondence: zhangyijie@ysu.edu.cn 1 School of Economics and Management, Yanshan University, Qinhuangdao 066004, China; nanyi@stumail.ysu.edu.cn (Y.W.) 2 Hebei Construction Material Vocational and Technical College, Qinhuangdao 066004, China * Correspondence: zhangyijie@ysu.edu.cn 2 Hebei Construction Material Vocational and Technical College, Qinhuangdao 066004, China * Correspondence: zhangyijie@ysu.edu.cn Abstract: Supply chain coordination has been a research hot spot in supply chain management. This paper constructs a secondary supply chain system. Taking the abatement of the bullwhip effect and the double marginal effect as the coordination objective, a simulation study of supply chain decision coordination was conducted using system dynamics. First, by controlling the lead time, it was found that in the decentralized decision-making model, the profit of the supplier and the whole supply chain increases with the shortening of the lead time, and vice versa for the retailer. In the centralized decision-making model with the addition of information sharing and contract, it was found that the retailer’s profit is consistent with the trend of the supplier and the supply chain as a whole, and the supplier’s profit is lower than that of decentralized decision making in the pre-cooperation period. In addition, it is also found that adjusting the contract parameters can effectively improve the situation. Finally, the above models were analyzed for supply chain coordination decisions based on two scenarios: “cooperative stability” or “balance of effects”. Keywords: lead time; centralized mode; the bullwhip effect; the double marginal effect; supply chain coordination; system dynamics Article Research on Supply Chain Coordination Decision Making under the Influence of Lead Time Based on System Dynamics Mingli Zhang 1,2, Yanan Wang 1 and Yijie Zhang 1,* 1. Introduction Innovations in information technology and the rapid development of market globaliza- tion have led to the gradual lengthening of supply chains. Adopting advanced management decisions to achieve a stable and coordinated development of the supply chain is a key concern for enterprises. The current development of the global industry is characterized by shorter and shorter product life cycles and rapid price declines. Meanwhile, driven by the “horizontal integration” management model of the supply chain, along with the optimization of logistics and transportation, as well as information transfer channels, the market competition for products has become more and more intense, requiring enterprises to make rapid and effective responses to the ever-changing market. Time-based compe- tition has evolved to become a major mode of competition among firms today, and firms themselves should pay attention to the impact of ordering time in addition to quantity- related order lot sizes [1]. Research has shown that lead time compression reduces the inventory levels of supply chain node firms, enabling them to forecast the market more accurately and respond more quickly to user demand [2]. The current theoretical research on lead times is too homogenous. Only the regulating role of lead time itself in supply management is considered, as well as how different types of lead time function differently. Consideration of the time factor is necessary and not exclusive. It may be necessary to consider the impact of lead time on the mode of operation of the entire supply chain system and its moderating role in multiple decision making, which has not been given much attention in previous studies. However, in the actual supply chain operation process, due to cooperative members having different interest subjects, inventory control, and decision Academic Editors: Omid Jadidi and Fatemeh Firouzi Academic Editors: Omid Jadidi and Fatemeh Firouzi Received: 18 December 2023 Revised: 7 January 2024 Accepted: 17 January 2024 Published: 18 January 2024 2.1. Lead Time With the spread of just-in-time production methods, lead time has become a key factor in supply chain competition [5]. While researchers have introduced fixed lead times into the model structure to refine inventory costing [6,7], due to changes in the real market environment and asymmetry in the flow of information about product orders, emergency stock-outs often occur, and fixed settings for order and flow times are no longer sufficient to meet the actual modeling needs. The stochastic nature of lead times and the impact of replenishment strategies have been gradually incorporated into system simulations [8–10]. Meanwhile, in the study of how to formulate optimal ordering and inventory cost control strategies to enhance the overall performance of the supply chain, it was found that the reasonable control of lead time shortened the lead time, and this shorter lead time allowed retailers to place orders closer to the beginning of the peak selling season, which effectively lowered the inventory level [11,12]. On the other hand, determining the right delivery time facilitates the supplier to have more sufficient time to supply the product and its initial cost will be reduced, which will ultimately realize an increase in profit for both parties [13,14]. The rationalization of lead time control is usually based on the cross impacts from factors such as changing demand patterns in the input system, customer channel preferences, etc. [15–18]. Some scholars have also classified lead times and viewed them as decision variables in order to analyze the impact of the distributional state of lead times on cost optimization and order quality on turnaround times [19,20]. Planning for lead times depends on the stochastic variability of the activity’s resource requirements, as well as the flexibility and utilization of the resources associated with the activity [21]. Moreover, there is a limit to the reduction and increase in lead times, beyond which there is a loss of performance or a doubling of the burden on practitioners [22]. Most of the literature focuses on the impact of lead time and other prefabricated times on supply chain performance and market demand. The time variable is often associated with many uncertainties. Each stage of supply chain operation is also subject to the compounding of multiple factors. Perhaps the key to realizing a virtuous cycle is how decision makers judge the impact of time factors and adopt production cooperation strategies to maintain efficiency. Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/systems Systems 2024, 12, 32. https://doi.org/10.3390/systems12010032 Systems 2024, 12, 32 2 of 20 making and the existence of information asymmetry, overall optimal joint decision making is a more ideal model, as only considering the lead time of the decision making may lead to supply chain dysfunction [3]. In this complex and changing environment and increas- ingly competitive background, effective order time management and the decision-making structure of synergistic optimization is important for enhancing the competitiveness of the market. Additionally, achieving the coordinated development of the supply chain is the key to a solid partnership, mutual benefit, and a win-win situation [4]. Therefore, this paper takes the mitigation of the bullwhip effect and double marginal effect as the coordination orientation. Under the consideration of the effect of lead time, the data related to the inventory and profit of the node enterprises of the supply chain are compared to find the problems arising from the cooperation of the supply chain members. Then, the adjustment of the decision-making structure of the supply chain is carried out in order to arrive at a strategy that can optimize the overall efficiency of the supply chain and the relative coordination between the node enterprises. 2. Literature Review In this paper, we first consider the effect of lead time in decentralized supply chain coordination. Then, it takes the mitigation of the bullwhip effect and double marginal effect as the coordination orientation and explores the effects of different decision structures on the two effects. Finally, the adjustment effects from lead time and decision structure are considered simultaneously. Therefore, this research is highly relevant to lead time and decision structure, and the literature related to these areas will be reorganized below. 2.1. Lead Time However, there are few studies on how lead time affects supply chain management decisions and how the coupling with the decision-making structure is considered. Systems 2024, 12, 32 3 of 20 2.2. Decision-Making Structures 2.2. Decision-Making Structures 2.2. Decision-Making Structures Based on the uncertainty of market demand, in order to avoid production risk and resource waste to a certain extent, supply chain coordination and internal decision-making structure are often accompanied in the research. There is no lack of exploration of inventory management methods to achieve supply chain coordination. For example, multi-objective mixed integer linear programming is used to improve inventory management efficiency and supply chain resilience [23]. Distributionally robust optimization methods are applied to optimize the dual-channel inventory warehouse structure [24]. The combined inventory method is applied to coordinate direct market demand and replenishment orders from downstream firms [25]. The bullwhip effect is an important cause of production risk and resource waste and is also one of the indicators affecting supply chain coordination. Scholars have argued that the role of differences in decision-making styles on the bullwhip effect is reflected in inventory. It was found that centralization enhanced behaviors such as inventory target tracking in the supply chain [26]. The inventory variance dimension can also be analyzed to show that order splitting and retail splitting are detrimental to the reduction in the bullwhip effect [27]. Inventory variance is also often used to characterize the impact of the bullwhip effect and the causes of supply chain inefficiencies analyzed [28]. Of course, many factors contribute to the bullwhip effect. Competitive market demand, firm characteristics, and the holistic thinking of decision makers have all been identified as “correlating variables” that contribute to the bullwhip effect [29–31]. On the other hand, studies have shown that coordination in decentralized supply chains is often difficult to maintain. This may be explained by another process: double marginal effects [32]. The negative impact of the decision structure on the level of cooperative effort can easily lead to the erosion of supply chain members’ profits [33]. In order to ensure the coordination of interests to cut down the double marginal effect, it is expedient to introduce a benefit- sharing mechanism or a commitment-penalty contract in decentralized supply chains [34]. This behavioral approach not only takes into account the bargaining power of members but also reveals firms’ private production cost information to avoid misrepresentation of information [35]. 3. Supply Chain Coordination Foundation Model Analysis The double marginal effect occurs when the goals of enterprises at different nodes in the supply chain are inconsistent. By setting their own optimal solutions, products are repeatedly priced during the circulation process, ultimately leading to the disruption of supply and demand balance and poor overall supply chain efficiency. The bullwhip effect occurs when the demand information transmission of upstream and downstream enterprises in the supply chain lags behind, making it difficult for enterprises to establish reasonable inventory, resulting in increased resource waste and cost consumption which affects the coordinated development of the The supply chain’s dual marginal effect and bullwhip effect caused by external random demand fluctuations and upstream/downstream information asymmetry (Figure 2) are both manifestations of this internal dynamism. The double marginal effect occurs when the goals of enterprises at different nodes in the supply chain are inconsistent. By setting their own optimal solutions, products are repeatedly priced during the circulation process, ultimately leading to the disruption of supply and demand balance and poor overall supply chain efficiency. The bullwhip effect occurs when the demand information transmission of upstream and downstream enterprises in the supply chain lags behind, making it difficult for enterprises to establish reasonable inventory, resulting in increased resource waste and cost consumption, which affects the coordinated development of the supply chain. dom demand fluctuations and upstream/downstream information asymmetry (Figure 2 are both manifestations of this internal dynamism. The double marginal effect occur when the goals of enterprises at different nodes in the supply chain are inconsistent. B setting their own optimal solutions, products are repeatedly priced during the circulatio process, ultimately leading to the disruption of supply and demand balance and poo overall supply chain efficiency. The bullwhip effect occurs when the demand informatio transmission of upstream and downstream enterprises in the supply chain lags behind making it difficult for enterprises to establish reasonable inventory, resulting in increase resource waste and cost consumption, which affects the coordinated development of th supply chain. p ,f supply chain. Figure 2. “Bullwhip effect” in the supply chain. Figure 2. “Bullwhip effect” in the supply chain. Figure 2. “Bullwhip effect” in the supply chain Figure 2. “Bullwhip effect” in the supply chain. Figure 2. “Bullwhip effect” in the supply chain. Generally speaking, the construction of the model reflects the research idea of the researcher, and system dynamics is no exception. 3. Supply Chain Coordination Foundation Model Analysis The bullwhip effect occurs when the demand information transmission of upstream and downstream enterprises in the supply chain lags behind, making it difficult for enterprises to establish reasonable inventory, resulting in increased resource waste and cost consumption, which affects the coordinated development of the Figure 1. Model structure analysis diagram. The supply chain’s dual marginal effect and bullwhip effect caused by external random demand fluctuations and upstream/downstream information asymmetry (Figure 2) are both manifestations of this internal dynamism. The double marginal effect occurs when the goals of enterprises at different nodes in the supply chain are inconsistent. By setting their own optimal solutions, products are repeatedly priced during the circulation process, ultimately leading to the disruption of supply and demand balance and poor overall supply chain efficiency. The bullwhip effect occurs when the demand information transmission of upstream and downstream enterprises in the supply chain lags behind, making it difficult for enterprises to establish reasonable inventory, resulting in increased resource waste and cost consumption, which affects the coordinated development of the supply chain. Figure 1. Model structure analysis diagram. The supply chain’s dual marginal effect and bullwhip effect caused by external ran dom demand fluctuations and upstream/downstream information asymmetry (Figure 2 are both manifestations of this internal dynamism. The double marginal effect occur when the goals of enterprises at different nodes in the supply chain are inconsistent. B setting their own optimal solutions, products are repeatedly priced during the circulatio process, ultimately leading to the disruption of supply and demand balance and poo overall supply chain efficiency. The bullwhip effect occurs when the demand informatio transmission of upstream and downstream enterprises in the supply chain lags behind making it difficult for enterprises to establish reasonable inventory, resulting in increase resource waste and cost consumption, which affects the coordinated development of th supply chain. Figure 1. Model structure analysis diagram. Figure 1. Model structure analysis diagram. Figure 1. Model structure analysis diagram. The supply chain’s dual marginal effect and bullwhip effect caused by external ra d d d fl d /d f Figure 1. Model structure analysis diagram. Figure 1. Model structure analysis diagram. The supply chain’s dual marginal The supply chain’s dual marginal effect and bullwhip effect caused by external ran- dom demand fluctuations and upstream/downstream information asymmetry (Figure 2) are both manifestations of this internal dynamism. 3. Supply Chain Coordination Foundation Model Analysis For example, FMCG supply chain market pathways are short and wide, and the operation process is cross-influenced by external market factors and internal dynamics. As the fluctuating demand for manufactured goods is highly influenced by factors such as buyer preference and ease of purchase coupled with the market penetration of imitations, consumers are prone to switching to different brands in similar products, i.e., brand loyalty is not high. Therefore, in order to maintain a certain amount of market share, manufacturers have to make frequent product upgrades to expand sales varieties, and ultimately, its value characteristics are mainly reflected in two aspects: timeliness and diversity. At the same time, since external conditions cannot be completely controlled, we pay more attention to the regulation of the internal dynamics from the supply chain. A single supply chain linking the different participants, including the supply chain network node enterprises, will be established based on factors such as quality control, cost and profitability, and other relevant considerations in selecting the object of cooperation and its relevant products. During the process of the formation of the supply chain, it is important to consider the direction of the information and financial flow from downstream to upstream of the direction, and the direction of the product flow from upstream to downstream (as in Figure 1). However, the interactive process is subject to the potential for mutual promotion and mutually beneficial outcomes but may also lead to a decline in individual firm or supply chain overall profits due to factors such as information asymmetry. The uncertainty in this internal collaboration is indicative of its internal dynamism. Systems 2024, 12, 32 4 of 20 uncer- Figure 1. Model structure analysis diagram. The supply chain’s dual marginal effect and bullwhip effect caused by external ran- dom demand fluctuations and upstream/downstream information asymmetry (Figure 2) are both manifestations of this internal dynamism. The double marginal effect occurs when the goals of enterprises at different nodes in the supply chain are inconsistent. By setting their own optimal solutions, products are repeatedly priced during the circulation process, ultimately leading to the disruption of supply and demand balance and poor overall supply chain efficiency. 3. Supply Chain Coordination Foundation Model Analysis In this paper, the research ideas of using this method are to (1) clarify the problem and determine the system boundary; (2) put forward the dynamic hypothesis; (3) analyze the system structure and write the equations; i Generally speaking, the construction of the model reflects the research idea of th researcher, and system dynamics is no exception. In this paper, the research ideas of usin this method are to (1) clarify the problem and determine the system boundary; (2) pu forward the dynamic hypothesis; (3) analyze the system structure and write the equation (4) test the model; and (5) simulate and analyze. The following will be a specific study i relation to the research object of this paper. Generally speaking, the construction of the model reflects the research idea of the researcher, and system dynamics is no exception. In this paper, the research ideas of using this method are to (1) clarify the problem and determine the system boundary; (2) put forward the dynamic hypothesis; (3) analyze the system structure and write the equations; (4) test the model; and (5) simulate and analyze. The following will be a specific study in relation to the research object of this paper. (4) test the model; and (5) simulate and analyze. The relation to the research object of this paper. 4. Assumption Constraints and System Modeling 4 1 Basic Assu ptio s for Model Co structio 4. Assumption Constraints and System Modeling 4.1. Basic Assumptions for Model Construction relation to the research object of this paper. 4. Assumption Constraints and System Modelin 4 1 Ba i A u tio fo Model Co t u tio y g 4.1. Basic Assumptions for Model Construction relation to the research object of this paper. 4. Assumption Constraints and System Modelin 4 1 B i A ti f M d l C t ti 4.1. Basic Assumptions for Model Construction The research object of this article is a secondary short-life-cycle product supply chain composed of a supplier and a retailer, and it is only a single supply chain. Since this paper focuses on the coordination of internal members of the supply chain as well as the whole, it does not deal with the influence of special events as well as external factors in special periods. Therefore, complex market demand types are not considered, and the demand is set as a random fluctuation input. Assumption 2 is as follows. Assumption 2. Market demand is stochastic, with no extreme conditions such as demand interruption. The conclusions drawn from studying relatively simple chain structures tend to have optimization and morphing potential. Therefore, the starting point of the research in this paper is set to be decentralized and the decentralized vs. centralized explored in the paper will not involve structures such as supplier-controlled inventory, joint inventory, and so on. Assumption 3 is as follows. Assumption 3. Inventory is set as independent inventory for suppliers and retailers, and the inventory inspection method is periodic inventory inspection. A decentralized structure generally means that the members are only responsible for their own operations, and the benefit objectives are considered relatively individual. And it is not exactly equivalent to the meaning of centralized in the broad sense. In this paper, the centralized structure is based on the goal of optimal development of the overall efficiency of the supply chain while focusing on the coordination of the micro-structure. Among them, having sufficient consultation as well as the exchange of resources and information are the key features of the centralized style. Combined with previous studies in the literature, Assumption 4 is as follows. Assumption 4. Centralization is achieved by incorporating information-sharing mechanisms and revenue-sharing contracts. Simulation models tend to ignore the feasibility of realistic factors. The expected limits for equilibrium gains and the cooperation preferences of the firm’s decision makers should also have been considered. Therefore, taking into account the feedback from the research participants as well as their experiences, we limit the upper limit of revenue sharing in the contract. Assumption 5 is as follows. Assumption 5. Considering practical factors, the maximum sharing limit for retailers under the revenue-sharing system is 20% of their own profits. relation to the research object of this paper. 4. Assumption Constraints and System Modelin 4 1 B i A ti f M d l C t ti 4.1. Basic Assumptions for Model Construction 4. Assumption Constraints and System Modeling 4.1. Basic Assumptions for Model Construction In order to fully reflect the actual situation and simplify the model as much as possi 4.1. Basic Assumptions for Model Construction In order to fully reflect the actual situation and simplify the model as much as poss ble for more accurate analysis, the following research hypotheses are made based on th In order to fully reflect the actual situation and simplify the model as much as possible for more accurate analysis, the following research hypotheses are made based on the research scope of the model. In order to fully reflect the actual situation and simplify the model as much as possi- ble for more accurate analysis, the following research hypotheses are made based on the research scope of the model. Based on the coordination goal of this paper, the performance of product perfor- mance is different; furniture, electrical appliances, automobiles, and other long-cycle life research scope of the model. Based on the coordination goal of this paper, the performance of product perfor mance is different; furniture, electrical appliances, automobiles, and other long-cycle lif Based on the coordination goal of this paper, the performance of product performance is different; furniture, electrical appliances, automobiles, and other long-cycle life products are updated and iterated quickly. However, for the retail industry, the order status of durable goods is relatively holistic and systematic, and behavioral strategies such as pre- sale and replenishment are often present in the sales process. Market competition is more of a long-term strategic nature. For daily necessities, tobacco, food and beverages, and other short-life-cycle products, they are more sensitive to short-term changes in market Systems 2024, 12, 32 5 of 20 demand. Their market categories are richer, and their information transfer and response speed are faster. Therefore, it is more suitable to discuss the coordination of the supply chain in a short cycle. On the other hand, this paper focuses on the problem of cooperative decision making among members of a supply chain strip. The impact of too many structural disturbances should be minimized. Therefore, Assumption 1 is as follows. Assumption 1. The research object of this article is a secondary short-life-cycle product supply chain composed of a supplier and a retailer, and it is only a single supply chain. Assumption 1. 4.2. Construction of Model System Flow Diagram The random input of market demand and the characteristics of short inventory and fast selling of goods require retailers and suppliers to effectively reduce inventory levels and maintain stable inventory fluctuations and respond quickly to market changes, improve prediction accuracy, and stabilize the dynamic balance of input and output. Meanwhile, as the supply chain continues to develop and mature, exploring the coordinating role of time variables has gradually become a focus of research for scholars. To this end, the model Systems 2024, 12, 32 6 of 20 introduces a time variable of order lead time internally. The system flow diagram is shown in the following Figure 3. introduces a time variable of order lead time internally. The system flow diagram is shown in the following Figure 3. Figure 3. Decentralized supply chain system flow diagram. Figure 3. Decentralized supply chain system flow diagram. Figure 3. Decentralized supply chain system f Figure 3. Decentralized supply chain system flow diagram. In decentralized decision making in the supply chain, participating mem decisions independently, and upstream behavior is often triggered by downs sions. Usually, upstream enterprises lack global information (such as retail cu mand patterns and information from different time points in the supply chain to rely on information they can obtain, such as production efficiency, their ow situation, and order information issued by downstream units. The variable sy parison table is shown in the following Table 1: In decentralized decision making in the supply chain, participating members make de- cisions independently, and upstream behavior is often triggered by downstream decisions. Usually, upstream enterprises lack global information (such as retail customer demand patterns and information from different time points in the supply chain) and have to rely on information they can obtain, such as production efficiency, their own inventory situation, and order information issued by downstream units. The variable symbol comparison table is shown in the following Table 1. Table 1. Variable symbo Table 1. Variable symbol comparison. Table 1. Variable symbol comparison. 4.3. Important Parameters and Equation Explanations In order to ensure that the test results are in line with reality, this article takes dairy beverages in fast-moving consumer products as an example for simulation analysis. The simulation data and variable relationships included in the model refer to relevant informa- tion on dairy beverages in Z supermarket and model materials in references [12–14]. The model system includes three types of variables: state variables, rate variables, and general variables. There are four stocks such as S-I, R-I, etc. There are nine types of traffic such as S-PR, S-SR, etc. General variables also include auxiliary variables and constants, including 41 such as P-R, R-IG, S-IG, etc. The variable names and equation designs are shown in Table 2 below. Table 2. Variable equations. Variable Type Equation Design Unit State variable S-I = INTEGER(S-PR −S-SR −S-L, initial time) kg R-I = INTEGER(S-SR −R-SR −R-L, initial time) kg S-P = INTEGER(S-RGR −S-CGR, initial time) yuan R-P = INTEGER(R-RGR −R-CGR, initial time) yuan Rate variable S-SR = IF THEN ELSE(R-OQ ≤S-I, SMOOTH(R-OQ, L-T + R-OT), SMOOTH(S-I, L-T + R-OT)) kg/Day R-SR=DELAY1(M-D, S-D) kg/Day S-L = S-I * L-R1 kg/Day R-L = R-I * L-R2 kg/Day S-CGR = S-PC + S-IC + S-TC + S-LC yuan/Day R-CGR = R-OC + R-IC + R-TC + R-LC yuan/Day Auxiliary variable M-D = RANDOM UNIFORM(200, 350, 280) kg/Day R-SF = SMOOTH(R-SR, R-DST) kg/Day R-DI = R-SF * R-OT * (1−R-SOR) kg/Day R-IG = MAX(0, (R-DI −R-I)/I-AC) kg/Day R-OQ = MAX(0, R-SF + R-IG) kg/Day S-DI = S-SF * S-SCT kg/Day S-IG = MAX(0, (S-DI −S-I)/I-AC) kg/Day P-R = MAX(0, S-SF + S-IG) kg/Day S-DR = R-UOP * R-OQ yuan/Day S-PC = S-UPC * S-PR yuan/Day S-IC = S-I * S-UIC yuan/Day S-TC = S-UTC * S-SR yuan/Day S-LC = S-L * S-ULC yuan/Day S-CP = S-P + R-P yuan/Day 4.2. Construction of Model System Flow Diagram Variable Name Symbol Variable Name Supplier Inventory S-I Supplier Inventory Cost Retailer Inventory R-I Supplier Unit Transport Cost Supplier Profit S-P Supplier Transport Cost Retailer profit R-P Retailer Order Time Supply Chain Profit S-CP Retailer Order Quantity Supplier Purchase rate S-PR Lead Time S-Shipment Rate S-SR Retailer Desired Inventory Retailer Sales Rate R-SR Retailer Inventory Gap Supplier Revenue Growth Rate S-RGR Retailer Sales Forecast Supplier Cost Growth Rate S-CGR Retailer Demands Smooth Time Retailer Revenue Growth Rate R-RGR Retailer Stock-out Rate Retailer Cost Growth Rate R-CGR Market Demand Supplier Sales Forecast S-SF Sales Delay Supplier Demands Smooth Time S-DST Retailer Losses Inventory Adjustment Cycle I-AC Loss Rate 2 Supplier Desired Inventory S-DI Retailer Loss Cost Supplier Safety Coverage Time S-SCT Retailer Unit Loss Cost Supplier Inventory Gap S-IG Product Price Production Requirement P-R Retail Sales Production Delay P-D Retailer Unit Inventory Cost Table 1. Variable symbol comparison. Variable Name Symbol Variable Name Symbol Supplier Inventory S-I Supplier Inventory Cost S-IC Retailer Inventory R-I Supplier Unit Transport Cost S-UTC Supplier Profit S-P Supplier Transport Cost S-TC Retailer profit R-P Retailer Order Time R-OT Supply Chain Profit S-CP Retailer Order Quantity R-OQ Supplier Purchase rate S-PR Lead Time L-T S-Shipment Rate S-SR Retailer Desired Inventory R-DI Retailer Sales Rate R-SR Retailer Inventory Gap R-IG Supplier Revenue Growth Rate S-RGR Retailer Sales Forecast R-SF Supplier Cost Growth Rate S-CGR Retailer Demands Smooth Time R-DST Retailer Revenue Growth Rate R-RGR Retailer Stock-out Rate R-SOR Retailer Cost Growth Rate R-CGR Market Demand M-D Supplier Sales Forecast S-SF Sales Delay S-D Supplier Demands Smooth Time S-DST Retailer Losses R-L Inventory Adjustment Cycle I-AC Loss Rate 2 L-R2 Supplier Desired Inventory S-DI Retailer Loss Cost R-LC Supplier Safety Coverage Time S-SCT Retailer Unit Loss Cost R-ULC Supplier Inventory Gap S-IG Product Price P-P Production Requirement P-R Retail Sales R-S Production Delay P-D Retailer Unit Inventory Cost R-UIC Supplier Losses S-L Retailer Inventory Cost R-IC Loss Rate 1 L-R1 Retailer Unit Transport Cost R-UTC Supplier Loss Cost S-LC Retailer Transport Cost R-TC Supplier Unit Loss Cost S-ULC Wholesale Discount Coefficient W-DC Supplier Delivery Revenue S-DR Retailer Unit Order Price R-UOP Supplier Unit Production Cost S-UPC Retailer Order Cost R-OC Supplier Production Cost S-PC Revenue Sharing Contract Coefficient R-SCC Supplier Unit Inventory Cost S-UIC Systems 2024, 12, 32 7 of 20 4.3. Important Parameters and Equation Explanations 5. Model Simulation and Analysis 5.1. Decentralized Supply Chain Coordination 5.1. Decentralized Supply Chain Coordination Before simulation, the model was successively tested for structural soundness and extreme conditions. For space considerations, we will not repeat them here. Firstly, consider a supply chain system with independent decision making by suppliers and retailers regard- ing lead time. At this point, retailers use product sales rates to make sales forecasts, while suppliers can only produce and ship products based on the orders submitted by retailers and predict sales for the next batch. Table 3 shows the assignment of constant variables. p g The simulation cycle of the model is 100 days, with a simulation step size of 1 day. Under the condition that the above constants remain unchanged, the lead time of the retailer’s order is adjusted to 1 day (1 d), 3 days (3 d), and 5 days (5 d) to obtain the inventory situation of the supplier and retailer as follows: (1) Analysis of the Bullwhip Effect Inventory is a key factor in regulating supply and demand balance and is a state variable that members of the supply chain must focus on in various cooperation modes. A reasonable inventory level can reduce holding costs while quickly responding to market Systems 2024, 12, 32 8 of 20 demand and reducing unnecessary waste. The fluctuation status of inventory can reflect the strength of the bullwhip effect. This section conducts a 100-day simulation analysis on the inventory level when suppliers and retailers make decentralized decisions under the influence of lead time. R-I(initial) 200 Kg The simulation cycle of the model is 100 days, with a simulation step size of 1 da Under the condition that the above constants remain unchanged, the lead time of the tailer’s order is adjusted to 1 day (1 d) 3 days (3 d) and 5 days (5 d) to obtain the invento Table 3. Constant assignment table. Constant Name Constant Value Unit S-DST 2 Day R-OT 3 Day I-AC 4 Day R-DST 2 Day S-SCT 3 Day P-D 1 Day S-I(initial) 150 Kg R-I(initial) 200 Kg tailer s order is adjusted to 1 day (1 d), 3 days (3 d), and 5 days (5 d) to obtain the invento situation of the supplier and retailer as follows: (1) Analysis of the Bullwhip Effect Inventory is a key factor in regulating supply and demand balance and is a state v iable that members of the supply chain must focus on in various cooperation modes. reasonable inventory level can reduce holding costs while quickly responding to mark demand and reducing unnecessary waste. The fluctuation status of inventory can refle the strength of the bullwhip effect. This section conducts a 100-day simulation analysis the inventory level when suppliers and retailers make decentralized decisions under t influence of lead time. It can be clearly seen from Figure 4 that in a decentralized supply chain system t Table 3. Constant assignment table. j y situation of the supplier and reta It can be clearly seen from Figure 4 that in a decentralized supply chain system, the peak levels of retailer inventory with lead times of 1 d, 3 d, and 5 d are reached in 22 d, 33 d, and 40 d, respectively, while those of suppliers are 26 d, 42 d, and 46 d. (1) Analysis of the Bullwhip Effect Both retailer and supplier inventory will enter a stable and lower level faster as the lead time shortens. At the same time, it also reflects that the node firms in the supply chain will respond more quickly and accurately to the external demand as the lead time is compressed. The speed of stabilizing their own state also increases. peak levels of retailer inventory with lead times of 1 d, 3 d, and 5 d are reached in 22 d, d, and 40 d, respectively, while those of suppliers are 26 d, 42 d, and 46 d. Both retai and supplier inventory will enter a stable and lower level faster as the lead time shorten At the same time, it also reflects that the node firms in the supply chain will respond mo quickly and accurately to the external demand as the lead time is compressed. The spe of stabilizing their own state also increases. (a) (b) Figure 4. The inventory under a decentralized mode, considering the influence of lead time. (a) T inventory of retailer. (b) The inventory of supplier. R-I 4,000 2,850 1,700 550 -600 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) kg "R-I" : L-T5 1 1 1 1 "R-I" : L-T3 2 2 2 2 2 "R-I" : L-T1 3 3 3 3 S-I 4,000 3,000 2,000 1,000 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) kg "S-I" : L-T5 1 1 1 1 "S-I" : L-T3 2 2 2 2 2 "S-I" : L-T1 3 3 3 3 Figure 4. The inventory under a decentralized mode, considering the influence of lead time. (a) The inventory of retailer. (b) The inventory of supplier. (1) Analysis of the Bullwhip Effect L-T1 L-T3 L-T5 Retailer inventory variance sum 1,484,498.76 3,336,775.92 4,859,699.88 1,484,498.76 3,336,775.92 4,859,699.88 (2) Analysis of double marginal effects As can be seen from Figure 5, the overall profit of the supply chain shows a clear i As can be seen from Figure 5, the overall profit of the supply chain shows a clear increasing trend after 15 d as the lead time is shortened. In this case, the retailer’s profit starts to rise after a short break-even period. The profit curve with a lead time of 1 d is consistently higher than that with lead times of 3 d and 5 d, and the rate of rise continues to accelerate, with the difference in profit potentials becoming progressively larger. Suppliers show the opposite trend, with shorter lead times keeping their profits at a low level. However, it is also observed that the difference between the three curves decreases over time. increasing trend after 15 d as the lead time is shortened. In this case, the retailer s profit starts to rise after a short break-even period. The profit curve with a lead time of 1 d is consistently higher than that with lead times of 3 d and 5 d, and the rate of rise continues to accelerate, with the difference in profit potentials becoming progressively larger. Sup- pliers show the opposite trend, with shorter lead times keeping their profits at a low level. However, it is also observed that the difference between the three curves decreases over time. (a) (b) (c) Figure 5. Profitability of members in a decentralized supply chain. (a) The profits of the retailer. (b) The profits of the supplier. (c) The profits of the supply chain. (1) Analysis of the Bullwhip Effect (b) R-P 4 M 3 M 2 M 1 M 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) CNY "R-P" : L-T5 1 1 1 1 "R-P" : L-T3 2 2 2 2 "R-P" : L-T1 3 3 3 3 R-P (a) S-P 6 M 4.5 M 3 M 1.5 M 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) CNY "S-P" : L-T5 1 1 1 1 "S-P" : L-T3 2 2 2 2 "S-P" : L-T1 3 3 3 3 (b) (a) (c) S-CP 8 M 6 M 4 M 2 M 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) CNY "S-CP" : L-T5 1 1 1 1 "S-CP" : L-T3 2 2 2 2 "S-CP" : L-T1 3 3 3 3 (c) Figure 5. Profitability of members in a decentralized supply chain. (a) The profits of the retailer. (b) The profits of the supplier. (c) The profits of the supply chain. Figure 5. Profitability of members in a decentralized supply chain. (a) The profits of the retailer. (b) The profits of the supplier. (c) The profits of the supply chain. In the actual supply chain system, the shortening of lead time will weaken the lag in market judgment of enterprises in the supply chain. At the same time, the sensitivity and accuracy of forecasting will be enhanced. This characteristic substantially eliminates the uncertainty of market demand or the ambiguity of excessively long lead times. (1) Analysis of the Bullwhip Effect S-P 6 M 4.5 M 3 M 1.5 M 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) CNY "S-P" : L-T5 1 1 1 1 "S-P" : L-T3 2 2 2 2 "S-P" : L-T1 3 3 3 3 R-P 4 M 3 M 2 M 1 M 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) CNY "R-P" : L-T5 1 1 1 1 "R-P" : L-T3 2 2 2 2 "R-P" : L-T1 3 3 3 3 S-CP 8 M 6 M 4 M 2 M 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) CNY "S-CP" : L-T5 1 1 1 1 "S-CP" : L-T3 2 2 2 2 "S-CP" : L-T1 3 3 3 3 Figure 5. Profitability of members in a decentralized supply chain. (a) The profits of the retailer. (b) The profits of the supplier. (c) The profits of the supply chain. (1) Analysis of the Bullwhip Effect (b) l S-I 4,000 3,000 2,000 1,000 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) kg "S-I" : L-T5 1 1 1 1 "S-I" : L-T3 2 2 2 2 2 "S-I" : L-T1 3 3 3 3 (a) R-I 4,000 2,850 1,700 550 -600 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) kg "R-I" : L-T5 1 1 1 1 "R-I" : L-T3 2 2 2 2 2 "R-I" : L-T1 3 3 3 3 (b) (a) Figure 4. The inventory under a decentralized mode, considering the influence of lead time. (a) T inventory of retailer. (b) The inventory of supplier. Figure 4. The inventory under a decentralized mode, considering the influence of lead time. (a) The inventory of retailer. (b) The inventory of supplier. In order to better measure the impact of lead time on the “bullwhip effect”, the variance suns is used to measure the inventory fluctuation after entering the steady state. Observe that stocks under all three lead times go to a relative plateau after 40 d of simulation length. As shown in Tables 4 and 5, both retailers and suppliers follow the rule that the shorter the lead time, the smaller the inventory variance sum. Combined with the above findings, it can be seen that the “bullwhip effect” in decentralized decision making is mitigated with the reduction in lead time. (2) Analysis of double marginal effects Table 4. Supplier inventory variance sum at different lead times (decentralized supply chain). L-T1 L-T3 L-T5 Supplier inventory variance sum 1,195,714.23 1,765,058.86 2,139,915.70 Table 4. Supplier inventory variance sum at different lead times (decentralized supply chain). Systems 2024, 12, 32 9 of 20 Table 5. Retailer inventory variance sum at different lead times (decentralized supply chain). 5.2. Centralized Supply Chain Coordination pp y In contrast to a decentralized supply In contrast to a decentralized supply chain, this model changes how suppliers predict the market via retailers’ orders instead of sharing retailers’ sales forecast information with suppliers. A wholesale discount factor and a revenue-sharing contract factor are introduced into the cost-income subsystem. The supplier gives the retailer a discounted price for wholesale orders, while the retailer promises to give the supplier a portion of the shared revenue after making a profit in order to improve the operational efficiency of the supply chain, harmonize the win-win relationship, and enhance the overall profitability of the supply chain.The model is shown in Figure 6. pp y g pp p the market via retailers’ orders instead of sharing retailers’ sales forecast information wit suppliers. A wholesale discount factor and a revenue-sharing contract factor are intr duced into the cost-income subsystem. The supplier gives the retailer a discounted pric for wholesale orders, while the retailer promises to give the supplier a portion of th shared revenue after making a profit in order to improve the operational efficiency of th supply chain, harmonize the win-win relationship, and enhance the overall profitabilit of the supply chain.The model is shown in Figure 6. Figure 6. Centralized supply chain system flow diagram. S-I R-I S-PR S-SR R-OQ R-DI M-D R-SF R-IG R-OT I-AC R-SOR R-DST S-SF S-DI S-IG S-SCT S-DST S-D P-R S-P R-P S-RGR S-CGR R-CGR R-RGR S-DR S-PC S-IC S-TC S-UPC <S-PR> S-UIC <S-I> S-UTC <S-SR> S-L R-L L-R1 L-R2 S-LC <R-OQ> P-UOP <P-UOP> R-S R-OC <R-OQ> R-TC R-UTC R-IC R-UIC <R-I> S-ULC R-LC R-ULC P-P S-CP L-T P-D <L-T > W-DC R-SCC <R-P > R-SR <R-SF> <R-SR> <R-SR> Figure 6. Centralized supply chain system flow diagram. S-UIC S-UPC Figure 6. Centralized supply chain system flow diagram Figure 6. Centralized supply chain system flow diagram. Since the demand information starts from downstream and reaches upstream v multiple levels of delayed transmission, the accuracy of the information received by th suppliers at the top of the upstream supply chain is most obviously disturbed. We observ Since the demand information starts from downstream and reaches upstream via multiple levels of delayed transmission, the accuracy of the information received by the suppliers at the top of the upstream supply chain is most obviously disturbed. We observe the impact of lead time decisions on the bullwhip effect in the supply chain from the fluctuation of suppliers’ inventory. (1) Analysis of the Bullwhip Effect It mitigates the risk of having to increase inventory holdings to cope with unforeseen events such as stock-outs. At the same time, the cost of inventory holding is also reduced with the reduction in inventory levels. Since retailers belong to the downstream enterprises of the chain, they are directly facing the customers’ demand, and the shortening of lead time has a more obvious effect of improving their operation compared with the middle and upstream enterprises. The supplier, as the source of the supply chain, is actually uncertain in the process of obtaining order information and reproducing. On one hand, the rationality of orders is strongly biased in favor of retailers, and this decentralized decision-making approach does not provide a more direct and effective way for suppliers to predict the market. On the other hand, suppliers plan production in accordance with the orders of downstream firms, and while the shortening of the lead time for ordering may mitigate Systems 2024, 12, 32 10 of 20 of down tigate th 10 of 20 of dow tigate th the asymmetry in the information transfer process, it may also result in the inability of suppliers to reasonably set safety stock standards in the short term. The randomness of demand inputs prevents them from responding adequately to sudden changes in order levels, which may be detrimental to their short-term profits. pliers to reasonably set safety stock standards in the short term. The randomness of d mand inputs prevents them from responding adequately to sudden changes in order le els, which may be detrimental to their short-term profits. 5 2 Centralized Supply Chain Coordination the asymmetry in the information transfer process, it may also result in the inability of suppliers to reasonably set safety stock standards in the short term. The randomness of demand inputs prevents them from responding adequately to sudden changes in order levels, which may be detrimental to their short-term profits. pliers to reasonably set safety stock standards in the short term. The randomness of d mand inputs prevents them from responding adequately to sudden changes in order le els, which may be detrimental to their short-term profits. 5 2 Centralized Supply Chain Coordination 5.2. Centralized Supply Chain Coordination pp y In contrast to a decentralized supply (a) S-I 4,000 3,000 2,000 1,000 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) kg "S-I" : L-T5 1 1 1 1 "S-I" : L-T3 2 2 2 2 2 "S-I" : L-T1 3 3 3 3 (b) f S-I 1,000 750 500 250 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) kg "S-I" : L-T5(C) 1 1 1 1 "S-I" : L-T3(C) 2 2 2 2 "S-I" : L-T1(C) 3 3 3 3 S-I (a) (b) Figure 7. Supplier inventory under different modes. (a) Decentralized mode. (b) Centralized mode Figure 7. Supplier inventory under different modes. (a) Decentralized mode. (b) Centralized mode. Table 6 shows that lead time significantly affects the supply chain bullwhip effe The longer the lead time, the larger the supplier inventory variance sum, which is man fested by the larger curve fluctuation, indicating a more serious bullwhip phenomeno In order to ensure the coordinated operation of the supply chain, in addition to controlli the inventory level and its smooth operation, it is also necessary to mitigate the phenom enon that the overall efficiency of the supply chain is lower than the sum of the interes of both sides of the supply chain due to the lopsided pursuit of their own interests by bo the supplier and the retailer—the double marginal effect. For this purpose, we compa the profits of decentralized and centralized for lead times of 1 d 3 d and 5 d respective Table 6 shows that lead time significantly affects the supply chain bullwhip effect. The longer the lead time, the larger the supplier inventory variance sum, which is manifested by the larger curve fluctuation, indicating a more serious bullwhip phenomenon. 5.2. Centralized Supply Chain Coordination pp y In contrast to a decentralized supply In order to ensure the coordinated operation of the supply chain, in addition to controlling the inventory level and its smooth operation, it is also necessary to mitigate the phenomenon that the overall efficiency of the supply chain is lower than the sum of the interests of both sides of the supply chain due to the lopsided pursuit of their own interests by both the supplier and the retailer—the double marginal effect. For this purpose, we compare the profits of decentralized and centralized for lead times of 1 d, 3 d, and 5 d, respectively, as shown in Tables 7–9. as shown in Tables 7–9. Table 7. Comparison of profitability of parties with L-T1. as shown in Tables 7 9. Table 6. Supplier inventory variance sum at different lead times (centralized supply chain). L-T1 L-T3 L-T5 Supplier inventory variance sum 65,226.27 124,165.08 240,838.47 Table 7. Comparison of profitability of parties with L-T1. Time Retailer Profit (D) Retailer Profit (C) Supplier Profit (D) Supplier Profit (C) Supply Chain Profit (D) Supply Chain Profit (C) 10 d 82,371 282,552 706,267 474,348 788,638 756,900 20 d 296,621 672,655 1,293,040 948,545 1,589,660 1,621,200 30 d 647,070 1,057,620 1,643,840 1,455,970 2,290,910 2,513,590 40 d 927,040 1,374,020 1,963,180 1,998,000 2,890,220 3,372,020 50 d 1,292,230 1,773,400 2,300,920 2,619,700 3,593,150 4,393,100 Table 7. Comparison of profitability of parties with L-T1. Time Retailer Profit (D) Retailer Profit (C) Supplier Profit (D) Supplier Profit (C) Supply Chain Profit (D) Supply Chain Profit (C) 10 d 82,371 282,552 706,267 474,348 788,638 756,900 20 d 296,621 672,655 1,293,040 948,545 1,589,660 1,621,200 30 d 647,070 1,057,620 1,643,840 1,455,970 2,290,910 2,513,590 40 d 927,040 1,374,020 1,963,180 1,998,000 2,890,220 3,372,020 50 d 1,292,230 1,773,400 2,300,920 2,619,700 3,593,150 4,393,100 60 d 1,642,760 2,133,700 2,671,840 3,360,170 4,314,600 5,493,880 70 d 2,009,560 2,513,740 3,045,940 4,158,260 5,055,490 6,671,990 80 d 2,383,450 2,901,030 3,410,370 5,007,320 5,793,820 7,908,350 90 d 2,742,950 3,271,580 3,794,120 5,941,920 6,537,070 9,213,500 100 d 3,090,690 3,633,620 4,164,990 6,925,260 7,255,670 10,558,900 The results are compared below when the lead time is 1 d: The profit of the retailer is always higher in the centralized than decentralized supply chain. The supplier has higher profits under decentralized in the early period, but after 43 d, profits under centralized exceed decentralized, and the gap gradually widens over time. The total supply chain profit is higher in centralized than decentralized after 22 d, and the gap is increasing. 5.2. Centralized Supply Chain Coordination pp y In contrast to a decentralized supply (In Table 6, (D) stands for decentralized decision making, and (C) stands for centralized decision making.) Table 6. Supplier inventory variance sum at different lead times (centralized supply chain). Table 6. Supplier inventory variance sum at different lead times (centralized supply chain). L-T1 L-T3 L-T5 Supplier inventory variance sum 65,226.27 124,165.08 240,838.47 In Figure 7, we can see that regardless of the length of the lead time, the inventory level of centralized suppliers is lower than that of the decentralized suppliers under the same lead time condition. This indicates that centralized decision making enables the upstream enterprises in the supply chain to obtain demand information more directly and accurately and respond quickly, and their own inventory levels can be kept low. In addition to the horizontal data comparison, the supplier inventory volatility under different lead time levels should also be considered. Here, we select the supplier inventory variance after 20 d of the centralized supply chain for comparison, as shown below: Systems 2024, 12, 32 11 of 20 ry varian (a) (b) Figure 7. Supplier inventory under different modes. (a) Decentralized mode. (b) Centralized mode. S-I 4,000 3,000 2,000 1,000 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) kg "S-I" : L-T5 1 1 1 1 "S-I" : L-T3 2 2 2 2 2 "S-I" : L-T1 3 3 3 3 S-I 1,000 750 500 250 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) kg "S-I" : L-T5(C) 1 1 1 1 "S-I" : L-T3(C) 2 2 2 2 "S-I" : L-T1(C) 3 3 3 3 Figure 7. Supplier inventory under different modes. (a) Decentralized mode. (b) Centralized mode. 5.2. Centralized Supply Chain Coordination pp y In contrast to a decentralized supply as shown in Tables 7 9. Table 6. Supplier inventory variance sum at different lead times (centralized supply chain). L-T1 L-T3 L-T5 Supplier inventory variance sum 65,226.27 124,165.08 240,838.47 Table 7. Comparison of profitability of parties with L-T1. Time Retailer Profit (D) Retailer Profit (C) Supplier Profit (D) Supplier Profit (C) Supply Chain Profit (D) Supply Chain Profit (C) 10 d 82,371 282,552 706,267 474,348 788,638 756,900 20 d 296,621 672,655 1,293,040 948,545 1,589,660 1,621,200 30 d 647,070 1,057,620 1,643,840 1,455,970 2,290,910 2,513,590 40 d 927,040 1,374,020 1,963,180 1,998,000 2,890,220 3,372,020 50 d 1 292 230 1 773 400 2 300 920 2 619 700 3 593 150 4 393 100 Table 7. Comparison of profitability of parties with L-T1. Time Retailer Profit (D) Retailer Profit (C) Supplier Profit (D) Supplier Profit (C) Supply Chain Profit (D) Supply Chain Profit (C) 10 d 82,371 282,552 706,267 474,348 788,638 756,900 20 d 296,621 672,655 1,293,040 948,545 1,589,660 1,621,200 30 d 647,070 1,057,620 1,643,840 1,455,970 2,290,910 2,513,590 40 d 927,040 1,374,020 1,963,180 1,998,000 2,890,220 3,372,020 50 d 1,292,230 1,773,400 2,300,920 2,619,700 3,593,150 4,393,100 60 d 1,642,760 2,133,700 2,671,840 3,360,170 4,314,600 5,493,880 70 d 2,009,560 2,513,740 3,045,940 4,158,260 5,055,490 6,671,990 80 d 2,383,450 2,901,030 3,410,370 5,007,320 5,793,820 7,908,350 90 d 2,742,950 3,271,580 3,794,120 5,941,920 6,537,070 9,213,500 100 d 3,090,690 3,633,620 4,164,990 6,925,260 7,255,670 10,558,900 , , , , , , , , , , The results are compared below when the lead time is 1 d: The profit of the retailer is always higher in the centralized than decentralized supply chain. The supplier has higher profits under decentralized in the early period, but after 43 d, profits under centralized exceed decentralized, and the gap gradually widens over time. The total supply chain profit is higher in centralized than decentralized after 22 d, and the gap is increasing. , , , , , , , , , , The results are compared below when the lead time is 1 d: The profit of the retailer is always higher in the centralized than decentralized supply chain. The supplier has higher profits under decentralized in the early period, but after 43 d, profits under centralized exceed decentralized, and the gap gradually widens over time. The total supply chain profit is higher in centralized than decentralized after 22 d, and the gap is increasing. 5.2. Centralized Supply Chain Coordination pp y In contrast to a decentralized supply The profit of the retailer is always higher in the centralized than decentralized supply chain. The supplier has higher profits under decentralized in the early period, but after 55 d, profits under centralized exceed decentralized, and the gap gradually widens over time. The total supply chain profit is higher in centralized than decentralized supply chain after 30 d, and the gap is increasing. Overall, it is divided into two dimensions: firstly, whether supplier, retailer, or the l h i h l th fit d th t li d d ill l b hi h th Systems 2024, 12, 32 12 of 20 Table 8. Comparison of profitability of parties with L-T3. Table 8. Comparison of profitability of parties with L-T3. Time Retailer Profit (D) Retailer Profit (C) Supplier Profit (D) Supplier Profit (C) Supply Chain Profit (D) Supply Chain Profit (C) 10 d 71,910 280,697 719,936 472,875 791,846 753,572 20 d 152,529 653,845 1,474,070 946,289 1,626,600 1,600,130 30 d 499,741 1,017,920 1,819,920 1,448,650 2,319,660 2,466,570 40 d 756,598 1,313,140 2,128,100 1,981,960 2,884,700 3,295,100 50 d 1,095,150 1,691,040 2,453,650 2,591,660 3,548,800 4,282,700 60 d 1,421,350 2,056,890 2,813,130 3,293,590 4,234,470 5,350,480 70 d 1,766,840 2,439,950 3,176,690 4,064,340 4,943,540 6,504,290 80 d 2,121,320 2,829,140 3,531,170 4,893,800 5,652,490 7,722,940 90 d 2,474,480 3,202,740 3,894,580 5,811,170 6,369,060 9,013,900 100 d 2,811,070 3,564,930 4,253,870 6,781,780 7,064,950 10,346,700 The results are compared below when the lead time is 3 d: The profit of the retailer is always higher in the centralized than decentralized supply chain. The supplier has higher profits under decentralized in the early period, but after 50 d, profits under centralized exceed decentralized, and the gap gradually widens over time. The total supply chain profit is higher in centralized than decentralized supply chain after 27 d, and the gap is increasing. Table 9. Comparison of profitability of parties with L-T5. Table 9. Comparison of profitability of parties with L-T5. 5.2. Centralized Supply Chain Coordination pp y In contrast to a decentralized supply , , , , , , , , , , The results are compared below when the lead time is 1 d: The profit of the retailer is always higher in the centralized than decentralized supply chain. The supplier has higher profits under decentralized in the early period, but after 43 d, profits under centralized exceed decentralized, and the gap gradually widens over time. The total supply chain profit is higher in centralized than decentralized after 22 d, and the gap is increasing. Systems 2024, 12, 32 12 of 20 Table 8. Comparison of profitability of parties with L-T3. Time Retailer Profit (D) Retailer Profit (C) Supplier Profit (D) Supplier Profit (C) Supply Chain Profit (D) Supply Chain Profit (C) 10 d 71,910 280,697 719,936 472,875 791,846 753,572 20 d 152,529 653,845 1,474,070 946,289 1,626,600 1,600,130 30 d 499,741 1,017,920 1,819,920 1,448,650 2,319,660 2,466,570 40 d 756,598 1,313,140 2,128,100 1,981,960 2,884,700 3,295,100 50 d 1,095,150 1,691,040 2,453,650 2,591,660 3,548,800 4,282,700 60 d 1,421,350 2,056,890 2,813,130 3,293,590 4,234,470 5,350,480 70 d 1,766,840 2,439,950 3,176,690 4,064,340 4,943,540 6,504,290 80 d 2,121,320 2,829,140 3,531,170 4,893,800 5,652,490 7,722,940 90 d 2,474,480 3,202,740 3,894,580 5,811,170 6,369,060 9,013,900 100 d 2,811,070 3,564,930 4,253,870 6,781,780 7,064,950 10,346,700 The results are compared below when the lead time is 3 d: The profit of the retailer is always higher in the centralized than decentralized supply chain. The supplier has higher profits under decentralized in the early period, but after 50 d, profits under centralized exceed decentralized, and the gap gradually widens over time. The total supply chain profit is higher in centralized than decentralized supply chain after 27 d, and the gap is increasing. Table 9. Comparison of profitability of parties with L-T5. Time Retailer Profit (D) Retailer Profit (C) Supplier Profit (D) Supplier Profit (C) Supply Chain Profit (D) Supply Chain Profit (C) 10 d 64,329 280,183 729,189 470,830 793,518 751,013 20 d 59,553 647,018 1,588,410 945,736 1,647,960 1,592,750 30 d 389,344 999,712 1,969,840 1,444,430 2,359,180 2,444,150 40 d 631,794 1,281,510 2,270,370 1,973,760 2,902,160 3,255,270 50 d 946,507 1,644,840 2,584,930 2,577,160 3,531,440 4,221,990 60 d 1,248,040 1,997,140 2,932,860 3,269,720 4,180,900 5,266,860 70 d 1,571,040 2,368,660 3,285,550 4,028,320 4,856,590 6,396,970 80 d 1,905,010 2,748,040 3,629,790 4,843,490 5,534,800 7,591,530 90 d 2,239,220 3,125,240 3,983,440 5,734,290 6,222,660 8,859,530 100 d 2,558,210 3,485,330 4,333,450 6,686,510 6,891,660 10,171,800 The results are compared below when the lead time is 5 d. 5.3. Centralized Supply Chain Coordination under Target Heterogeneity To date, research results generated under the condition that the parameters of the revenue-sharing contract are fixed. In order to enrich the research conclusions, contribute to the cooperation and stabilization of inter-firms with higher probability, and find the optimal decision domain of the lead time, we will adjust the key parameters of the centralized decision structure in the next step. We will observe and compare the stage-by-stage profit direction of supply chain parties’ interactions to find more specific paths and countermea- sures to drive the coordinated development of the supply chain. First of all, based on the above simulation results, it can be concluded that the retailer’s profit in the simulation period is all higher than the decentralized mode when the length of the lead time is 1 d, 3 d, and 5 d for the centralized at the coefficient of a contract of 0.018 and the coefficient of wholesale discount of 0.95, and the supplier and the supply chain as a whole are also superior to the centralized in terms of profit in the long run. However, the reality is that some suppliers may mind short-term profit and loss because they do not see the future trend of profit, thus hindering the smooth achievement of the second level of cooperation mode; for this reason, this paper uses Tpd (meaning: time of profit disadvantage) to indicate the time when the supplier profit under the centralized mode at the beginning of the simulation is briefly lower than that under the decentralized mode. Then, the further “coordination condition” is to keep the retailer’s profit level within the permissible range and simultaneously shorten the Tpd to help the supplier obtain a satisfactory level of benefits as soon as possible. In other words, combining contract coefficients and wholesale discount coefficients to shorten the Tpd to a suitable area will be the key to stabilizing the cooperative relationship. In the parameter exploration stage, the following laws are found: 1⃝when the whole- sale discount coefficient is set to 0.8, it is necessary to adjust the coefficient of the revenue- sharing contract to about 0.3 at the same time in order to achieve the above coordination conditions, but at this time, the assumptions are no longer satisfied; 2⃝in the value domain, the larger the coefficient of revenue-sharing contract and the coefficient of wholesale dis- count are, the better the coordination effect is. 5.2. Centralized Supply Chain Coordination pp y In contrast to a decentralized supply Time Retailer Profit (D) Retailer Profit (C) Supplier Profit (D) Supplier Profit (C) Supply Chain Profit (D) Supply Chain Profit (C) 10 d 64,329 280,183 729,189 470,830 793,518 751,013 20 d 59,553 647,018 1,588,410 945,736 1,647,960 1,592,750 30 d 389,344 999,712 1,969,840 1,444,430 2,359,180 2,444,150 40 d 631,794 1,281,510 2,270,370 1,973,760 2,902,160 3,255,270 50 d 946,507 1,644,840 2,584,930 2,577,160 3,531,440 4,221,990 60 d 1,248,040 1,997,140 2,932,860 3,269,720 4,180,900 5,266,860 70 d 1,571,040 2,368,660 3,285,550 4,028,320 4,856,590 6,396,970 80 d 1,905,010 2,748,040 3,629,790 4,843,490 5,534,800 7,591,530 90 d 2,239,220 3,125,240 3,983,440 5,734,290 6,222,660 8,859,530 100 d 2,558,210 3,485,330 4,333,450 6,686,510 6,891,660 10,171,800 The results are compared below when the lead time is 5 d. The profit of the retailer is always higher in the centralized than decentralized supply chain. The supplier has higher profits under decentralized in the early period, but after 55 d, profits under centralized exceed decentralized, and the gap gradually widens over time. The total supply chain profit is higher in centralized than decentralized supply chain after 30 d, and the gap is increasing. Overall, it is divided into two dimensions: firstly, whether supplier, retailer, or the supply chain as a whole, the profit under the centralized mode will always be higher than the decentralized mode after a certain length of time; secondly, with the shortening of the lead time, the slopes of the profit curves of the retailer and supplier are increasing, and the profits of the suppliers and the supply chain as a whole can reach the intersection point of the two decision-making modes more quickly. This shows that centralized decision making Systems 2024, 12, 32 13 of 20 13 of 20 can improve the cooperative enterprise, which not only deepens the suppliers’ willingness to cooperate but also promotes the coordinated development of the whole supply chain. 5.3. Centralized Supply Chain Coordination under Target Heterogeneity Therefore, based on the research pattern and assumption constraints, we set the wholesale discount coefficient between 0.85 and 0.95 and the shared covenant coefficient between 0.005 and 0.2 to determine the adjustment range. (Note: the following for example (a, b) is interpreted as the case where the wholesale discount coefficient is (a) while the revenue sharing coefficient is (b)). The simulation results are shown below. Step 1: As shown in Figure 8, Iconsider the case where the lead time is 1 d, and the parameter combination is (0.85, 0.005). It is observed that the curve declines day by day, and the supply chain profit system collapses completely. When the coefficient of the revenue-sharing contract is adjusted to 0.006, the system returns to normal and is in the “coordination condition” state. This indicates a critical point between the two combinations that affects the system’s stability. According to research rule 2⃝, the lower limit of parameter combination to reach the “coordination condition” is (0.85, 0.006). Step 2: According to Law 2⃝, when the wholesale discount coefficient is set to 0.95, Table 10 shows that the retailer’s profit level is lower than the decentralized decision under the conditions of 1 d lead time and the parameter combination of (0.95, 0.17), which means that this scheme harms the retailer’s profit and is not adopted. By adjusting the gain-sharing contract factor to 0.16, the system returns to normal and is in a “harmonized state”. At this point, it is found that when the wholesale discount coefficient takes the maximum value of 0.95 in the range, the contractual coordination coefficient of 0.16 < 0.2 (assuming constraints), and for the time being, it is not possible to use (0.95,0.16) as the upper limit of parameter combinations. Since other parameter combinations such as (0.94, Systems 2024, 12, 32 14 of 20 pper lim 14 of 20 pper lim 0.17) may result in better supplier profitability under the “coordination condition”, the parameter combinations need to be re-selected for comparative analysis. result in better supplier profitability under the coordination condition , the paramet combinations need to be re-selected for comparative analysis. (a) (b) (c) Figure 8. The profits under the parameter combination (0.85, 0.005) and (0.85, 0.006). (a) The prof of the retailer. (b) The profits of the supplier. (c) The profits of the supply chain. Figure 8. The profits under the parameter combination (0.85, 0.005) and (0.85, 0.006). 5.3. Centralized Supply Chain Coordination under Target Heterogeneity (a) The profits of the retailer. (b) The profits of the supplier. (c) The profits of the supply chain. (a) (b) (a) (b) (c) (b) (a) (c) Figure 8. The profits under the parameter combination (0.85, 0.005) and (0.85, 0.006). (a) The prof of the retailer. (b) The profits of the supplier. (c) The profits of the supply chain. Figure 8. The profits under the parameter combination (0.85, 0.005) and (0.85, 0.006). (a) The profits of the retailer. (b) The profits of the supplier. (c) The profits of the supply chain. Table 10. Retailer’s profit under different decision structure and parameter combinations with L Table 10. Retailer’s profit under different decision structure and parameter combinations with L-T1. Table 10. Retailer s profit under different decision structure and parameter combinations with L-T Centralized Mode Decentralized Mode (0.95,0.16) (0.95,0.17) 18,831 18,607 18,822 1,516,960 1,398,900 1,491,120 3,108,190 3,071,180 3,090,690 Table 10. Retailer s profit under different decision structure and parameter combinations with L-T1. Simulation Time Centralized Mode Decentralized Mode (0.95,0.16) (0.95,0.17) 1 d 18,831 18,607 18,822 50 d 1,516,960 1,398,900 1,491,120 100 d 3,108,190 3,071,180 3,090,690 Step 3: Repeat step 2 with the help of the “dichotomy” research idea and adjust th five parameter combinations as shown in Figure 9 without harming the interests of retai ers and the supply chain as a whole. Due to the influence of assumption constraints, onc the coefficient of revenue sharing contract in the parameter combinations reaches 0.2, n further adjustment can be made, and the upper limit of the parameter combinations t reach the “coordination conditions” is finally determined to be (0 91 0 2) Step 3: Repeat step 2 with the help of the “dichotomy” research idea and adjust the five parameter combinations as shown in Figure 9 without harming the interests of retailers and the supply chain as a whole. Due to the influence of assumption constraints, once the coefficient of revenue sharing contract in the parameter combinations reaches 0.2, no further adjustment can be made, and the upper limit of the parameter combinations to reach the “coordination conditions” is finally determined to be (0.91,0.2). reach the coordination conditions is finally determined to be (0.91,0.2). 5.3. Centralized Supply Chain Coordination under Target Heterogeneity Similarly, we can find the domain of values of parameter combinations when th ime is 3 d and 5 d based on the above three steps as ((0.85,0.006), (0.95,0.2) (0 85 0 006) (0 95 0 2)) respectively From Table 11, it can be seen that when the lead time is 1 d, the shortest Tpd of the parameter combination “after improvement” is 15 d, which means that the supplier’s profit can reach the intersection of centralized and decentralized mode the fastest when the lead time is 1 d. Meanwhile, via the difference in Tpd, we find that the smallest change in the direction of the supplier’s profit before and after the improvement in parameter combinations is when the lead time is 3 d, with a Tpd difference of 58 d, and the largest change in the direction is when the lead time is 5 d, with a Tpd difference of 78 d. g pp pif p Step 4: Via steps 1, 2, and 3, it can be finally determined that when the lead tim d, the parameter combinations that can reach the “coordination conditions” unde ralized mode are ((0.85,0.006), (0.91,0.2)). Similarly, we can find the domain of values of parameter combinations when th ime is 3 d and 5 d based on the above three steps as ((0.85,0.006), (0.95,0.2) (0 85 0 006) (0 95 0 2)) respectively From Table 11, it can be seen that when the lead time is 1 d, the shortest Tpd of the parameter combination “after improvement” is 15 d, which means that the supplier’s profit can reach the intersection of centralized and decentralized mode the fastest when the lead time is 1 d. Meanwhile, via the difference in Tpd, we find that the smallest change in the direction of the supplier’s profit before and after the improvement in parameter combinations is when the lead time is 3 d, with a Tpd difference of 58 d, and the largest change in the direction is when the lead time is 5 d, with a Tpd difference of 78 d. According to the above conclusion, the Tpd and the supplier’s profit when the time is 1 d, 3 d, and 5 d are compared and analyzed, and the lower limit of the para combination is called “before improvement”, and the upper limit is called “after imp ment”. The simulation results are shown in the figure below. 5.3. Centralized Supply Chain Coordination under Target Heterogeneity i y ( ) Step 4: Via steps 1, 2, and 3, it can be finally determined that when the lead time is 1 d, the parameter combinations that can reach the “coordination conditions” under centralized mode are ((0.85,0.006), (0.91,0.2)).i Similarly, we can find the domain of values of parameter combinations when the lead time is 3 d and 5 d based on the above three steps as ((0.85,0.006), (0.95,0.2)) and ((0.85,0.006), (0.95,0.2)), respectively. p y According to the above conclusion, the Tpd and the supplier’s profit when the lead time is 1 d, 3 d, and 5 d are compared and analyzed, and the lower limit of the param- eter combination is called “before improvement”, and the upper limit is called “after improvement”. The simulation results are shown in the figure below. According to the above conclusion, the Tpd and the supplier’s profit when the lead time is 1 d, 3 d, and 5 d are compared and analyzed, and the lower limit of the param- eter combination is called “before improvement”, and the upper limit is called “after improvement”. The simulation results are shown in the figure below. Systems 2024, 12, 32 tems 2024, 12, x FOR 15 of 20 15 Figure 9. Supplier’s profit for different parameter combinations. Step 4: Via steps 1, 2, and 3, it can be finally determined that when the lead tim d, the parameter combinations that can reach the “coordination conditions” under tralized mode are ((0.85,0.006), (0.91,0.2)). Similarly, we can find the domain of values of parameter combinations when the time is 3 d and 5 d based on the above three steps as ((0.85,0.006), (0.95,0.2)) ((0.85,0.006), (0.95,0.2)), respectively. 5.3. Centralized Supply Chain Coordination under Target Heterogeneity i 供应链总利润 40 M 30 M 20 M 10 M 0 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) 元 供应链总利润 : 提前期c1契约（0.91，0.2） 1 1 1 1 供应链总利润 : 提前期c1契约（0.92，0.19） 2 2 2 2 供应链总利润 : 提前期c1契约（0.93，0.18） 3 3 3 3 供应链总利润 : 提前期c1契约（0.94，0.17） 4 4 4 4 供应链总利润 : 提前期c1契约（0.95，0.16） 5 5 5 5 5 S-CP 20 M 15 M 10 M 5 M 0 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) 元 "S-CP" : L-T1&（0.91，0.2） 1 1 1 1 1 1 "S-CP" : L-T1&（0.92，0.19） 2 2 2 2 2 2 "S-CP" : L-T1&（0.93，0.18） 3 3 3 3 3 3 "S-CP" : L-T1&（0.94，0.17） 4 4 4 4 4 4 "S-CP" : L-T1&（0.95，0.16） 5 5 5 5 5 5 5 S-CP 20 M 15 M 10 M 5 M 0 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) 元 "S-CP" : L-T1&（0.91，0.2） 1 1 1 1 1 1 "S-CP" : L-T1&（0.92，0.19） 2 2 2 2 2 2 "S-CP" : L-T1&（0.93，0.18） 3 3 3 3 3 3 "S-CP" : L-T1&（0.94，0.17） 4 4 4 4 4 4 "S-CP" : L-T1&（0.95，0.16） 5 5 5 5 5 5 5 Figure 9. Supplier’s profit for different parameter combinations. From Table 11, it can be seen that when the lead time is 1 d, the shortest Tpd of the parameter combination “after improvement” is 15 d, which means that the supplier’s profit can reach the intersection of centralized and decentralized mode the fastest when the lead time is 1 d. 5.3. Centralized Supply Chain Coordination under Target Heterogeneity 供应链总利润 40 M 30 M 20 M 10 M 0 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) 元 供应链总利润 : 提前期c1契约（0.91，0.2） 1 1 1 1 供应链总利润 : 提前期c1契约（0.92，0.19） 2 2 2 2 供应链总利润 : 提前期c1契约（0.93，0.18） 3 3 3 3 供应链总利润 : 提前期c1契约（0.94，0.17） 4 4 4 4 供应链总利润 : 提前期c1契约（0.95，0.16） 5 5 5 5 5 S-CP 20 M 15 M 10 M 5 M 0 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) 元 "S-CP" : L-T1&（0.91，0.2） 1 1 1 1 1 1 "S-CP" : L-T1&（0.92，0.19） 2 2 2 2 2 2 "S-CP" : L-T1&（0.93，0.18） 3 3 3 3 3 3 "S-CP" : L-T1&（0.94，0.17） 4 4 4 4 4 4 "S-CP" : L-T1&（0.95，0.16） 5 5 5 5 5 5 5 S-CP 20 M 15 M 10 M 5 M 0 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) 元 "S-CP" : L-T1&（0.91，0.2） 1 1 1 1 1 1 "S-CP" : L-T1&（0.92，0.19） 2 2 2 2 2 2 "S-CP" : L-T1&（0.93，0.18） 3 3 3 3 3 3 "S-CP" : L-T1&（0.94，0.17） 4 4 4 4 4 4 "S-CP" : L-T1&（0.95，0.16） 5 5 5 5 5 5 5 Figure 9. Supplier’s profit for different parameter combinations. Figure 9 Supplier’s profit for different parameter combinatio Figure 9. Supplier’s profit for different parameter combinations. g pp pif p Step 4: Via steps 1, 2, and 3, it can be finally determined that when the lead tim d, the parameter combinations that can reach the “coordination conditions” unde ralized mode are ((0.85,0.006), (0.91,0.2)). 5.3. Centralized Supply Chain Coordination under Target Heterogeneity From Table 11, it can be seen that when the lead time is 1 d, the shortest Tpd parameter combination “after improvement” is 15 d, which means that the supp profit can reach the intersection of centralized and decentralized mode the fastest Table 11. Tpd values at different lead times. Lead Time Tpd (Before Improvement) Tpd (After Improvement) Tpd (Gap) L-T1 80 d 15 d 65 d L-T3 75 d 17 d 58 d L-T5 97 d 19 d 78 d According to the above conclusio i i 1 d 3 d d 5 d d Table 11. Tpd values at different lead times. the lead time is 1 d. Meanwhile, via the difference in Tpd, we find that the smallest ch in the direction of the supplier’s profit before and after the improvement in para combinations is when the lead time is 3 d, with a Tpd difference of 58 d, and the l change in the direction is when the lead time is 5 d, with a Tpd difference of 78 d. As shown in Figure 10, the trend of profit level to suppliers before and after the improvement is different; in order to further study the improvement in parameter combi- nations under different lead times to bring different impact effects on suppliers and the supply chain as a whole, the statistics are as follows: W 16 t lead times. nt) Tpd (After Improvement) Tp 15 d 17 d 19 d the trend of profit level to suppliers before and der to further study the improvement in param mes to bring different impact effects on supplier tistics are as follows: (b) f Table 11. Tpd values at different lead times. ead Time Tpd (Before Improvement) Tpd (After Improvement) Tpd (gap) L-T1 80 d 15 d 65 d L-T3 75 d 17 d 58 d L-T5 97 d 19 d 78 d As shown in Figure 10, the trend of profit level to suppliers before and after th provement is different; in order to further study the improvement in parameter com tions under different lead times to bring different impact effects on suppliers and the ply chain as a whole, the statistics are as follows: (a) (b) Figure 10. Supplier’s profit with different lead times. (a) before improvement; (b) after improve Figure 10. Supplier’s profit with different lead times. (a) before improvement; (b) after improvement. Table 11. 5.3. Centralized Supply Chain Coordination under Target Heterogeneity Meanwhile, via the difference in Tpd, we find that the smallest change in the direction of the supplier’s profit before and after the improvement in parameter combinations is when the lead time is 3 d, with a Tpd difference of 58 d, and the largest change in the direction is when the lead time is 5 d, with a Tpd difference of 78 d. 5.3. Centralized Supply Chain Coordination under Target Heterogeneity Figure 9 Supplier’s profit for different parameter combinations 供应链总利润 40 M 30 M 20 M 10 M 0 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) 元 供应链总利润 : 提前期c1契约（0.91，0.2） 1 1 1 1 供应链总利润 : 提前期c1契约（0.92，0.19） 2 2 2 2 供应链总利润 : 提前期c1契约（0.93，0.18） 3 3 3 3 供应链总利润 : 提前期c1契约（0.94，0.17） 4 4 4 4 供应链总利润 : 提前期c1契约（0.95，0.16） 5 5 5 5 5 S-CP 20 M 15 M 10 M 5 M 0 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) 元 "S-CP" : L-T1&（0.91，0.2） 1 1 1 1 1 1 "S-CP" : L-T1&（0.92，0.19） 2 2 2 2 2 2 "S-CP" : L-T1&（0.93，0.18） 3 3 3 3 3 3 "S-CP" : L-T1&（0.94，0.17） 4 4 4 4 4 4 "S-CP" : L-T1&（0.95，0.16） 5 5 5 5 5 5 5 S-CP 20 M 15 M 10 M 5 M 0 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 Time (Day) 元 "S-CP" : L-T1&（0.91，0.2） 1 1 1 1 1 1 "S-CP" : L-T1&（0.92，0.19） 2 2 2 2 2 2 "S-CP" : L-T1&（0.93，0.18） 3 3 3 3 3 3 "S-CP" : L-T1&（0.94，0.17） 4 4 4 4 4 4 "S-CP" : L-T1&（0.95，0.16） 5 5 5 5 5 5 5 Figure 9. Supplier’s profit for different parameter combinations. Figure 9. Supplier’s profit for different parameter combinations. 5.3. Centralized Supply Chain Coordination under Target Heterogeneity Tpd values at differe ead Time Tpd (Before Improvem L-T1 80 d L-T3 75 d L-T5 97 d As shown in Figure 1 provement is different; in o tions under different lead t ply chain as a whole, the st (a) (b) (a) igure 10. Supplier’s profit with different lead times. (a) before improvement; (b) after improv Figure 10. Supplier’s profit with different lead times. (a) before improvement; (b) after improvement. Tables 12 and 13 show the specific data of the profit value of the supplier and th whole supply chain before and after the improvement in the parameter combination and it is found via the comparative analysis that ① before the improvement in the p Tables 12 and 13 show the specific data of the profit value of the supplier and the whole supply chain before and after the improvement in the parameter combination, and it is found via the comparative analysis that 1⃝before the improvement in the parameter Tables 12 and 13 show the specific data of the profit value of the supplier and th whole supply chain before and after the improvement in the parameter combination and it is found via the comparative analysis that ① before the improvement in the p Tables 12 and 13 show the specific data of the profit value of the supplier and the whole supply chain before and after the improvement in the parameter combination, and it is found via the comparative analysis that 1⃝before the improvement in the parameter Systems 2024, 12, 32 16 of 20 16 of 20 combination, the profit level of the supplier in each stage of the simulation cycle is not strictly in accordance with the trend of the higher profit level of the supplier with the shorter lead time, and the profit level is the highest in the case of the lead time of 3 d. For the overall profit level of the supply chain, there is also a disorder in the later stage when the lead time is 5 d, which is higher than 3 d. 2⃝With the parameter combination “after improvement”, both the supplier and the whole supply chain profits at each stage of the simulation period increase with the shortening of lead time. 3⃝The supplier’s profit level and the supply chain’s overall profit level after improvement are much higher than before improvement, and the advantage gradually increases with the simulation time. 5.3. Centralized Supply Chain Coordination under Target Heterogeneity Table 12. Supplier’s profit at different lead times before and after improvement. Simulation Time Before Improvement After Improvement L-T1 L-T3 L-T5 L-T1 L-T3 L-T5 20 d 791,911 882,397 790,631 1,762,700 1,722,700 1,718,040 50 d 1,941,820 2,116,960 1,927,250 8,892,750 8,360,210 8,234,390 80 d 3,420,190 3,619,750 3,338,640 21,818,200 20,376,000 19,934,200 100 d 4,519,960 4,746,310 4,400,190 33,660,100 31,516,700 30,802,000i Table 12. Supplier’s profit at different lead times before and after improvement. Table 13. Supply chain profit at different lead times before and after improvement. Table 13. Supply chain profit at different lead times before and after improvement. Simulation Time Before Improvement After Improvement L-T1 L-T3 L-T5 L-T1 L-T3 L-T5 20 d 1,569,780 1,544,230 1,542,440 2,341,910 2,255,370 2,245,150 50 d 3,953,970 3,828,670 3,809,170 10,407,400 9,737,840 9,574,380 80 d 6,706,270 6,483,460 6,465,390 24,294,100 22,680,800 22,172,900 100 d 8,634,270 8,354,800 8,357,860 36,760,700 34,420,900 33,641,300 Table 14 compares the total profit of the supply chain under centralized decision making and decentralized decision making when the lower limit of parameter combination is taken. It finds that, regardless of whether the lead time is 1 d, 3 d, or 5 d, the total profit of the supply chain is higher under the decentralized mode when compared with the centralized mode “before improvement”. However, there is a short period of time in the early stage, but the overall point of view is still in a disadvantageous position. It is further validated from the perspective of the coordinated supply chain to verify the feasibility of the centralized mode under the decision-making domain of parameter combination. Table 14. Supply chain profit at different lead times before and after improvement. Simulation Time Before Improvement Decentralized Mode L-T1 L-T3 L-T5 L-T1 L-T3 L-T5 20 d 1,569,780 1,544,230 1,542,440 1,589,660 1,626,600 1,647,960 50 d 3,953,970 3,828,670 3,809,170 3,593,150 3,548,800 3,531,440 80 d 6,706,270 6,483,460 6,465,390 5,793,820 5,652,490 5,534,800 100 d 8,634,270 8,354,800 8,357,860 7,255,670 7,064,950 6,891,660 In summary, there are two models of cooperation for the centralized supply chain oriented to goal heterogeneity. Table 14. Supply chain profit at different lead times before and after improvement. In summary, there are two models of cooperation for the centralized supply chain oriented to goal heterogeneity. In summary, there are two models of cooperation for the centralized supply chain oriented to goal heterogeneity. (1) With cooperative stability as the primary goal (1) With cooperative stability as the primary goal (1) With cooperative stability as the primary goal (1) With cooperative stability as the primary goal This model aims to help the supplier obtain a stronger willingness to cooperate, which can weaken part of the retailer’s revenue at an appropriate range. In the pre-cooperation period between the supplier and the retailer, the order lead time is compressed to 1 d, while the parameter combination is set to (0.91.0.2) so that the supplier’s Tpd value is Systems 2024, 12, 32 17 of 20 17 of 20 the shortest. In this state, the retailer’s profit is still higher than that of a decentralized company, while the supplier’s benefit reaches a satisfactory level in the shortest time, which maximizes the stability of the cooperative relationship. However, the first middle stage will cause the retailer to be unable to achieve more profits due to the adoption of centralized decision-making mechanisms that are more favorable for suppliers to participate in the cooperation. Therefore, the decision-making model can be adjusted when the profit level of the supplier is higher than the decentralized decision making after entering the middle stage of cooperation. According to the duration of cooperation, within the scope of “coordi- nation conditions”, the coefficient of revenue sharing contract should be reduced, and the wholesale discount coefficient should be increased, in order to regulate the profit leverage; the benefits of the advantage will gradually shift to the retailer, mobilizing the enthusiasm of both parties to cooperate. At this time, the overall profit level of the supply chain is also relatively high, and the two sides will maintain a friendly situation of “profitable, win-win cooperation”, which is conducive to the overall coordinated and sustainable development of the supply chain. the shortest. In this state, the retailer’s profit is still higher than that of a decentralized company, while the supplier’s benefit reaches a satisfactory level in the shortest time, which maximizes the stability of the cooperative relationship. However, the first middle stage will cause the retailer to be unable to achieve more profits due to the adoption of centralized decision-making mechanisms that are more favorable for suppliers to participate in the cooperation. Therefore, the decision-making model can be adjusted when the profit level of the supplier is higher than the decentralized decision making after entering the middle stage of cooperation. (1) With cooperative stability as the primary goal According to the duration of cooperation, within the scope of “coordi- nation conditions”, the coefficient of revenue sharing contract should be reduced, and the wholesale discount coefficient should be increased, in order to regulate the profit leverage; the benefits of the advantage will gradually shift to the retailer, mobilizing the enthusiasm of both parties to cooperate. At this time, the overall profit level of the supply chain is also relatively high, and the two sides will maintain a friendly situation of “profitable, win-win cooperation”, which is conducive to the overall coordinated and sustainable development of the supply chain. For FMCG supply chains, such as dairy products, there may be challenges in adopting such a cooperative model. Prior to profit leverage, retailers were in a relatively passive position in the partnership. Pricing was less flexible and could not be changed at will in response to real-time market changes due to the strategic goal of partnering first. For example, stepping outside of the hypothetical constraints, the retailer is most likely to be at the secondary crossroads of the horizontal chain due to the wide and short distribution channels of FMCG. When unexpected events occur, the cooperation mode of the chain may affect the adjustment of the overall strategic layout of the dairy enterprise. Therefore, before the cooperation is reached, it should be matched with detailed market research data and contingency plans. So, FMCG retailers can not only coordinate in the vertical chain but also in the fierce horizontal market, competition can still stand firm. (2) With balanced development with benefits as the primary goal (2) With balanced development with benefits as the primary goal 6. Conclusions In this paper, with the help of Vensim software, a system dynamics simulation study was conducted to investigate the coordination problem of decentralized and centralized supply chains under the influence of lead time, and the following conclusions were found: (1) The decentralized mode of the secondary supply chain composed of suppliers and retailers slowed down the bullwhip effect in the process of lead time shortening, and the overall profit is gradually optimized. However, there is a localized situation that is not conducive to cooperation and stability, which is manifested in the fact that the supplier’s profit decreases with the shortening of lead time. This change is contrary to the trend of retailers and the supply chain as a whole, i.e., with the shortening of lead time, the decision-making orientation of all parties in the decentralized supply chain is inconsistent, and the supply chain coordination is ineffective. pp y (2) The centralized approach is more effective than the decentralized one in mitigating the bullwhip and double marginal effects. Moreover, the centralized approach not only conforms to the rule that the shorter the lead time, the more coordinated the supply chain as a whole is, but also eliminates the unfavorable factors that may lead to unsuccessful cooperation among local members in the decentralized approach, which positively promotes win-win cooperation between suppliers and retailers as well as the stability of the supply chain’s long-term sustainable development. (3) (3) After comparing the centralized and decentralized approaches, it is found that the parameter combinations of the centralized contractual coordination mechanism need to be within the appropriate range of values in order to achieve the coordination effect. After repeated testing of the adjustment variables, we find the parameter combinations under different lead times. Furthermore, via comparison, it is found that with the shortening of the lead time, there are parameter combinations in the parameter range that enable suppliers to obtain a satisfactory level of benefits faster, i.e., the shorter the lead time, the higher the probability of stabilizing the situation of cooperation. In addition, based on the profit development trajectory of suppliers, retailers, and the whole supply chain, and the heterogeneous demand within the supply chain, we find a coordinated development path based on the primary goal of “cooperative stabilization” or “balanced effect”. (2) With balanced development with benefits as the primary goal This model aims to achieve satisfactory benefits for both the supplier and the retailer under centralized decision making and only ensures that the supplier’s profit level is higher than that of decentralized decision making. Considering the possibility that retailers may not be willing to give up part of their profits in order to have a higher probability of cooperation, the effect of the Tpd value is not considered in the first place. Via the study, during the period of cooperation between suppliers and retailers, a variety of parameter combinations within the range of “coordination conditions” can be selected, based on which the overall profit level of the supply chain is fully considered. It is found that when the lead time is 1 d, and the parameter combinations take the values around the lower limit, in the pre-simulation period, there is a short period of time that is lower than the total profit of the supply chain under decentralized decision making, and as the lead time increases, the time of this situation will be longer. In order to shorten this time and consider the respective profits of suppliers and retailers, it is more reasonable to choose a lead time of 1 d and take the value of the parameter combination interval. In this way, it can ensure the benefit level of suppliers and retailers and promote the coordinated development of the supply chain as a whole. Unlike the cooperative-led model, this model emphasizes autonomy while ensuring that the overall efficiency of the supply chain is optimized. The practical challenges are therefore different. Since the focus is more on self-efficiency, at the micro level, the degree of information sharing about the retailer’s operations and the value of the interval for the combination of the above parameters will depend on the firm’s managerial preference decisions. The assumption that firms possess overly rational traits may weaken the degree of information sharing. However, the cooperative goal of overall profit optimization cannot be violated, in which case the retailer may need to adjust the thresholds of the parameter combinations, and the selection of the interval values may affect the retailer’s original profit advantage. Therefore, in-depth discussions on the types as well as the Systems 2024, 12, 32 18 of 20 18 of 20 modalities of centralized cooperation models may be worth investigating in order to address the challenges. 6. Conclusions pp p The limitations of this paper and future research directions are as follows: (1) In the discussion of decentralized vs. centralized, there should be more discussion on several types of centralized decision making, pointing out the advantages and disadvantages and other different conclusions. Maybe in the future research can be further refined the centralized research. i (2) Since this paper only considers the influencing role of lead time, it does not address issues such as multi-cycle environment and time compression cost. Scholars often ignore issues such as the cycle correlation of information and updating mechanisms when considering supply and demand change studies. Setting the length of the study to be multi-cycle is the only way to achieve satisfactory and real results under the influence of multi-node change factors. Dynamic pricing, multi-cycle inventory, and other research combined with the lead time have to be studied in depth. (3) (3) The model construction of this paper is limited to the proposed assumptions, and there is still a big gap between the structure and comprehensiveness of the variables covered by the model and the reality. With the development of economic globalization, the supply chain structure has become more and more complex, often not limited to parallel supply chain competition. The situation of overlapping nodes between supply chains is increasing. And the connotation of outsourcing services is becoming more and more broad, which gives supply chain managers stronger control over demand information. Extending the boundary of the theoretical system to include richer elements has yet to be studied. Author Contributions: Conceptualization, M.Z. and Y.Z.; methodology, Y.W.; software, Y.W.; validation, M.Z.; investigation, Y.W. and Y.Z.; resources, M.Z. and Y.Z.; writing—original draft preparation, Y.W.; writing—review and editing, M.Z. and Y.W.; supervision, Y.Z.; project administration, Y.Z.; funding acquisition, M.Z. and Y.W. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, M.Z. and Y.Z.; methodology, Y.W.; software, Y.W.; validation, M.Z.; investigation, Y.W. and Y.Z.; resources, M.Z. and Y.Z.; writing—original draft preparation, Y.W.; writing—review and editing, M.Z. and Y.W.; supervision, Y.Z.; project administration, Y.Z.; funding acquisition, M.Z. and Y.W. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the General Program of the National Social Science Foundation of China (Grant No. 23BJY214), the Research Project on the Development of Social Sciences in Hebei Province in 2021 (Grant No. 6. Conclusions The supply chain coordination model in this paper also discusses the cooperative behavior of suppliers and retailers after random demand inputs from the market from a micro perspective. It can be seen that the reduction in lead time is a double-edged sword. Just making unilateral adjustments is not necessarily favorable. The supply chain ben- efits can only be accomplished with a reasonable decision-making structure. Therefore, it is recommended that managers should have a detailed and sufficient knowledge of the overall operation structure of the supply chain and their own development status when considering the strategic choice of the time factor. In addition, the study concludes that improving the transmission speed of information is conducive to the consistency of supply chain coordination and development goals. Whether as a supplier or retailer, in the actual supply chain operation process, we should pay attention to the mastery of order information at all levels. The research content of enterprises should not only focus on the market situation, but also on the relevance of upstream and downstream docking units. This is important to enhance their own chain embeddedness as well as survival position. In the exploration of revenue-sharing contracts, we find that suppliers and retailers are generally fully rational. The balance of responsibilities and benefits is frequently consid- ered by managers. In choosing the appropriate revenue-sharing parameters, state policy, and concepts, management personnel from all parties should increase their frequency of communication. After a real-time understanding of the global impact of market changes, Systems 2024, 12, 32 19 of 20 19 of 20 the parameters should be revised to the extent allowed by all parties. Tentative behavior should be avoided to prevent causing a backlash of two uncoordinated effects. As it is often said, competition and cooperation come hand in hand, and the period is full of games. But only with the overall coordination of the supply chain being the goal to operate their own units can this appear to reach a win-win path. the parameters should be revised to the extent allowed by all parties. Tentative behavior should be avoided to prevent causing a backlash of two uncoordinated effects. As it is often said, competition and cooperation come hand in hand, and the period is full of games. But only with the overall coordination of the supply chain being the goal to operate their own units can this appear to reach a win-win path. 6. Conclusions 20210101027), and Major humanities and social science project of Hebei Provincial Education Department (Grant No. ZD202209). Data Availability Statement: The original contributions presented in the study are included in the article. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Ye, F.; Xu, X. Cost allocation model for optimizing supply chain inventory with controllable lead time. Comput. Ind. Eng. 2010, 59, 93–99. 2. Li, Y.; Xu, X.; Ye, F. Supply chain coordination model with controllable lead time and service level constraint. Comput. Ind. Eng. 2011, 61, 858–864. [CrossRef] 2. Li, Y.; Xu, X.; Ye, F. Supply chain coordination model with controllable lead time and service level constraint. Comput. Ind. Eng. 2011, 61, 858–864. [CrossRef] , , [ ] 3. Yang, H.; Peng, J. 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[CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
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Arsenic trioxide, a potent inhibitor of NF-κB, abrogates allergen-induced airway hyperresponsiveness and inflammation
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Respiratory Research BioMed Central Open Access Research Arsenic trioxide, a potent inhibitor of NF-κB, abrogates allergen-induced airway hyperresponsiveness and inflammation Lin-Fu Zhou*1,2,3, Yi Zhu1, Xue-Fan Cui1, Wei-Ping Xie1, Ai-Hua Hu3 and KaiSheng Yin*1 Address: 1Department of Respiratory Medicine, The First Affiliated Hospital, Nanjing Medical University, Nanjing, China, 2Global Health Programs, University of Pennsylvania School of Medicine, Philadelphia, USA and 3Division of Pulmonary Medicine, Joseph Stokes Jr. Research Institute, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, USA Email: Lin-Fu Zhou* - lfzhou@njmu.edu.cn; Yi Zhu - zhuyi2000@citiz.net; Xue-Fan Cui - xuefancui@njmu.edu.cn; WeiPing Xie - wpxie@njmu.edu.cn; Ai-Hua Hu - hua@email.chop.edu; Kai-Sheng Yin* - yinks@126.com * Corresponding authors Published: 20 December 2006 Respiratory Research 2006, 7:146 doi:10.1186/1465-9921-7-146 Received: 19 July 2006 Accepted: 20 December 2006 This article is available from: http://respiratory-research.com/content/7/1/146 © 2006 Zhou et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Overactivation of nuclear factor κB (NF-κB) orchestrates airway eosinophilia, but does not dampen airway hyperresponsiveness in asthma. NF-κB repression by arsenic trioxide (As2O3) contributes to apoptosis of eosinophils (EOS) in airways. Here we provide evidence that As2O3 abrogates allergen (OVA)-induced airway eosinophilia by modulating the expression of IκBα, an NF-κB inhibitory protein, and decreases the airway hyperresponsiveness. Methods: Using a murine model of asthma, the airway hyperresponsiveness was conducted by barometric whole-body plethysmography. Airway eosinophilia, OVA-specific IgE in serum, and chemokine eotaxin and RANTES (regulated upon activation, normal T cell expressed and secreted) in bronchoalveolar lavage fluid were measured by lung histology, Diff-Quick staining, and ELISA. Chemokine-induced EOS chemotactic activity was evaluated using EOS chemotaxis assay. Electrophoretic mobility shift assay and Western blot analysis were performed to assess pulmonary NF-κB activation and IκBα expression, respectively. Results: As2O3 attenuated the allergen-induced serum IgE, chemokine expression of eotaxin and RANTES, and the EOS recruitment in bronchoalveolar lavage fluid, which is associated with an increased IκBα expression as well as a decreased NF-κB activation. Also, As2O3 suppressed the chemotaxis of EOS dose-dependently in vitro. Additionally, As2O3 significantly ameliorated the allergen-driven airway hyperresponsiveness, the cardinal feature underlying asthma. Conclusion: These findings demonstrate an essential role of NF-κB in airway eosinophilia, and illustrate a potential dissociation between airway inflammation and hyperresponsiveness. As2O3 likely exerts its broad anti-inflammatory effects by suppression of NF-κB activation through augmentation of IκBα expression in asthma. Page 1 of 12 (page number not for citation purposes) Respiratory Research 2006, 7:146 Background Asthma is now accepted as a T-helper type 2 (Th2) lymphocyte-mediated chronic inflammatory disorder, characterized by airway eosinophilia and airway hyperresponsiveness (AHR) [1]. Eosinophils (EOS) appear to play a crucial role in the ongoing inflammation due to either an impaired clearance or a delayed apoptosis in the airways, where accumulation of a number of EOS cytotoxic proteins including major basic protein, cationic proteins and peroxidase could occur [2]. Existing data support the notion that morphologic changes in airway tissue to the development and severity of AHR in asthma correlates with the presence of activated airway inflammatory cells, in particular EOS [3]. The molecular regulatory pathways in induction of chronic cytokine expression and recruitment/activation of inflammatory cells in asthma remain elusive. However, there is growing recognition that these processes involve increased transcription of inflammatory genes via transcription factors [4]. One such transcription factor, nuclear factor κB (NF-κB), is abundant of p50 (NF-κB1)/ p65 (RelA) heterodimer. In a latent state, NF-κB is sequestered as an inactive trimer by complexing with IκBα, a 37 kDa inhibitory protein, which promotes cytoplasmic retention and maintains a low basal transcriptional activity. IκBα consists of an N-terminal domain containing specific phosphorylation sites, five ankyrin repeat sequences, and a C-terminal domain of Pro-Glu-Ser-Thr polypeptides [5]. Upon stimulation, IκBα is phosphorylated by the IκB kinase, ubiquitinated and degraded through the 26S proteasome pathway [6]. Subsequently, the nuclear localization sequence of NF-κB is unmasked to allow its translocation into the nucleus, where it binds to DNA and initiates transcription of a wide range of NFκB-dependent genes in association with immune and inflammatory responses [7]. Arsenic compound has long been considered as a protoplasmic poison that can bind to human sulfydryl-containing proteins with high affinity. Arsenic trioxide (As2O3), extracted from arsenic compound, is a powerful ancient medication for a variety of ailments with the principle of "using a toxic against another toxic" in traditional Chinese medicine. Strikingly, As2O3 treatment in a regime of 10 mg/d of intravenous infusion for 28 to 60 days is effective in patients with acute promyelocytic leukemia (APL) without viable toxicity in refractory to the all-trans retinoic acid (ATRA) and the conventional chemotherapy by inducing apoptosis of APL cells [8]. Many studies have demonstrated that NF-κB overactivation underlines the chronicity of airway inflammation characteristic of asthma [9-12]. Recently, we have reported that As2O3mediated NF-κB repression in airways facilitated EOS apoptosis in a dose-dependent manner, contributing to http://respiratory-research.com/content/7/1/146 the resolution of airway eosinophilic inflammation [13]. In this study, we investigated the effects of As2O3 on allergen-induced AHR and NF-κB-mediated airway inflammation in a murine model of asthma. Our data indicate that inhibition of NF-κB activation through induction of IκBα expression may account for the broad anti-inflammatory action of As2O3. Methods Asthma modeling Specified pathogen-free female BALB/c mice, aged 6 to 8 weeks, were provided by the Chinese Academy of Medical Sciences (Beijing, China). The animal experiment was approved by Nanjing Medical University according to the guidelines of the Institutional Animal Care and Use Committee. A murine asthma model was established as described previously [14] with minor modifications. On days 0 and 7, mice received intraperitoneal injection of 20 µg of chicken ovalbumin (OVA, Grade V, SigmaAldrich, St. Louis, MO) adsorbed to 20 mg of aluminum hydroperoxide gel (Pierce, Rockford, IL). On days 14, mice were randomized to receive aerosol challenge with either 6% OVA in phosphate-buffered saline (PBS) or PBS alone via a nebula (1–5 µM particles, Bohringer Ingelheim, Germany) for 40 min per day up to 7 days. During the treatment period, As2O3 (Yida Pharmaceutics, Harbin, China) at dose of 0.5–4.5 mg/kg, dexamethasone (Dex, Phoenix Pharmaceutics, Belmont, CA) at dose of 2.5 mg/ kg or PBS alone was injected into the peritoneum 30 min before each airway challenge. After the last aerosol exposure, mice were sacrificed at designated timepoints. Airway physiology Baseline resistance and AHR induced by nebulized methacholine (Sigma-Aldrich, St. Louis, MO) at dose of 12.5– 100 mg/ml in conscious unrestrained-mice were assessed using barometric whole-body plethysmography (Buxco Electronics Inc., Troy, NY) as described previously [15]. Airway resistance is expressed as: Penh = [(Te/0.3 Tr)-1] × [2 Pef/3 Pif], where Penh = enhanced pause, Te = expiratory time (sec), Tr = relaxation time (sec), Pef = peak expiratory flow (ml/sec), and Pif = peak inspiratory flow (ml/sec). Bronchoalveolar lavage Four hours after the last airway challenge, mice underwent euthanasia and were cannulated in the trachea. The lungs were washed twice with 1 ml aliquots of PBS to collect the bronchoalveolar lavage fluid (BALF). Subsequently, the lungs were removed, quickly frozen in liquid nitrogen, and stored at -70°C. Additionally, the lungs were collected at 1, 12, and 24 hrs post the last airway challenge to study the kinetics of pulmonary NF-κB activation. Page 2 of 12 (page number not for citation purposes) Respiratory Research 2006, 7:146 Lung histology Paraffin embedded lung sections (5 µm) collected 24 hrs after airway challenge were stained with hemotoxylin & eosin (Sigma-Aldrich, St. Louis, MO) for examination of histology. Diff-Quick staining Diff-Quick staining is a modified Wright's staining [16]. Centrifuged at 300 × g for 10 min, the pelleted cells of BALF were suspended in a serum-free RPMI 1640 medium. The cell viability, evaluated by the trypan blue exclusion method, was over 95%. Total and differential cell counts were enumerated on cytospins (Thermo Shandon, Pittsburgh, PA) in compliance with the Diff-Quick staining profile (Merck, Germany) by counting at least 200 to 500 cells in cross-section. Enzyme-linked immunosorbant assay (ELISA) Serum levels of OVA-specific immunoglobulin E (IgE) were analyzed by ELISA using samples collected 24 hrs after the last OVA challenge. Briefly, 96-well plates were coated with either purified anti-mouse IgE (5 µg/ml, BD PharMingen, San Diego, CA) or OVA (100 µg/ml). After addition of serum samples, OVA-specific IgE was detected using horseradish peroxidase (HRP)-conjugated sheep anti-IgG (Calbiochem, La Jolla, CA). Arbitrary units (AU) were calculated according to OD50 of the standard curve. Murine chemokines, eotaxin and RANTES (regulated upon activation, normal T cell expressed and secreted), in the BALF samples were measured by utilizing paired antibodies following the manufacturer's recommendations. The ELISA kits were purchased from R&D Systems (Minneapolis, MN) with a minimum detectable levels of 3 and 5 pg/ml for eotaxin and RANTES, respectively. EOS chemotaxis assay (ECA) Interleukin (IL)-5 transgenic mice (CBA/CaH-TnN) were provided by the Institute of Chemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). EOS (~98% purity) were derived from spleen of IL-5 transgenic mice with depletion of B, T, and antigen-presenting cells using anti-B220, anti-CD4, anti-CD8 and anti-class II, as well as rat anti-mouse Ig-conjugated magnetic beads (Miltenyi Biotec, Auburn, CA) as described previously [17]. EOS were seeded at 5 × 104 density in triplicate and preincubated for 15 min at room temperature with 0.25– 2 µM of As2O3 prior to chemotaxis measurement. Chemotaxis was assessed in 48-well micro-Boyden chambers using polyvinylpyrrolidone-free polycarbonate membranes (NeuroProbe, Bethesda, MD). Cell suspension and diluted chemokines of eotaxin or RANTES (PeproTech, London, UK) were added into the chamber with RPMI 1640 containing 25 mM N-2-hydroxyethylpiperazine-N'- http://respiratory-research.com/content/7/1/146 2-ethanesulfonic acids (HEPES, pH 7.4) and 0.05% bovine serum albumin. The plates were incubated for 60 min at 37°C under 5% CO2. The migrated cells were counted in five randomly selected high-power fields (magnification was × 1,000). Spontaneous migration was evaluated in the absence of chemoattractant. Extraction of nuclear and total proteins Nuclear and total proteins of lung tissue were collected as described previously [18]. Briefly, aliquots of liquid nitrogen-frozen tissue were pulverized and lysed in 200 µl of cold Buffer A [10 mM Tris-HCl (pH7.5), 150 mM NaCl, 1.5 mM MgCl2, 0.65% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and 0.5 mM dithiothreitol (DTT)] for 3 min. After centrifugation at 10,000 × g for 1 min at 4°C, the nuclear pellets were extracted with 20 µl of Buffer B [20 mM HEPES (pH7.9), 1.5 mM MgCl2, 420 mM NaCl, 0.5 mM DTT, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM PMSF and 25% glycerol] for 30 min with intermittent mixing on ice. The supernatant containing nuclear proteins was collected by centrifugation at 12,000 × g for 5 min. The total proteins were prepared by addition of Buffer A to the lung powder and subjected to two freeze/thaw cycles to fracture the nuclear membranes. After centrifugation, the supernatant was collected. The nuclear and total proteins were quantitated using the Bradford assay (BioRad, Hercules, CA), aliquoted and stored at -70°C until use. Electrophoretic mobility shift assay (EMSA) EMSA analysis was performed using a commercial kit (Promega, Madison, WI). Double-stranded oligonucleotide probe (5'-AGTTGAGGGGACTTTCCCAGGC-3') containing a consensus NF-κB sequence (underlined) was end-labelled with [γ-32P]-adenosine triphosphate (Furui Biotechnology, Beijing, China) by T4 polynucleotide kinase and purified by chromatography. The binding reaction was conducted in a final volume of 20 µl containing 5 µg of nuclear proteins and 30 fmol of 32P-labelled oligonucleotide probe. Protein-DNA complexes were separated by electrophoresis on a 5% native polyacrylamide gel (37:1 acrylamide:bis-acrylamide) in a 0.5 × Tris-borateEDTA running buffer. The dried gel was exposed to PhosphorImager (Molecular Dynamics) using ImageQuant software (Amersham Life Science, Arlington Heights, IL). For competition assay, a 100-fold excess of unlabelled NFκB or activator protein 1 (AP-1) oligonucleotide probe was added to the reaction mixture 10 min before addition of the labelled probe. For supershift assay, a 0.5 µg of antip50 or anti-p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the reaction mixture prior to the labelled probe for 30 min. Page 3 of 12 (page number not for citation purposes) Respiratory Research 2006, 7:146 Western blot analysis Denatured samples (100 µg of total proteins) were fractionated by 10% sodium dodecyl sulfate polyacrylamide gel eletrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Blots were blocked with 5% milk containing 1 × TBST [40 mM Tris-HCl (pH7.6), 300 mM NaCl and 0.1% Tween-20] at 4°C overnight. Thereafter the blot was probed with primary antibodies of anti-IκBα (1:1,000 dilution) or anti-β-actin antibody (1:800 dilution) for 1 hr. After an HRP-conjugated goat anti-rabbit IgG (1:5,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) incubation, the immunoblots were visualized by an enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL) according to the manufacture's instructions. Data analysis Statistical analysis was performed by one-way analysis of variance (ANOVA) and q test with SPSS 11.0 software package (SPSS Inc., Chicago, IL). The negative relationship was evaluated by Pearson correlation analysis. Data were expressed as mean ± SEM, and p < 0.05 was considered statistically significant. Results Attenuation of airway EOS recruitment by As2O3 OVA-challenged mice in response to 0.5–4.5 mg/kg of As2O3 reduced the number of EOS in BALF in a dosedependent manner (Fig. 1). Since the anti-inflammatory effects of As2O3 were similar at the doses of 4 and 4.5 mg/ kg, and it was comparable to the effect of 2.5 mg/kg of Dex (p > 0.05), the 4 mg/kg of As2O3 was herein chosen as the effective dosage in the rest of experiments. This dosage was also proved to be relatively safe based on our previous experiments [13,14]. Histological analysis of the OVAchallenged mice lung revealed an enhanced airway eosinophilia as compared to the naïve control mice that were treated with PBS (Fig. 2A). Conversely, pretreatment of As2O3 protected mice from developing the allergeninduced peribronchial inflammation (Fig. 2A). Examination of BALF collected from mice at 24 hrs after OVA challenge showed a marked influx of inflammatory cells into the airways, including EOS, lymphocytes, macrophages and neutrophils (Fig. 2B–C). The increased EOS in the BALF was correlated with an increase of EOS recruitment by the Diff-Quick analysis in OVA-challenged mice (Fig. 2B). The number of EOS in BALF from naïve mice was less than 1%, whereas that of OVA-challenged mice was about 49% (p < 0.01). Pretreatment of As2O3 dramatically attenuated the airway eosinophilia in the OVA-challenged mice (p < 0.01; Fig. 2A–C; Table 1). Amelioration of AHR by As2O3 Penh, relative to the measured airway resistance, was obtained as an index and was normalized to the postsaline – Penh. This readout was used as a measure of AHR. http://respiratory-research.com/content/7/1/146 Mice previously sensitized and challenged with OVA developed a dose-dependent methacholine-induced bronchospasm as compared to the naïve mice that were treated with PBS. As2O3 treatment significantly reduced the effect (p < 0.01; Fig. 3). Reduction of serum IgE and BALF chemokines by As2O3 IgE can augment allergic airway responses in a high affinity receptor-dependent manner. Serum levels of OVA-specific IgE were elevated in OVA-challenged mice compared with the naïve control mice (p < 0.01), whereas pretreatment with As2O3 resulted in a 4.8-fold decrease to the levels of the OVA mice (p < 0.01; Fig. 4A). Eotaxin and RANTES play a critical role in inducing chemotaxis of EOS [19]. ELISA analysis showed that levels of eotaxin and RANTES in BALF were markedly increased in OVA-challenged mice in comparison with the control mice (p < 0.01). However, these chemokine levels were largely reduced by pretreatment with As2O3 (p < 0.05 or 0.01; Fig. 4B). Ablation of EOS chemotaxis by As2O3 Eotaxin and RANTES with respective concentrations of 1 (100) and 103 nM reached a maximal chemotaxis response indicating that eotaxin is a more active chemotaxin to EOS than RANTES (Fig. 5A). As2O3 significantly inhibited the EOS chemotaxis mediated by eotaxin or RANTES in a dose-dependent manner (p < 0.05 or 0.01; Fig. 5B). Inhibition of pulmonary NF-κB activation by As2O3 The OVA challenged mice showed a sharp increase in the pulmonary DNA binding activity of NF-κB at various timepoints as compared to the unchallenged mice lung. Indeed, NF-κB activity was increased within 1 hr (p < 0.01), peaked at 4 hrs (p < 0.01), and decreased by 12 (p < 0.01) to 24 hrs (p < 0.05). This effect of OVA challenge was clearly ameliorated by pretreatment with As2O3 (p < 0.01; Fig. 6, lane 6 as compared to lane 3; Table 1). In the competition assay, addition of 100-fold excess of unlabelled NF-κB, but not AP-1, oligonucleotide probe competed away the NF-κB-DNA complexes, verifying the specificity of NF-κB binding. In the supershift assay, addition of antibodies against p50 and p65 resulted in retardation of supershifted bands, with reciprocal decreases in the intensity of the NF-κB bands, confirming the classic subunits of NF-κB heterodimer (Fig. 6). Augmentation of pulmonary IκBα expression by As2O3 The pulmonary IκBα expression in the lung lysate was relatively decreased in OVA-challenged mice (p < 0.01; Fig. 7; Table 1) compared to the control lung. In contrast, pretreatment of As2O3 accumulated the pulmonary IκBα (p < 0.01). Furthermore, there was a tight negative correlation between EOS recruitment in the BALF or the pulmonary Page 4 of 12 (page number not for citation purposes) Respiratory Research 2006, 7:146 http://respiratory-research.com/content/7/1/146 Figure 1 EOS recruitment in BALF in a dose-dependent manner As2O3 decreases As2O3 decreases EOS recruitment in BALF in a dose-dependent manner. Intraperitoneal administration of OVAchallenged mice with As2O3 (0.5–4.5 mg/kg) reduced the EOS in BALF, in which both 4, 4.5 mg/kg of As2O3 and 2.5 mg/kg of Dex achieved the similar anti-inflammatory effects. BALF EOS, stained with Diff-Quick solution, were counted using a hematocytometer, and expressed as a percentage in total leukocytes. Data represent the mean ± SEM of four separate experiments (n = 6 per group). # p < 0.05, *p < 0.01, vs the control mice; ‡ p < 0.05, † p < 0.01, vs the OVA-challenged mice. NF-κB activation and IκBα expression (r = -0.82 and 0.94, respectively; p < 0.01). Discussion Multiple upstream signal events converge on the NF-κBinducing kinase (NIK) [20]. Activation of NIK results in phosphorylation of IκB kinases, which render the phosphorylation of IκBα at N-terminal serines 32 and 36 (Ser32 and Ser36) residues, leading to a proteolytic degradation of IκBα. Consequently, the activated NF-κB translocates to the nucleus, where it bonds to specific κB sites to facilitate the transcription of target genes. This results in expression of numerous pro-inflammatory cytokines, chemokines and adhesion molecules [21]. These proinflammatory mediators are essential in the recruitment of airway inflammatory cells, including EOS and CD4+ T lymphocytes, which in turn secret Th2 cytokines [22]. Therefore, NF-κB repression in airways via suppression of IκBα degradation or augmentation of IκBα synthesis would decrease the transcription of a myriad of NF-κBdependent genes. This strategy proved to be more effective than that of blocking a single downstream inflammatory or an immune gene among the inflammatory cascade [23,24]. Several lines of evidence suggest a central role of NF-κB in the pathogenesis of asthma. Activated NF-κB has been identified in sputum-induced macrophages and bronchial biopsy specimens of asthmatic patients [25]. Agents such as allergens, ozone and viral infections, which are associated with exacerbation of asthma, stimulate activation of NF-κB [26]. As the major effective treatment for asthma, Page 5 of 12 (page number not for citation purposes) Respiratory Research 2006, 7:146 http://respiratory-research.com/content/7/1/146 As Figure 2 ameliorates allergic airway inflammation 2O3 markedly As2O3 markedly ameliorates allergic airway inflammation. (A) Lung tissues of naïve mice, untreated OVA-challenged mice, and OVA-challenged mice treated with 4 mg/kg of As2O3 were subjected to histological analysis by staining with hematoxylin & eosin. Magnification was × 400. (B) BALF was collected 24 hrs after the final OVA challenge, and stained with DiffQuick for microscopic detection of EOS dyed in orangeophil red with cytoplasmic acidophil granules (arrows). Magnification was × 200. (C) Total and differential cell counts in BALF are plotted for each group. Data represent the mean ± SEM of three independent experiments (n = 6 per group). # p < 0.05, * p < 0.01, vs the control mice; † p < 0.01, vs the OVA-challenged mice. Page 6 of 12 (page number not for citation purposes) Respiratory Research 2006, 7:146 http://respiratory-research.com/content/7/1/146 Table 1: Effect of As2O3 on EOS recruitment in BALF (%), pulmonary NF-κB activity (relative intensity units) and IκBα expression (IκBα/β-actin). Asthma EOS NF-κB IκBα Control 4 hrs 1 hr 4 hrs 12 hrs 24 hrs As2O3 4 hrs 0.56 ± 0.22 51.47 ± 4.53 0.80 ± 0.25 5.08 ± 1.37* 162.31 ± 9.46* 0.45 ± 0.04* 11.12 ± 1.93* 255.74 ± 11.10* 0.23 ± 0.10* 20.25 ± 2.99* 127.59 ± 8.72* 0.36 ± 0.03* 48.72 ± 5.38* 80.97 ± 6.15# 0.54 ± 0.07# 4.69 ± 1.21*† 75.80 ± 9.33*† 1.56 ± 0.34*† Data represent the mean ± SEM of four independent experiments (n = 6 per group). # p < 0.05, * p < 0.01, vs the control mice; † p < 0.01, vs the OVA-challenged mice at 4 hrs. As Figure 3 allergen-induced AHR 2O3 prohibits As2O3 prohibits allergen-induced AHR. Mice were placed in whole-body plethysmographs and underwent varying methacholine challenge 24 hrs after the last airway challenge of OVA or PBS. The OVA-challenged mice exhibited remarkable bronchial reactivity to inhaled methacholine, compared with control mice or mice challenged with OVA in the presence of 4 mg/kg of As2O3. Data represent the mean ± SEM of four independent experiments (n = 5 per group). # p < 0.05, * p < 0.01, vs the control or OVA-challenged mice. Page 7 of 12 (page number not for citation purposes) Respiratory Research 2006, 7:146 http://respiratory-research.com/content/7/1/146 Figure As2O3 alleviates 4 OVA-specific IgE in serum and eotaxin and RANTES in BALF of allergen-sensitized mice As2O3 alleviates OVA-specific IgE in serum and eotaxin and RANTES in BALF of allergen-sensitized mice. Serum and BALF were collected 24 hrs after the last OVA challenge. Levels of (A) OVA-specific IgE in serum and (B) chemokine eotaxin and RANTES in BALF were analyzed by ELISA. Data represent the mean ± SEM of three independent experiments (n = 6 per group). # p < 0.05, * p < 0.01, vs the control mice; † p < 0.01, vs the OVA-challenged mice. Page 8 of 12 (page number not for citation purposes) Respiratory Research 2006, 7:146 http://respiratory-research.com/content/7/1/146 As Figure 5 EOS chemotaxis 2O3 ablates As2O3 ablates EOS chemotaxis. (A) Eotaxin and RANTES induced chemotaxis of EOS, in which eotaxin was more potent than RANTES. The numbers of migrating cells per five high-power fields (magnification was × 1,000) are shown. (B) Pretreatment of EOS with As2O3 15 min before transferring to the chemotaxis chamber greatly suppressed the eotaxin or RANTESinduced migration in a dose-dependent manner. Data represent the mean ± SEM of three independent experiments (n = 5 per group). # p < 0.05, * p < 0.01, vs the control (medium alone); † p < 0.01, vs the prestimulation with medium plus stimulation with 1 nM of chemokines. Page 9 of 12 (page number not for citation purposes) Respiratory Research 2006, 7:146 http://respiratory-research.com/content/7/1/146 As Figure 6 pulmonary NF-κB activation in OVA-sensitized and challenged mice 2O3 inhibits As2O3 inhibits pulmonary NF-κB activation in OVA-sensitized and challenged mice. Nuclear extracts of lung tissue were prepared and subjected to EMSA analysis of NF-κB activity. Lane 1: Naïve control mice; Lanes 2–5: OVA-sensitized mice 1, 4, 12, and 24 hrs after the final OVA challenge; Lane 6: OVA-sensitized mice treated with As2O3 4 hrs after the final OVA challenge; Lanes 7–8: Specific (cold) and nonspecific (NS) competition; Lanes 9–10: Supershifts of p50 and p65. Nuclear extracts of lanes 7 to 10 were derived from those of lane 3. Free DNA probe is not shown. The arrows indicate the specific NF-κBDNA complexes, p50 dimer, and supershifts, respectively. One of four independent experiments is shown. Figure As2O3 augments 7 pulmonary IκBα expression in OVA-sensitized and challenged mice As2O3 augments pulmonary IκBα expression in OVA-sensitized and challenged mice. Total proteins of lung tissue were extracted 4 hrs after the final OVA challenge, and subjected to Western blot analysis of IκBα. β-Actin was utilized as the standard control. Lane 1: Naïve control mice; Lane 2: OVA-sensitized and challenged mice; Lane 3: OVA-sensitized and challenged mice treated with 4 mg/kg of As2O3. The positions of molecular size standards (in kDa) are indicated by arrows. One of three separate experiments is shown. Page 10 of 12 (page number not for citation purposes) Respiratory Research 2006, 7:146 glucocorticoids are potent blockers of NF-κB activation [27]. Furthermore, mice lacking the NF-κB subunits p50 or c-Rel develop less airway inflammation upon antigen challenge [28]. Nevertheless, NF-κB activation orchestrates allergen-induced inflammation and subsequent adaptive responses, but does not appear to modulate AHR, the cardinal feature that underlies asthma, signifying a potential dissociation between airway inflammation and AHR [29]. Clearly, additional airway signaling pathways activated, residual NF-κB activity or other inflammatory processes may be responsible for the AHR. Alternatively, events localized more distally within the alveolar compartments, such as microvasculature leakage of macromolecules, alveolar injury or surfactant dysfunction might dominate the genesis of AHR [30-32]. As2O3 (1–2 µM) induces the apoptosis in t (15;17) APL cell line NB4 in vitro and in APL patients without significant myelosuppression in vivo [8]. We and others have confirmed that inhibition of NF-κB was essential to arsenic-induced apoptosis [13,33]. In this report, despite a decreased serum OVA-specific IgE production, we demonstrated an inhibitory effect of As2O3 on EOS recruitment from OVA-challenged BALF, in agreement with our previous observation that As2O3 promoted EOS apoptosis in the airway eosinophilic inflammation [13]. Additionally, both eotaxin and RANTES, downstream genes of NFκB, demonstrated potent chemoattractants to EOS and Th2 lymphocytes [34]. Presumably, the ablation of airway eosinophilia by As2O3 results from a collective effects of NF-κB inhibition such as a reduced specific IgE secretion, chemokine expression and Th2 cytokine production as well as an altered eosinophilic cytoskeletal rearrangement [35,36]. Overall, As2O3 might exert its multiple antiinflammatory action through augmentation of IκBα expression and suppression of NF-κB activation in the airways. This is partially in accordance with the therapeutic role of glucocorticoid-mediated NF-κB repression in asthma [37,38]. Interestingly, in this model of asthma, As2O3 abrogated both allergic airway inflammation and AHR in contrast with the previous report [29], suggesting a specific effect of As2O3 besides NF-κB suppression. Taken together, these findings not only prove an essential role of NF-κB-mediated airway inflammation, but also illustrate the importance of alternative signaling pathway and additional cell types in the airways, and the complicated interactions between them in dictating the pathophysiology of asthma. Conclusion Our data demonstrate that a broader anti-inflammatory activity of As2O3 lies in the inhibition of NF-κB activation through induction of IκBα expression in the airways. Clinically, low dosage of As2O3 may have a potential benefit in treating patients with asthma, especially in those with steroid-dependent and -resistant asthma [8,13]. It is http://respiratory-research.com/content/7/1/146 anticipated that specific inhibitors of NF-κB may be developed by modifying the poisonous group(s) of As2O3 and screen As2O3 analogues in the libraries of chemical compounds. Moreover, novel nondegradable IκBα mutant, namely super-repressor of NF-κB, may be achieved by completely deleting the phosphorylation sites of Ser32 and Ser36 residues [18,37]. This will offer promising strategies for future immunotherapy of asthma as well as the infectious, inflammatory, cancerous and autoimmune diseases associated with aberrant NF-κB activation [1,5,39-42]. Abbreviations AHR, Airway hyperresponsiveness; ANOVA, One-way analysis of variance; APL, Acute promyelocytic leukemia; As2O3, Arsenic trioxide; ATRA, All-trans retinoic acid; BALF, Bronchoalveolar lavage fluid; ECL, Enhanced chemiluminescence; EOS, Eosinophils; ECA, EOS chemotaxis assay; ELISA, Enzyme-linked immunosorbant assay; EMSA, Electrophoretic mobility shift assay; HRP, Horseradish peroxidase; IL, Interleukin; IκB, Inhibitor of NF-κB; NF-κB, Nuclear factor κB; OVA, Ovalbumin; PBS, Phosphate-buffered saline; RANTES, Regulated upon activation, normal T cell expressed and secreted; SEM, Standard error of the mean; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel eletrophoresis; Th2, T-helper type 2. Competing interests The author(s) declare that they have no competing interests. Authors' contributions LFZ conceived and designed the study, carried out all experiments, analyzed the data, and drafted the manuscript. YZ participated in the animal experiments, BALF cell counts, ECA, and ELISA. XFC performed the EMSA and Western blot analysis. WPX conducted the airway physiology, lung histology, and partial data analysis. AHH gave helpful advice for data analysis and interpretation. KSY coordinated most of the experiments and advised on data analysis. All authors read and approved the final manuscript. Acknowledgements We thank Drs. Heng-Jiang Zhao, Jing-Xu Zhu (The Hospital of University of Pennsylvania), Ruth He and Chyze-Whee Ang for thoughtful comments, and Guang Yang, Hakon Hakonarson and Michael M. Grunstein (The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine) for critical review of the manuscript. This work was supported by grants from the National Youth Natural Science Foundation of China 30400191 (LFZ), National Natural Science Foundation of China 30570797 (KSY and LFZ), Key Subject of Project "135" of Jiangsu Province 20013102 (KSY), Jiangsu Provincial Administration Bureau of Traditional Chinese Medicine 9974 (KSY), and Summit Project of Jiangsu Personnel 06B035 (LFZ). Page 11 of 12 (page number not for citation purposes) Respiratory Research 2006, 7:146 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. Zhou LF, Zhang MS, Yin KS, Ji Y, Xie WP, Cui XF, Ji XH: Effects of adenoviral gene transfer of mutated IκBα, a novel inhibitor of NF-κB, on human monocyte-derived dendritic cells. Acta Pharmacol Sin 2006, 27:609-616. Trautmann A, Schmid-Grendelmeier P, Kruger K, Crameri R, Akids M, Akkaya A, Brocker EB, Blaser K, Akdis CA: T cells and eosinophils cooperate in the induction of bronchial epithelial cell apoptosis in asthma. 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German
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Dial-Ciba und dialcibismus
Zeitschrift für die Gesamte Neurologie und Psychiatrie
1,918
public-domain
3,870
1) Einiges N/ihere fiber die Verbindung findet sich in der yon der Fabrik (Ge- sellschaft fiir chemische Industrie in Basel) den Versuchsmustern beigegebeacu Literatur. (Aus der psychiatrischcn Klinik uad Poliklinik Z/iricb [Direktor: Prof. E. Bleuler].) (Aus der psychiatrischcn Klinik uad Poliklinik Z/iricb [Direktor: Prof. E. Bleuler].) (Aus der psychiatrischcn Klinik uad Poliklinik Z/iricb [Direktor: Prof. E. Bleuler].) Z. f. d. ges. Neut. u. Psych. O. XLIII. (Einffegangen ant 11. Juni 191~'.) Dial-Ciba ist als Derivat der Barbiturshure ein naher Verwandter des Veronals. Es soll sich unter anderem dadureh auszeiehnen, dais kumulative Wirkungen ausbleibenl). Wir stellten zun~chst im Laufe der letzten zwei Jahre Versuehc rein praktischer Natur an; es gait tins, zu prtifen, ob und wie Dial-Ciba als Hypnotieum zu gebrauchen und mit welchen eventuellen Neben- wirkungen zu reehnen sei. Bei 24 weiblichen Geisteskranken der m lruhigen Abteilungen machten wir Vorversuche. Die einzelne Patientin erhielt bis zu llmal naeh- einander Dial-Ciba (2 Fhlle) ; in weiteren 2 Fifilen wurde es 6-, in je 3 FSllen 5- und 4 real, in 5 F~llen 3- in eincm Falle 2- und in 8 F~llen nur eiulaal verabreicht. Die kiirzeste, auf die Zeit vor und nach Verabreichung des Mittels ausgedehnte Beobachtungsdauer war 10, die l~ngste 34 Tage. Die Doseil betrugen 0,2 bis 0,4, gew6hnlich 0,3 g Dial; racist begnfigten wir uns mit einmaliger Verabreiehung pro Naeht; wo nieht, wurde doch die Gesamtdosis yon 0,4 g in 24 Stunden nicht iiberschritten. Das Mittel wurde jeweilen mit Wasser, worin die Tabletten leicht zerfallen, in einem L6ffel gereieht; seine Aufnahme begegnete keinen Sehwierig- keiten. Wir brachten Dial in Anwendung bei Patientinnen vom jugend- lichen bis zum Greisenalter in Erregungszustanden yon Paralyse, De- mentia senilis, Epilepsie, Schizophrenie und maniseh-depressivem Irresein. In 2/a der 86 Verabreiehungen trat Sehlaf prompt ein; sein Beginn fiel meist 1/~ Stunde nach Aufnahme des Mittels; ausnahms- weise schliefen die Kranken schon in 1/~ oder erst in 3/4 Stunden ein. Bei den Obrigen F~llen lief~ oft der Schlafeintritt eine Stunde nnd langer Z. f. d. ges. Neut. u. Psych. O. XLIII. 4 II. Christoffel: 50 auf sich ~artell: dal.~ aber auch (laml me|st der Schlaf (lurch das Medi- kament vertieft wurde, gitlg aus dem Vergleich mit dialcibafreien Niichteu hervor. Die Schlafdauer betrug in ebenfalls 2/a der Verabreichuugeu die gauze Nacht, d.h. yon abends 8 Uhr bis morgens um 6 [;hr. Kfirzere Schlafdauer als 3 Stut~den wurde fiber- haupt mlr eilimalnoticrt. WarderSehlafl)eginnprompt, so war me|st auch die Schlafwirk~lng ausge(tehnt: ganz ausnahmsweise trat erst 2 Stunde~l nach Aufnahme des Dial kriiftiger Schlaf ein. Nachsehl~ifrigkeit am Morgen war auch txei Sl)iiter Verabreichuug nichg auffSllig. Zwisehell (let" Menge des Dial uud seiner Wirksamkeit zeigte sich ein deutlicher Zusammenhang: so betrug x~iederholt bet einer Maniaca (lie Schlafdauer mit 0,2 Dial mlr 6--7 Stuuden, w~Lhrend 0,3 Dial jeweilel~ fiir die gauze Nacht (10sti:ndig) genSgten. (Einffegangen ant 11. Juni 191~'.) Ei ne Ku- m ulationswirku ng beobaehteten wir in keiuerWeise. Ander u- tells t~ahm aber aueh (lie Wirksamkeit bet regelmii[~iger Ver- abreichu rig nicht ab. Seehsmal bet im ganzen 4 Patieutinnen ver- sagte Dial vollstiindig. Bet der einen, ether del(ressiven bejahrten Kata- ton|ca, tat (las Mittel in der folgend~l Nacht seine Wirkung wieder wie sonst. [m zweite~ I:alle, bet ether Presbyophrenen, spie]te deut.lich die Zeit der Einnahme eine Rolle : mit 0,2 Dial, die sie um 128 Uhr abends erhalten hatte, schlief sie durch : mit 0,3 um 91/4 abends dauerie (let" Schlaf blo[t einige Stunden; mit 0,3 um l l Uhr war kein Schlaf mehr zu er- zielen. ]~hnlich verhielteu sich andere Fiille. Es best~itigte sich wohl blol.~ die allgemeiue Regel, dal~ das Schlafmitte] am besten quasi pro- phylaktisch gegebel~ wird. -- Auf zwei weitere Patient|mien, bet denen das Mittel zeitweise versagte, |st spiiter zuriickzukommem Nach diesel~ \'orversuehet~ wandten wir Dial-Ciba in ausgedehutem Mal.~e bci Patienten u~serer Klbdk und Poliklinik au. Bet den letzteren, ~mgef~ihr 30 F~illen, handelte es sich haupts/iehlich u~n Depressionen und uerv6se Zustandsbilder bet verschiedeuen Krallkheiten. Besonders (lann, welm die Kranken (lurch (lie im Vordergrund stehende Schlaf- losigkeit sichtlieh heruutergekommt, l~ waren, lieB sich der Circulus vitiosus iu ausgezeiehneter Wt'ise dutch Dial-(!iba unterbrechen. Bet diesen poliklinisehen Fi~llen verwal/(ltell wir gew6hnlieh mlr 0,1 und 0.2 g, stiegen auf je(leu Fall erst dam~ auf 0,3, wem~ erstere Dosen sich als zu gering erwiese~: nach zwei- |)is dreimaliger Medikation erfolgte jeweilen Kontrolle in der S1)rechsttm(le ; gew6hnlich wurde nach 3---6 Tagen das Narkotieum wieder ausgesetzt, lnsbesoudere ftir die mit den typischen Symptomen des K]imakteriums vergesellsehaftete Schlaflosigkeit erwies sich diese Theral)ie als gtinstig; hatten (lie Frauen wieder einmal tiichtig geschlafen, so lieften sieh die tibrigen Symptone viel leichter bekiimpfen, ohne dal$ mit Dial-Ciba mehr fortgefahren worden w~ire. Ganz ghnlich verhielten ~ieh FSlle der bet Frauen der untereu Klassen nieht seltenen Dial-('iba und I)ialcibismus. 51 wirkliehen (Tberarbeitung. in der genannten Weise als Gelegenheits-, nieht als Gewohnheitsmittel angewan(It, leistete uns l)ial-('iba poli- klinisch gute Dienste. Nebst den Fiillen yon Sehlaflosigkeit behandelten ~)'ir im Verlaufe eines Jahres Aufregungszust~inde bei fiber 5<) ~veiblichen Geisteskranken ,nit Dial-C:iba. Das Resultat deckte sich mit (lemjenigen der Vorver- 9 suchc. Die anfiingliche Maximaldosis yon 0,4 steigerten wit, ohne daIl es zu Nebenwirkungen gekommen wiire, bis zu 0,9 in 24 St unden. (Einffegangen ant 11. Juni 191~'.) Christoffel: Dose Veronalnatrium (0,25) gleieh wie die 0,1 Dial-Ciba gewirkt hi, tie; eine gewisse Euphorie scheint aber doch als Nebenwirkung des Dial vorzukommen; ich beobachtete sie auch bei einer i~ltlichen Wi~rterin, die nach l~ngerer Schlaflosigkeit unbekannter Genese mit 0,2 Dial ausgezeichnet geschlafen hatte. Diese F/~lle allein wfirden keinerlei Beweiskraft haben, wenn nicht die Euphorie bei toxischen Dosen tiber- aus stark hervortri~te. Wir werden unten darauf zurtickkommen. Besonders bei katatonen Aufregungen versagte Dial- C ib age leg e n tlic h. Es zeigte sich dann gewOhnlich, dag die Patienten gegen andere Schlafmittel auch refraktiir waren. So schlief z. B. einc jugendliche Hebephrene nach 0,3 Dial zusammen mit einer lnjektion yon 0,01 Mo. und 0,00l Scopolamin nicht nut keinen Moment, sondern sic wurde auch kaum dadurch beruhigt. 9--10 Stunden spi~ter, abends 8 Uhr erhielt sie 0,6 Dial-Ciba; auch damit dauerte der Schlaf nur his morgens 3 Uhr. Dann erwachte Patientin aufgeregt wie vorher. Die gleiche Patientin verhielt sich in ihren Aufregungszust~tnden gegen Sulfonal oder Chloral in Einzeldosen yon 4 g ebenfalls refrakt~r, war auch ohne Schlafmittel fast a nalgetisch, so dag sie sich schwere Selbst- besch~idigungen schmcrzlos zufiigen konnte, hu Anschlug an (lie Verab- reichung yon 0,4 und 0,3 Dial trat t)ei zwei jugendlichen Katatonike- rinnen, yon (lenen (lie eine sich sonst einfach refrakt4tr gegen die Schlaf- wirkungverhalten, lallen(le Sprache und taumeln(ler Gang auf. Psychisch hatte sich zu (lem sonstigen Zustand eine schlaffe E upho- r i e gesellt. Die eine Kra nke m u gte wegen ihrer liirmenden Aggressiviti~t, die sich nut wenig yon ihrem sonstigen Wesen unterschied, in einen Wickel gelegt werden; nach 7 Stunden schlief sic ein. Bei der anderen, gegen Schlafmittel refrakti~ren Patientin schwand das Lallen uach 4 Stunden, w~hrend die Gehst6rung 3 Stunden lhnger zu beobaehten war; auch diese Patientin versank nacix 7 Stunden in Schlaf, ai)er l)lo[.i ftir 2 Stunden; dann kam sie wieder auger Bert, war laut und wollte das Wachbuch zerreigen. Weder w~thrend, noch nach dem einem Alkoholrausch ~thnlichen Zustand waren auff~illige KSrpersym- ptome auger dem Lallen und Taumeln nachweisbar; die Sehncnreflexe waren gesteigert. Der Rausch t rat bei den beiden im gleichen Saale untergebrachten Mi~dchen in derselben Nacht auf. Die vorhergehenden wie die folgenden Niichte war bei ihnen hie ein abnormer Dial- oder sonstiger Schlafmitteleffekt zu beobachten. Eine kumulative Wirkung ist also bei beiden Patientinnen auszuschliel~en. Zwei weitere F~tlle, welche die Nebenwirkungen des Dial-Ciba illustrieren, verdanke ich /-Ierrn Dr, Ch. (Einffegangen ant 11. Juni 191~'.) Es zeigte sich auch, da[.~ das Mittel iiber 4 Monatc lang sozusagen t~glich (w~hrend l l0 Tagen) gegeben werdcn kom~te. Es betraf (lies eine 77jiih- rige Senile in einem heftigen Verwirrtheitszustande. Die Kranke erhielt his jetzt 38,8 g Dial-(~il)a. Wiederholte Urinuntersuchungen auf Eiwei6 und Zueker ergaben stets normalen Befund. Aueh sonst waren keine Nebenerscheimmgen zu beobachten. Die Dosierung konnte sich be- liebig nach dem Gra(le der Erregu ng richten; 0,2 Dial wirkten be- ruhigend, nachdem i) Tagc vorher noch 0,8 n6tig gewesen; es konnte aueh Tage zwischenhinein geben, wo das Mitt el ganz ausgesetzt werden konnte. Al)stinenzerseheinungen zeigten sieh keine. --Die Kranke, dcrcn Zustan(1 sieh gcgenwiirtig so welt gebessert hat, (lab sic au6erhall) (Ics Wachsaals gchalien werden kamh steht immer m>ch unter kleineren [)<)sen Dial. An(leren Sehlafmitteln in entsl~rcehen(I gr(il.~eren I)osen zeigte cs sich gleichwertig (mlgef:,ihr 0,2 g Dial-('iba -- 0,5 g Veronal = 2 g ('hloral.) lmmerhin sehien eine gewisse indivi(1 uelle ]{ca ktionsart zu existieren in der Weise, (la6 das eine oder andere Schlafmittel besser wirkte oder besser vertragen wurde. Wo z. B. ('hloral genfigend hypnagog und ohne Nachsehlitfrigkeit wirkte, wnrde nach der entspreehenden Dosis Dial fiber schweren Kol)f in den ersten Morgenstunden ge- klagt. An<lerentcils t)cohachtete ich cinen jungen Mensehen, der nae, h 0,5 V('ronahmtrium, das (.r al)en(ls spfitestens i) Uhr genommen hatte am niichsten Vormittag iiber hcnommenen Kopf klagte, w:~thrend er mit 0, l Dia]-('iba, zur n,imlichen Zeit eingcnommen, einen so erfrischen- den Schlaf hatte, (la/.~ er lnorgens start tun S Uhr sehou um 7 Uhr auf- gestanden war : es han(lelte sich um einenjugendlichen Psychopathen, der infolgc einer leichten Fu[lverletzung das Zimmer hiiten mul3te und dabei an Schlaflosigkeit litt. Er selbst beriehtet: D'aprgs l'dxp6rience que j'ai faite sur moi, chaque fois que j'ai pris du Dial-Ciba avant de me eoucher, en petite close (une pastille on m6me deux) mon sommeil venut tr6s naturellement 1/2 hem'e environ apr6s l'absorption, a 6t6 t rhs agr6able, ldger, sans rO, ves violents ni canchemars, it quoi je suis sl,jet, et ]e matin je me suis r6veilld facilement, la t6te trbs 16g~,re et I)lus ddgagde que d'autres lois off j'avais eu du vdrona] ou mOme quand je n'avais rich l)ris du tout. --Es wiire m/iglich, dab eine kleinere 4* 52 H. (Einffegangen ant 11. Juni 191~'.) Strasser in Ziirich: ,,Der erste Fall, M. kam am 26. Fe- bruar 1918 zu mir in (lie Sprechstunde und machte auf reich den Ein- druek einer progressiven Paralyse: Koordinationsstbrungen- Euphorie. Daneben war das Bild tiiuschend einem schweren Rausch- Dial-Ciba und Dialcibismus. 53 zustand i~hnlich. Patient hatte von einem hiesigen Arzte, zu dem er wegen (wie ich jetzt annehme) psychogener Schlafst6rungen gegangen war, Dial-Ciba erhalten. Um schneller gesund zu werden, nahm er etwas mehr als ibm verordnet war: in 4 Tagen 13 Tabletten ~ 0,1. Am ftinften Morgen, nachdem er schon die vorhergehenden Tage all Sehwin- delgeffihlen gelitten halle, fiel er beim Aufstehen neben das Bett der Lhnge nach bin, schrieb es von selbst der Dial-Ciba-Wirkung zu und warf den Rest seiner Tabletten in dell ()fen. Patient. +ersicherte mir, es gehe, seit er mit den Tabletten ausgesetzt babe, etwas besser. Das Bild war mir so ungew6hn]ich, dai~ ich es doch ftir richtig hie]t, einen Wasser- mann machen zu lassen, der dann v6llig negativ ausfiel. Die schmie- rige Sprache besserte sich am zweiten Tage meiner Beobachtung. Romberg blieb noch 4--5 Tage positiv. Dann i~nderte sich der psy- chische Habitus des Patienten zusehends, so dai~ ich den anscheinend verbl6deten, dumm-euphorischen Menschen nachtr:,iglich als einen sehr lebhaften und intelligenten Arbeiter kennenlernte. -- Der zweite Fall war wegen Schlafst6rungen zum ,Nervenarzt' Dr. X. gegangen, der ihn sofort mit seine:n nervenstarkenden Einspritzungen beh~,ndelte und ihm au[~erdem Dial-Ciba verschrieb. Patient nahm 7 Tabletten ~t 0,1 innerhalb 3 Tagen. Aucher klagte dann fiber schwere Schwindel- anfhlle, wie wcn ncr bel'a use ht gewesen wgre, konnte nicht richtig gehen, schwankte. Die Erscheinul)gen verschwanden sofort nach Aus- setzen des Dial-Ciba." -- (~ber einen weiteren Fall yon Dial-Ciba- Vergiftung sind wir nur ungenfigend nnterrichtet. Ein ca. 50jithriger Taboparalytiker nahm 12 Tal)letten = 1,2 g Dial-Ciba zusammen mit ,,2 Fl~tschchen" ()piumtropfen. Er war eine Woche lang bewnl3tlos, hatte w~ihrend dieser Zeit derartige Kr~impfe, (tall er aus dem Belle fiel. ]rgendwelche weiteren Folgeerscheinungen traten nieht auf. Die Para- ]yse fiihrte crst iu (le]l folgenden Jahren zur Interllierung. -- Ein ffiiffter Fall betrifft einen 25jahrigen Kollegen, bei dem sich auf das Staats- examen bin eine neurotische I)epression eingeste]lt hatte. Hartnackige Schlaflosigkeit, Gewissenskonflikte bei Verlust jeglicher Entschlu[L f~ihigkeit und des Selbstvertrauen~ lie[]en ihn einige Stunden vor Be- ginn der Prfifhng l0 Dial-Ciba-Tabletten zu sich nehmen. Ob das Mittel unmittelbar zu Schlaf gefiihrt oder eine Aufregung vorhergegangen war, ]~il~t sich nicht mehr sicher eruieren. (Einffegangen ant 11. Juni 191~'.) Stundenlange Bewul~tlosig- keit war nnterbrochen von Delirien ;im Laufe des ersten Tages erfolgte zweimaliges Erbrechen. Im Laufe des zweiten trat ein so heftiges Delir ein, dal] Patient yon ffinf Mann gehalten und aus dem Spital in die Irrenanstalt iibergeffihrt werden mu~te. Gegen Abend dieses zweiten Tages klang das Delirium ab. Von k6rperlichen Symptomen war blol3 eine gewisse Pupi]lenverengerung bei guter Lichtreaktion, sowie das Tau meln auff~illig Aueh am dritteli Tage bestand noeh Nach- zustand i~hnlich. Patient hatte von einem hiesigen Arzte, zu dem er wegen (wie ich jetzt annehme) psychogener Schlafst6rungen gegangen war, Dial-Ciba erhalten. Um schneller gesund zu werden, nahm er etwas mehr als ibm verordnet war: in 4 Tagen 13 Tabletten ~ 0,1. Am ftinften Morgen, nachdem er schon die vorhergehenden Tage all Sehwin- delgeffihlen gelitten halle, fiel er beim Aufstehen neben das Bett der Lhnge nach bin, schrieb es von selbst der Dial-Ciba-Wirkung zu und warf den Rest seiner Tabletten in dell ()fen. Patient. +ersicherte mir, es gehe, seit er mit den Tabletten ausgesetzt babe, etwas besser. Das Bild war mir so ungew6hn]ich, dai~ ich es doch ftir richtig hie]t, einen Wasser- mann machen zu lassen, der dann v6llig negativ ausfiel. Die schmie- rige Sprache besserte sich am zweiten Tage meiner Beobachtung. Romberg blieb noch 4--5 Tage positiv. Dann i~nderte sich der psy- chische Habitus des Patienten zusehends, so dai~ ich den anscheinend verbl6deten, dumm-euphorischen Menschen nachtr:,iglich als einen sehr lebhaften und intelligenten Arbeiter kennenlernte. -- Der zweite Fall war wegen Schlafst6rungen zum ,Nervenarzt' Dr. X. gegangen, der ihn sofort mit seine:n nervenstarkenden Einspritzungen beh~,ndelte und ihm au[~erdem Dial-Ciba verschrieb. Patient nahm 7 Tabletten ~t 0,1 innerhalb 3 Tagen. Aucher klagte dann fiber schwere Schwindel- anfhlle, wie wcn ncr bel'a use ht gewesen wgre, konnte nicht richtig gehen, schwankte. Die Erscheinul)gen verschwanden sofort nach Aus- setzen des Dial-Ciba." -- (~ber einen weiteren Fall yon Dial-Ciba- Vergiftung sind wir nur ungenfigend nnterrichtet. Ein ca. 50jithriger Taboparalytiker nahm 12 Tal)letten = 1,2 g Dial-Ciba zusammen mit ,,2 Fl~tschchen" ()piumtropfen. Er war eine Woche lang bewnl3tlos, hatte w~ihrend dieser Zeit derartige Kr~impfe, (tall er aus dem Belle fiel. ]rgendwelche weiteren Folgeerscheinungen traten nieht auf. Die Para- ]yse fiihrte crst iu (le]l folgenden Jahren zur Interllierung. -- Ein ffiiffter Fall betrifft einen 25jahrigen Kollegen, bei dem sich auf das Staats- examen bin eine neurotische I)epression eingeste]lt hatte. (Einffegangen ant 11. Juni 191~'.) Hartnackige Schlaflosigkeit, Gewissenskonflikte bei Verlust jeglicher Entschlu[L f~ihigkeit und des Selbstvertrauen~ lie[]en ihn einige Stunden vor Be- ginn der Prfifhng l0 Dial-Ciba-Tabletten zu sich nehmen. Ob das Mittel unmittelbar zu Schlaf gefiihrt oder eine Aufregung vorhergegangen war, ]~il~t sich nicht mehr sicher eruieren. Stundenlange Bewul~tlosig- keit war nnterbrochen von Delirien ;im Laufe des ersten Tages erfolgte zweimaliges Erbrechen. Im Laufe des zweiten trat ein so heftiges Delir ein, dal] Patient yon ffinf Mann gehalten und aus dem Spital in die Irrenanstalt iibergeffihrt werden mu~te. Gegen Abend dieses zweiten Tages klang das Delirium ab. Von k6rperlichen Symptomen war blol3 eine gewisse Pupi]lenverengerung bei guter Lichtreaktion, sowie das Tau meln auff~illig. Aueh am dritteli Tage bestand noeh Nach- 54 I [. Christoffel: schli~frigkeit, Kopfweh un<l l)epression. Nach cinem Vierteljahre, w/thrend dessen Patient sein Examen gut bestanden hatte, konnte er yon der Depression geheilt entlassen wet'den. -- (~ber andersartige Bil- der yon I)ialcibism us berichtcte tler oben zitierte junge Mann, der so ziemlich in allen Ausschweifungen dutch war (('ocainismus, .~theris- mus), an (lessen Glaubwtirdigkeit aber bci der Ffille des Details und der ganzen Art. wie er seine Erfahrungen mir gelegentlich mitteilte, wenig gezweifelt werden diirfte: ,,J'ai comm des 1)ersonnes qui ~t force d'absor- ber du Dial ont fini par en (lcvenir maniaques, et non seulement en pre- naient par doses tr6s fortes (jusqu',~ 8 pastilles) mais encore avaient ]e besoin d'en prendre comme on a besoin (le pretl(Ire (le l'6ther ou de la cocaine lorsque on s'y ('st habitu(4. [ls nc faisaient pas cela pour dormir eependant, mais se procurer' des visions c( des jouissances qu'ils prdtendaient 6tre tr6s agrdables. Un de rues amis prenait souvent 7 pastilles en role fois et avant le sommeil qui ne vcnait que plus <l'une heure apr6s, disait avoir des visions, dprouver des jouis- sanccs, comme s'il avait pris de l'dther, voyait les objets en routes sortes de couleur, mais n'avait aucune envie (le parler pendant ce temps, ce qui est tr6s particnlier, car en gdndral l'dther et les autres stupdfiants pr<>v<)quent une grande surexeitation et une irrdsistible envie de parler et (l'entendre parler. Pour re'assurer de la chose, j'ai pris un soir se pt pastilles de Dial et m~e prise tr6s petite de cocaine afin-(le m'emp6cher (le (Iormir au cas <)h le I)ial in'endormirait. (Einffegangen ant 11. Juni 191~'.) Apr6s (li x minutes j'ai dt6 obligd de m'6ten(Ire sur un canapd 1)arceque 1~ t6t<~ me tournait comme si j'avais bu beaucoup de champagne ou rtspir6 de l'dther. Tout de suite j'ai eu dans les oreilles des sonneries (le cloches (encore (,omme avcc l'dther) et quan(I j'ouvrais les yeux je voyais tous lcs objets de couleur claire, en rouge, et tons ceux de couleur foncde, en violet presque lilas. Puis autour de moi des points et des cercles lumineux, des sortes (le serpents de feu de l'cffet le pins ddsagrdable. En m6me tcmps nne sensation d'audantissenmnt, (le tomber darts le vide, une impossibilitd de penser ct la crainte de mourir d touffd, car je scntais (lc tr6s forth bourdonnements dank les orei]les. Mais aucunc sorte de jouissance ni de plaisir physique. Apr6s une dcmic heure le sommcil est venu tr6s Iourd et brus- quement. Le lende main apr6s avoir dormi tr6s longtcmps jc me suis dveilld difficilemcnt avec une violente migraine, des naus6es et la t6tequi me tournait commc sij'dtaisivre. Puisj'aieu,'~pcine levd, (lc tr6s forts vomissements qui m'ont obligd h me recoucher et h rester (leux jours au lit shns aucun al)pdtit et ~moitid abruti. d'ai dt6 tr6s dt(mnd car j'avais vu bien d'autres personnes prendre (lu Died en plus grande quantild que je n'en avais pris, avoir des visions, s'en(Iormir ensnite et se rdveiller apr6s un tr6s long sommeil tr6s bien schli~frigkeit, Kopfweh un<l l)epression. Nach cinem Vierteljahre, w/thrend dessen Patient sein Examen gut bestanden hatte, konnte er yon der Depression geheilt entlassen wet'den. -- (~ber andersartige Bil- der yon I)ialcibism us berichtcte tler oben zitierte junge Mann, der so ziemlich in allen Ausschweifungen dutch war (('ocainismus, .~theris- mus), an (lessen Glaubwtirdigkeit aber bci der Ffille des Details und der ganzen Art. wie er seine Erfahrungen mir gelegentlich mitteilte, wenig gezweifelt werden diirfte: ,,J'ai comm des 1)ersonnes qui ~t force d'absor- ber du Dial ont fini par en (lcvenir maniaques, et non seulement en pre- naient par doses tr6s fortes (jusqu',~ 8 pastilles) mais encore avaient ]e besoin d'en prendre comme on a besoin (le pretl(Ire (le l'6ther ou de la cocaine lorsque on s'y ('st habitu(4. [ls nc faisaient pas cela pour dormir eependant, mais se procurer' des visions c( des jouissances qu'ils prdtendaient 6tre tr6s agrdables. (Einffegangen ant 11. Juni 191~'.) Un de rues amis prenait souvent 7 pastilles en role fois et avant le sommeil qui ne vcnait que plus <l'une heure apr6s, disait avoir des visions, dprouver des jouis- sanccs, comme s'il avait pris de l'dther, voyait les objets en routes sortes de couleur, mais n'avait aucune envie (le parler pendant ce temps, ce qui est tr6s particnlier, car en gdndral l'dther et les autres stupdfiants pr<>v<)quent une grande surexeitation et une irrdsistible envie de parler et (l'entendre parler. Pour re'assurer de la chose, j'ai pris un soir se pt pastilles de Dial et m~e prise tr6s petite de cocaine afin-(le m'emp6cher (le (Iormir au cas <)h le I)ial in'endormirait. Apr6s (li x minutes j'ai dt6 obligd de m'6ten(Ire sur un canapd 1)arceque 1~ t6t<~ me tournait comme si j'avais bu beaucoup de champagne ou rtspir6 de l'dther. Tout de suite j'ai eu dans les oreilles des sonneries (le cloches (encore (,omme avcc l'dther) et quan(I j'ouvrais les yeux je voyais tous lcs objets de couleur claire, en rouge, et tons ceux de couleur foncde, en violet presque lilas. Puis autour de moi des points et des cercles lumineux, des sortes (le serpents de feu de l'cffet le pins ddsagrdable. En m6me tcmps nne sensation d'audantissenmnt, (le tomber darts le vide, une impossibilitd de penser ct la crainte de mourir d touffd, car je scntais (lc tr6s forth bourdonnements dank les orei]les. Mais aucunc sorte de jouissance ni de plaisir physique. Apr6s une dcmic heure le sommcil est venu tr6s Iourd et brus- quement. Le lende main apr6s avoir dormi tr6s longtcmps jc me suis dveilld difficilemcnt avec une violente migraine, des naus6es et la t6tequi me tournait commc sij'dtaisivre. Puisj'aieu,'~pcine levd, (lc tr6s forts vomissements qui m'ont obligd h me recoucher et h rester (leux jours au lit shns aucun al)pdtit et ~moitid abruti. d'ai dt6 tr6s dt(mnd car j'avais vu bien d'autres personnes prendre (lu Died en plus grande quantild que je n'en avais pris, avoir des visions, s'en(Iormir ensnite et se rdveiller apr6s un tr6s long sommeil tr6s bien Diai-(;iba und Dialcil)ismus. (Einffegangen ant 11. Juni 191~'.) 55 p~wtants, et sans aucune fatigue ni ddpression morale apl)arente , et cela m'a d'autant plus 6tonnd que j'dtais moi-m6me beaucoup plus intoxiqud que ees gcns, et que je SUpl>ortais tr6s bien tout antre stup6- fiant, l,a seule consdquence remarquable chez ees l)ialcibatistes, dtait le manque total d'aplidtit et le ddgoft tie ioute nourriture pendant presquctoutelajourndequisuivaitla sdance. Quant h nioi, je ll'ai trouvd rich d'agrdable all Dial eit forte dose et je n'ai jainais recomlnencd l'expdrience laquelle ne lll'a lir(icurd qu'ulie seiisatiolL tr'6s courte el mddiocre et roll(hi inala(le i)ell(lant dellx ji)uvs. Mais par centre quand pour conll)att/'e l'insoinnie je lirelmis uiw I)astille ou mbme deux, j'ai toujours tr6s bien dormi d'un sommeil agrdable et naturel, et men rdveil a 6td facile et ldger le matill.'" Zusammenfassung: DiaI-Ciba ist in ka(im halt) so groBer I)osis wie Veronal, zehnmal kleinerer als Chloral eil~ wirksames Hyimoticum auch bei aufgeregten Geisteskranken. In 2/~ der Verabreichmlgen trat prompter, (tie ganze Nacht dauernder Schlaf ein. Bei fortgesetztem, ilberwachtem Gebrauch zeigten sich keine Sch:,idigutigen des Organis- mus; Kumulation, Abnahme (ler Wirksamkeit, Abstinenzerscheimmgen wurdela nieht beobachte[. Einzeldosen v<)n 0, lbis 0,2 hatten hie Neben- folgen. Bei Dosen von 0,3 und 0,4 wurde als Ausnahme eine Art Rausch beobachtet (sog. Dialcibismus). Dieser Zustand ist nieht als durch Kumulationswirkung entstanden zu betrachten. Vielmehr scheint er auf einer t emporhr uu(l individuell verschiedenen l)~eal<ti(msart zu be- ruhen. Dialcibismus (K<)ordinati<)nsst6rungen, liippische Euphoric) wurde ferner bei mehrtitgigem uniiberwachtem Dial-('iba-Gebrauch, wobei die durchschnittliche Tagcsdosis 0,4 g nicht iiberstieg, beobachtct. Die therapeutische Maximaleinzeldosis diirfte 0.6 ideht iibersteigeu. Bei star ken Erregungen kann Dial-(!iba in maximalen l)<)sen versagen; ein nach 1,0 g entstan(lenes gewaltt~tiges l)elirium ist mit (lem Mittel nicht in sicheren urs~t(:hlichen Zusammenhang zu bringen. Erbreehen nach Dosen yon 0,7 al~ scheint nieht selten, Appetitlosigkeit regehnitBig. Bei chronisch mit Ather und Cocain Vergifteten k6nnen nach Einzel- desert yon 0,7 an [)is zu ca. einer Stuude anhaltende, yon langem er- frischendem Schlaf gefolgte still euphorisehe Zust:,inde mit Gesichts- illusionen auftreten.
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© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Open Access Open Access The upper respiratory tract microbiota of healthy adults is affected by Streptococcus pneumoniae carriage, smoking habits, and contact with children A. Cristina Paulo1*†, João Lança1†, Sónia T. Almeida1, Markus Hilty2 and Raquel Sá‑Leão1* †A. Cristina Paulo and João Lança are co-first authors. †A. Cristina Paulo and João Lança are co-first authors. †A. Cristina Paulo and João Lança are co-first authors. *Correspondence: A. Cristina Paulo acpaulo@itqb.unl.pt Raquel Sá‑Leão rsaleao@itqb.unl.pt Full list of author information is available at the end of the article *Correspondence: A. Cristina Paulo acpaulo@itqb.unl.pt Raquel Sá‑Leão rsaleao@itqb.unl.pt Full list of author information is available at the end of the article Abstract Background  The microbiota of the upper respiratory tract is increasingly recognized as a gatekeeper of respiratory health. Despite this, the microbiota of healthy adults remains understudied. To address this gap, we investigated the composition of the nasopharyngeal and oropharyngeal microbiota of healthy adults, focusing on the effect of Streptococcus pneumoniae carriage, smoking habits, and contact with children. Results  Differential abundance analysis indicated that the microbiota of the oropharynx was significantly differ‑ ent from that of the nasopharynx (P < 0.001) and highly discriminated by a balance between the classes Negativi‑ cutes and Bacilli (AUC of 0.979). Moreover, the oropharynx was associated with a more homogeneous microbiota across individuals, with just two vs. five clusters identified in the nasopharynx. We observed a shift in the nasopharyn‑ geal microbiota of carriers vs. noncarriers with an increased relative abundance of Streptococcus, which summed up to 30% vs. 10% in noncarriers and was not mirrored in the oropharynx. The oropharyngeal microbiota of smokers had a lower diversity than the microbiota of nonsmokers, while no differences were observed in the nasopharyngeal microbiota. In particular, the microbiota of smokers, compared with nonsmokers, was enriched (on average 16-fold) in potential pathogenic taxa involved in periodontal diseases of the genera Bacillus and Burkholderia previously identi‑ fied in metagenomic studies of cigarettes. The microbiota of adults with contact with children resembled the micro‑ biota of children. Specifically, the nasopharyngeal microbiota of these adults had, on average, an eightfold increase in relative abundance in Streptococcus sp., Moraxella catarrhalis, and Haemophilus influenzae, pathobionts known to colonize the children’s upper respiratory tract, and a fourfold decrease in Staphylococcus aureus and Staphylococcus lugdunensis. Conclusions  Our study showed that, in adults, the presence of S. pneumoniae in the nasopharynx is associated with a shift in the microbiota and dominance of the Streptococcus genus. Furthermore, we observed that smoking habits are associated with an increase in bacterial genera commonly linked to periodontal diseases. Interestingly, our research also revealed that adults who have regular contact with children have a microbiota enriched in pathobionts Microbiome Microbiome Paulo et al. Microbiome (2023) 11:199 https://doi.org/10.1186/s40168-023-01640-9 †A. Cristina Paulo and João Lança are co-first authors. Backgroundh The microbiota of the human upper respiratory tract (URT) has an important role in human health since it modulates the colonization of commensal bacteria and provides colonization resistance against pathogens [1]. The URT comprises several structures, among which the nasopharynx and the oropharynx are distinctive, as they are the preferential niches of important human pathobi- onts, including Streptococcus pneumoniae (or pneumo- coccus). Pneumococcus is a gram-positive facultative anaerobe that is known to be the main cause of bacte- rial respiratory infections worldwide [2]. Risk groups for pneumococcal disease include young children, the elderly, and immunocompromised individuals of all ages [3]. Colonization is mostly asymptomatic and is very fre- quent in children under 5 years of age, in which it is often higher than 50% [4–6]. In contrast, in adults, it has been reported as being between 20 and 40% [7–9]. Two impor- tant factors that contribute to increased pneumococcal colonization and persistence in adults include contact with children and smoking [9–12]. To what extent the microbiota composition is a risk factor for pneumococ- cal colonization has not been explored. Nonetheless, to the best of our knowledge, while there are studies in chil- dren, the microbiota of the nasopharynx and oropharynx remains poorly characterized since the few microbiota studies of the upper respiratory tract in adults have been associated with disease status [13–16]. The study was nested in a prospective 6-month longi- tudinal study that aimed to characterize the dynamics of S. pneumoniae colonization in healthy adults [9]. The original study was conducted between February 2015 and December 2016 and enrolled 87 immunocompetent adults aged between 25 and 50 years old living in the Lis- bon metropolitan area, Portugal. Detailed information about sample collection and study design is described in the supplementary information (“Sample collection”) and in Almeida et  al. [9]. Briefly, nasopharyngeal and oropharyngeal samples were collected using appropriate swabs and were immediately stored in STGG medium. All samples were kept at − 80 °C. The presence of pneu- mococci was screened by classical culture based methods and real-time PCR targeting the genes lytA and piaB [9]. The study was approved by the ethical committee of Instituto de Higiene e Medicina Tropical, Universi- dade Nova de Lisboa, and was registered at the National Commission of Data Protection (ref. 3803/2014). DNA extraction Nasopharyngeal and oropharyngeal samples were main- tained at − 80 °C in STGG. Samples were thawed on ice, and for each sample, 200 μL was pipetted and added to 200 μL of lysis buffer (MagNA Pure Compact Nucleic Acid Isolation Kit, Roche Diagnostics, GmbH). Sam- ples were incubated at 37 °C for 20 min. DNA extraction was performed with the MagNA Pure Compact System (Roche) according to the manufacturer’s instructions. For Backgroundh Signed informed consent was obtained from all par- ticipants; samples and questionnaires were processed anonymously.i In the current study, cases were defined as individu- als who were found to carry pneumococci in the naso- pharynx and/or oropharynx at a minimum of three time points at least 1 month apart from each other. Controls were defined as individuals who were sampled at least five times (1  month apart from each other) during the 6-month period and were never colonized with pneu- mococci. For both cases and controls, samples collected within 1 month of antibiotic use were excluded. For both cases and controls, three samples per individual were selected for analyses. The three samples were, as much as possible, distant in time and covered different seasons. Understanding the factors that shape and characterize a healthy microbiota is a fundamental step in strategies aimed at promoting a healthy state, for example, through the use of live biotherapeuticals [17]. Here, we comprehensively analyzed the composition of the nasopharyngeal and oropharyngeal microbiota of immunocompetent healthy adults aged between 25 and 50 years old. The specific aims of our study were (i) to compare the microbiota of pneumococcal carriers vs. noncarriers in the nasopharynx and oropharynx and (ii) to understand how individual characteristics such as age, sex, contact with children, and smoking habits shape the microbiota of the nasopharynx and oropharynx. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 20 Paulo et al. Microbiome (2023) 11:199 Paulo et al. Microbiome frequently carried by children. These findings collectively contribute to a deeper understanding of how various factors influence the upper respiratory tract microbiota in adults. Keywords  Microbiota, Nasopharynx, Oropharynx, Streptococcus pneumoniae, Healthy adults Keywords  Microbiota, Nasopharynx, Oropharynx, Streptococcus pneumoniae, Healthy adults and oropharyngeal bacterial microbiota of immuno- competent healthy adults colonized with S. pneumoniae (cases) and noncolonized individuals (controls). Total bacterial load quantification i To prepare a standard curve for total bacterial load quan- tification, the method described by Bogaert et  al. [18] was followed with some modifications. Representative strains of eight bacterial species that commonly colo- nize the upper respiratory tract were used: Corynebac- terium accolens, Dolosigranulum pigrum, Haemophilus influenzae, Moraxella catarrhalis, Streptococcus mitis, Streptococcus oralis, S. pneumoniae, and Streptococcus pseudopneumoniae. First, serial dilutions of the frozen stocks of each strain were performed to quantify the bacterial load. Except for H. influenzae, which was cul- tured on chocolate agar, all other species were cultured on blood agar plates. Corynebacterium accolens cultures were incubated overnight at 37  °C in anaerobic condi- tions; Dolosigranulum pigrum cultures were incubated overnight at 37 °C in aerobic conditions; and the remain- ing bacterial species were incubated overnight at 37 °C in a 5% ­CO2 atmosphere. On the following day, CFU/ mL was estimated for each frozen stock. Afterwards, a mixture containing ­104  CFU/mL of each species was prepared. DNA extraction of the mixture was carried out as described above. DNA was quantified on a Nan- oDrop and used as a reference for total bacterial load quantification. Study population and study design A case–control study was designed with the aim of evalu- ating potential differences between the nasopharyngeal Paulo et al. Microbiome (2023) 11:199 Paulo et al. Microbiome (2023) 11:199 Page 3 of 20 A standard curve was considered valid when the differ- ence between Ct values of consecutive dilutions did not exceed three Ct values and the paired results obtained for a given dilution did not exceed 0.5 Ct. Samples with a bacterial load lower than the DNA extraction negative control were considered of low quality and, whenever possible, were replaced by other samples from the same individual following the inclusion and exclusion criteria described above. every run, water samples (used as negative controls) were extracted in parallel. DNA was stored at − 20 °C. Water samples were used as technical negative controls and were processed in parallel with biological samples to con- trol for potential contaminations arising during manipu- lation and testing. every run, water samples (used as negative controls) were extracted in parallel. DNA was stored at − 20 °C. Water samples were used as technical negative controls and were processed in parallel with biological samples to con- trol for potential contaminations arising during manipu- lation and testing. 16S rRNA gene amplicon sequencing For all samples, the V4 region of 16S rRNA was amplified using forward (5′-GTG​CCA​GCMGCC​GCG​GTAA-3′) and reverse (5′-GGA​CTA​CHVGGG​TWT​CTAAT-3′) primers previously described [19]. PCR was conducted in a final volume of 25 μL containing 10 μL of 2 × master mix, 2.5 μL of primer barcode (2 μM), 2.5 μL of univer- sal primer (2 μM), and 10 μL of DNA. The thermocycling conditions were 94 °C for 3 min, 35 amplification cycles of 94 °C for 1 min, 50 °C for 1 min, 72 °C for 105 s, and a final extension of 72 °C for 10 min. Each sample was run in triplicate. After that, triplicates were pooled and submitted to a next-generation sequencing platform for indexing and pair-end sequencing (2 × 250  bp) on a MiSeq platform. Amplification and sequencing were per- formed at the Genomics Unit of Instituto Gulbenkian da Ciência. Statistical analysis of nasopharyngeal and oropharyngeal microbiota profilesh The statistical analysis of the microbiota was performed using compositional data analysis methods. In brief, for each sample, count reads were normalized using the centered ­log2-ratio (CLR) transformation [26]. This transformation allows us to account for the complex compositional data structure of metagenomic studies and to reduce the likelihood of spurious correlations. The microbiome package for the CLR transformation, which replaces ASV read counts with exact zero relative abun- dance with a pseudocount before calculating the loga- rithms, was used [27]. Several additional approaches were used to remove potential contaminants by filtering ASVs and samples. First, ASVs attributed to Eukaryota and Archaea were excluded. Second, ASVs were filtered according to a fre- quency-based approach described by Davis et  al. [23]. This approach is based on the observation that the prob- ability of having contaminants is higher when the DNA concentration is lower. Briefly, for each ASV, a regres- sion line was fitted to the number of reads as a function of DNA concentrations measured by 16S qPCR in each sample. If the number of reads of an ASV was observed to decrease linearly with increased DNA concentration, it was considered a contaminant and was excluded. Oth- erwise, it was kept in the analysis. ASVs were filtered according to their relative abundance and were kept if they were present in at least two samples, with a relative abundance within each sample higher than 0.1% [24]. Finally, samples that had fewer than 1000 reads after all ASV filters were excluded [14, 25]. To identify homogeneous bacterial communities in the nasopharynx and oropharynx, a hierarchical clus- tering approach was employed. The samples were trans- formed using the CLR transformation, and the Euclidean distance between samples was used for clustering. The Ward’s minimum variance method [28] was used to agglomerate samples that share similar taxonomic pro- files. To determine the optimal number of clusters, the gap statistics proposed by Tibshirani et al. [29] were used. For cross-validation, a random forest model classifier with 500 trees was utilized. The out-of-bag error, repre- senting the percentage of misclassified samples, was esti- mated, and the confusion matrix was examined to assess the degree of cluster overlap. Each cluster was character- ized by the two most abundant genera it contained. i For ASVs that were shown to be significantly differ- ent in the differential abundance analysis, NCBI BLAST searches were performed (using MegaBLAST) to identify the presumptive species. Bioinformatic processing Di i i A li D Microbiome (2023) 11:199 of 0.05. Differences between strata were considered sta- tistically significant if the adjusted P-value was < 0.05. forward and reverse reads were ready for merging. The final step involved identifying and removing ASVs that could potential be chimeric sequences originating from defective PCR amplification (for example, resulting from pairing of incomplete parental sequences). Tax- onomy was assigned using the Silva v132 database as a reference [22]. Bioinformatic processing Di i i A li D Bioinformatic processing Divisive Amplicon Denoising Algorithm  2 (DADA2) [20] was used to denoise and taxonomically assign the 16S rRNA sequences following the authors’ online pipeline tutorial 1.16 (https://​benjj​neb.​github.​io/​dada2/​ tutor​ial.​html). DADA2 was run on R version 3.6.2 [21]. The parameters used in each step of the DADA2 work- flow were those predefined and recommended in the pipeline except for the parameters that are data driven, specifically trimming and inference of error rates. Sequences were trimmed in the position in which the 25th percentile of the quality score was above 30 (see supplementary information and Fig. S1); error infer- ence rates were calculated using the entire dataset and pooled sequences. In brief, reads were filtered and trimmed to remove sequencing errors, based on their quality scores (Phred scores) and on the identification of ambiguous bases in both forward and reverse reads. Subsequently, an estimation of the error rates (i.e., pos- sible transitions or transversion point mutations), made by MiSeq platform, was performed. This step aimed to achieve the following: (i) infer amplicon sequence vari- ants (ASVs) based on the estimated error rates men- tioned earlier, (ii) dereplicate reads to obtain unique sequences, and (iii) remove singletons. Afterward, the i To evaluate the quality of the nasopharyngeal and oro- pharyngeal samples, the total bacterial load of each sam- ple was quantified by qPCR using universal primers and probes that target the 16S rDNA gene [18]: 16Sfw-5′- CGA AAG CGT GGG GAG CAA A-3′, 16Srev-5′-GTT CGT ACT CCC CAG GCG G-3′, and FAM-ATT​AGA​ TAC​CCT​GGT​AGT​CCA-MGB. qPCRs were performed in a final volume of 25 μL containing 12.5 μl of 1 × mas- ter mix (FastStart TaqMan® Probe Master, Roche), 1 μL of each primer (0.4 μM), 1 μL of probe (0.2 μM), 7 μL of ­H2O, and 2.5 μL of DNA. DNA amplification was per- formed in CFX96™ Real-Time System Amplification (Bio-Rad). The thermocycling conditions were 50 °C for 2 min and 95 °C for 10 min followed by 45 amplification cycles of 95 °C for 15 s and 60 °C for 1 min. In each 16S qPCR run, multiple negative controls (one per every 16 reactions) and serial dilutions (in duplicate) of the DNA extracted from the species mixture ­(100 to ­10−5  ng/μL) were included. The latter was included to obtain a stand- ard curve for each qPCR. Page 4 of 20 Paulo et al. Microbiome (2023) 11:199 Paulo et al. Statistical analysis of the study populationh The baseline characteristics of the study population and samples were stratified by the presence/absence of pneu- mococci. To compare characteristics between strata, the chi-square or Student’s t-test was used in conform- ity with the type of data. Bacterial DNA quantification was stratified by the presence/absence of pneumococci and by the anatomical site (oropharynx or nasopharynx). Bacterial DNA quantification, per stratum, was summa- rized by their geometric mean and respective standard deviation (SD). The Wilcoxon rank-sum test with con- tinuity correction was used for multiple comparisons of groups two by two. The Benjamini–Hochberg procedure was used to control for the false discovery rate at the level Statistical analysis y All analyses described below were performed in R ver- sion 3.6.2 (Boston, MA, USA). Statistical analysis of nasopharyngeal and oropharyngeal microbiota profilesh Species assignment was based exclusively on hits with 100%, as differences in one or more nucleotides result in assignment of different ASVs. To study associations between clusters and the pneu- mococcal carrier state, mixed general linear models with a logit link function were used. A model was fit to each microbiota profile using the microbiota profile compris- ing the higher number of samples as a reference. Indi- viduals were introduced as a random variable to account for repeated measurements. In addition, models were adjusted for sociodemographic characteristics and envi- ronmental factors (individual’s age, gender, having con- tact with children, smoking habits, and season in which the sample was collected). Associations between vari- ables and the clusters were calculated using odds ratios (ORs) and corresponding confidence intervals (CIs) at 95%. A CI that did not include 1 was considered statisti- cally significant. Microbiota α‑diversityh The abundance-based diversity of the microbiota groups was estimated using Hill’s first five numbers [30]. Hill’s numbers have a scaling parameter, known as the order of diversity (q), that modulates sensitivity toward more Paulo et al. Microbiome (2023) 11:199 Paulo et al. Microbiome (2023) 11:199 Page 5 of 20 Page 5 of 20 Dynamics of microbiota carriageh abundant or rare taxonomic units. The higher the order, the higher the importance attributed to abundant taxo- nomic units. The Hill numbers of orders 0, 1, and 2 are related to three popular diversity indexes known as rich- ness, Shannon’s index, and Simpson’s index, respectively, with the advantage of having the replication principle (i.e., when doubling the number of taxonomic units in a system, the diversity is also doubled). Evenness was measured according to the steepness of the diversity pro- file from the Hill number of order 0 to the Hill number of order 1 (the higher the steepness, the lower the com- munity evenness). The Hill numbers were calculated for each sample using the abundance-based estimates at the taxa level of genus and then by calculating the geomet- ric mean for each group. Differences in Hill numbers between groups were calculated using the Mann‒Whit- ney test. abundant or rare taxonomic units. The higher the order, the higher the importance attributed to abundant taxo- nomic units. The Hill numbers of orders 0, 1, and 2 are related to three popular diversity indexes known as rich- ness, Shannon’s index, and Simpson’s index, respectively, with the advantage of having the replication principle (i.e., when doubling the number of taxonomic units in a system, the diversity is also doubled). Evenness was measured according to the steepness of the diversity pro- file from the Hill number of order 0 to the Hill number of order 1 (the higher the steepness, the lower the com- munity evenness). The Hill numbers were calculated for each sample using the abundance-based estimates at the taxa level of genus and then by calculating the geomet- ric mean for each group. Differences in Hill numbers between groups were calculated using the Mann‒Whit- ney test. The dynamics of individual nasopharyngeal and oro- pharyngeal clusters were represented by alluvial plots and stratified by pneumococcus carriage. The number of individuals who changed clusters was reported as proportions and compared using a chi-squared test. Microbiota α‑diversityh Temporal changes in the nasopharyngeal and oro- pharyngeal microbiota were analyzed at the genus level by comparing the microbiota of each individual in con- secutive samples (first and second, second and third). Differences between the composition of microbiota in consecutive samples were expressed as volatility (Aitch- ison distance), calculated using the Euclidean distance on the CLR transformed data [34]. The Wilcoxon rank- sum test was used to compare volatility values. Differential abundance analysis of microbiotaif Differential abundance analysis of microbiota To evaluate if there were significant differences between the microbiota of different groups, a permutational multivariate analysis of variance (PERMANOVA) [31] implemented on the Adonis algorithm of the R Vegan package was utilized. PERMANOVA was performed on the Euclidean distance matrix with CLR-transformed read counts. To ensure the reliability of the results, the assumption of variance homogeneity was checked. If a significant effect was found, a differential abundance analysis to determine which taxa were differentially abun- dant between groups of samples was performed. Since the data were very sparse, we made use of a zero-inflated Gaussian mixed model (ZIGMM) [32] implemented in R with metagenomeSeq [31]. The cumulative sum scaling method was used to normalize sequence counts based on the lower-quartile abundance of features. Data were also filtered to maintain a threshold of ASVs that were present in at least 75% of the samples, a step needed to avoid unreliable fold-change estimates [32]. Only ASVs with more than 1.5 log fold-change differences and an adjusted P-value ≤ 0.05 were considered [31]. Volcano plots of the ­log10 of statistical significance (P-value) vs. ­log2 of the magnitude of change (fold-change) were used to visualize the results. The records of 87 individuals who were followed-up for 6 months were reviewed retrospectively. Fifty-nine individuals met the following criteria to be included in the current study: 12 pneumococcal carriers with at least three samples (collected 1 month apart from each other) positive for pneumococci and 47 pneumococcal noncarriers (negative for pneumococci on at least five occasions separated 1  month apart from each other). None of the individuals included had samples collected within 1 month of antibiotic use. The baseline characteristics of the study population are summarized in Table  1. There were no significant differences between carriers and noncarriers when mean age, sex, smoking status, and antibiotic consump- tion in the previous six months were compared. Pneu- mococcal carriers were more likely to have regular contact with children than nonpneumococcal carriers (83.3% vs. 38.3%, P = 0.014) and to have received sea- sonal flu vaccination (33.2% vs. 7.4%, P = 0.015). Antibi- otic consumption prior to sample collection occurred in a minority of samples (and always in a period exceeding 1 month from sample collection), with no significant differences between carriers and noncarriers (Table 1). Samples analyzed The Wilcoxon rank-sum test with continuity correction and the Benjamini–Hochberg procedure were used to adjust for the false discovery rate Samples analyzed Microbial signatures, that is, groups of microbial taxa that are predictive of a phenotype of interest, were fur- ther identified using the algorithm selbal developed in R [33]. Briefly, this algorithm takes the log ratio of the geo- metric mean of the taxa from two groups and tests for association with the response variable by fitting a logis- tic model. The model that maximizes the area under the receiver operating characteristic (AUC) curve is then selected. For each of the 59 individuals, paired samples col- lected from the oropharynx and the nasopharynx at three time points were analyzed, resulting in a total of 354 samples. Antibiotic consumption between vis- its occurred 8.3% of the time. Samples were collected throughout the year with no significant differences between pneumococcal carriers and noncarriers by sampling season (Table 2). Paulo et al. Microbiome (2023) 11:199 Page 6 of 20 Table 1  Baseline characteristics of the study population stratified by the presence/absence of pneumococci Characteristics with a P-value less than 0.05 are highlighted in bold a Individual characteristics (with the exception of mean age) were compared with Pearson’s chi-squared test b Mean age between groups was compared with Student’s t-test Characteristics Carriers N = 12 Noncarriers N = 47 P-valuea Mean age ± standard deviation (years) 36.3 ± 5.7 37.5 ± 7.1 0.142b Gender male, n (%) 5 (41.7) 22 (46.8) 1.000 Living with children ≤ 18 years old, n (%) 10 (83.3) 18 (38.3) 0.014 Smoker, n (%) 5 (41.7) 20 (42.6) 1.000 Chronic diseases, n (%) 6 (50.0) 12 (25.5) 0.158 Seasonal flu vaccination, n (%) 4 (33.4) 2 (7.4) 0.015 Vaccination with PCV13, n (%) 4 (33.4) 6 (12.8) 0.189 Antibiotic consumption 6 months before enrollment, n (%) 3 (25) 8 (17) 0.679 Table 2  Characteristics of samples included in the study according to individuals’ pneumococcal carrier state a Sample characteristics were compared with Pearson’s chi-squared test Characteristics Pneumococcal carriers’ samples N = 72 Pneumococcal noncarriers’ samples N = 282 P-valuea Antibiotic consumption between sam‑ pling, n (%) 6 (8.3) 20 (7.1) 0.799 Sampling season, n (%) Spring 22 (30.6) 80 (28.4) 0.826 Summer 18 (25.0) 78 (27.7) 0.761 Autumn 6 (8.3) 50 (17.7) 0.077 Winter 26 (36.1) 74 (26.2) 0.130 ble 2  Characteristics of samples included in the study according to individuals’ pneumococcal carrier state 1) 74 (26.2) 0.130 Fig. 1  Total bacterial load of nasopharyngeal (NP) and oropharyngeal (OP) samples. Bacterial DNA quantification, processing of 16S rRNA gene data, and identification of clustersh i The geometric mean of the total bacterial load based on 16S rRNA gene quantification in the nasophar- ynx (19.58  pg/μL) was significantly lower (P < 0.001) than the bacterial DNA quantity in the oropharynx (961.27 pg/μL) independent of the pneumococcal car- rier state. In the nasopharynx, the geometric mean of the total bacterial load of pneumococcal carriers was higher than that of noncarriers (43.91  pg/μL vs. 15.89  pg/μL, P < 0.009). In the oropharynx, the corre- sponding numbers were 1237.57  pg/μL  (carriers) and 901.23 pg/μL (noncarriers, P = 0.233) (Fig. 1). Processing of the raw metagenomic sequencing data was performed for all 354 samples as detailed in the supplementary information (“Processing of raw metagenomic sequencing data” and Fig. S1 therein). Processing of the raw metagenomic sequencing data was performed for all 354 samples as detailed in the supplementary information (“Processing of raw metagenomic sequencing data” and Fig. S1 therein). A total of 9,027,200 reads were received. The aver- age number of reads per sample was 22,071 (range 2  and 38,536), which were clustered in 14,669 ASVs. After removing sequences from Eukarya and Archaea, there were a total of 8,079,020 reads with a medium number of 24,652 reads per sample (range between 2 and 38,536) and 187 singletons. The SILVA database assigned 6,589 ASVs to bacteria. A total of 108 reads A total of 9,027,200 reads were received. The aver- age number of reads per sample was 22,071 (range 2  and 38,536), which were clustered in 14,669 ASVs. After removing sequences from Eukarya and Archaea, there were a total of 8,079,020 reads with a medium number of 24,652 reads per sample (range between 2 and 38,536) and 187 singletons. The SILVA database assigned 6,589 ASVs to bacteria. A total of 108 reads Fig. 1  Total bacterial load of nasopharyngeal (NP) and oropharyngeal (OP) samples. The Wilcoxon rank-sum test with continuity correction and the Benjamini–Hochberg procedure were used to adjust for the false discovery rate Paulo et al. Microbiome (2023) 11:199 Page 7 of 20 Page 7 of 20 were unassigned, with the majority (105 reads) sampled from the nasopharynx. was the least diverse and least even of all nasopharyngeal clusters (Fig. S4A). The other clusters were named Corynebacterium-Moraxella (16.3%, n = 27), Streptococcus- Acinetobacter (13.3%, n = 22), Streptococcus-Pseudomonas (12.0%, n = 20), and Pseudomonas-Corynebacterium (10.2%, n = 17) (Fig. 3B–E). Bacterial DNA quantification, processing of 16S rRNA gene data, and identification of clustersh To identify groups of samples that shared closer bacte- rial taxonomic profiles with each other, hierarchical clus- tering was performed. Two main groups were identified, and these, with few exceptions, segregated nasopharyn- geal samples from oropharyngeal samples (Fig. 2). Clusters in which Streptococcus were not dominant had a higher effective number of genera (supplementary information “Bacterial profiles in the oropharynx and nasopharynx” and Fig. S4 therein). A total of eight clusters were determined. However, the random forest that we used as a cross-validation method showed a confusion matrix with an out-of-bag error of 15.0% due to an overlap between two oropharyngeal clus- ters, where 71.4% of the samples that belonged to one of the clusters were classified in another cluster. When analyzed further, we found that the ten most abundant genera in both clusters were identical; thus, we opted to merge these two clusters. Ultimately, five nasopharyngeal clusters and two oropharyngeal clusters were observed (Fig. 2). PERMANOVA indicated that the oropharynx microbiota was significantly different from the nasophar- ynx microbiota (P < 0.001), as detailed in the supplemen- tary information (“Bacterial profiles in the oropharynx and nasopharynx” and Fig. S2–S3 and Table S1 therein). Association between nasopharyngeal microbiota profiles and variables under study A mixed general linear model was used to investigate potential associations between the nasopharyngeal microbiota profiles and the pneumococcal carrier state and sociodemographic and environmental character- istics. The cluster Bacillus-Streptococcus was used as a reference since this cluster accounted for the high- est number of samples (Table  3). In winter, the naso- pharyngeal microbiota was less likely to be described by the Streptococcus-Acinetobacter cluster (OR = 0.18; 95% CI 0.02–0.85) and more likely to be described by the Corynebacterium-Moraxella cluster (OR = 28.1, 95% CI 4.4–307.2) compared to the reference. Males were more likely to have a nasopharyngeal microbiota described by clusters Corynebacterium-Moraxella (OR = 227.0, 95% CI 25.2–679.6) or Pseudomonas-Corynebacte- rium (OR = 12.5, 95% CI 1.5–310.3). Having contact with children increased the likelihood of having a nasopharyngeal microbiota described by the clus- ter Pseudomonas-Corynebacterium (OR = 15.6, 95% Characterization of the nasopharyngeal microbiota profilesii In each cluster, the relative abundance of the three most common genera is specified Fig. 3  Nasopharyngeal microbiota clusters. Taxonomic heat trees for the five clusters identified. A Bacillus-Streptococcus (48.2% of the total samples), B Corynebacterium-Moraxella in dark blue (16.3%), C Streptococcus-Acinetobacter (13.3%), D Streptococcus-Pseudomonas (12.0%), and E Pseudomonas-Corynebacterium (10.2%). The center of each tree represents the kingdom and has a relative abundance of 1. In the extremities, the relative abundances at the genus level are represented. From the center to the extremities, each taxonomic level from kingdom to genus is indicated. The gradient of colors represents relative abundance. In each cluster, the relative abundance of the three most common genera is specified Fig. 3  Nasopharyngeal microbiota clusters. Taxonomic heat trees for the five clusters identified. A Bacillus-Streptococcus (48.2% of the total samples), B Corynebacterium-Moraxella in dark blue (16.3%), C Streptococcus-Acinetobacter (13.3%), D Streptococcus-Pseudomonas (12.0%), and E Pseudomonas-Corynebacterium (10.2%). The center of each tree represents the kingdom and has a relative abundance of 1. In the extremities, the relative abundances at the genus level are represented. From the center to the extremities, each taxonomic level from kingdom to genus is indicated. The gradient of colors represents relative abundance. In each cluster, the relative abundance of the three most common genera is specified Characterization of the nasopharyngeal microbiota profilesii We identified five clusters in the nasopharynx and named them after the two most abundant genera found in each cluster. The most frequent cluster was named Bacillus- Streptococcus and included 48.2% (n = 80) of the total samples from the nasopharynx. The three most abundant genera in this cluster were Bacillus (27.7%), Streptococ- cus (14.3%), and Moraxella (8.7%) (Fig. 3A). This cluster Fig. 2  Oropharynx and nasopharynx microbiota profiles. Dendrogram showing the clusters identified by hierarchical cluster analysis performed on the Euclidean distance of the centered log-ratio transformed data (Aitchison distance). The gray lines surrounding clades represent the clusters identified by the Calinski and Harabasz index. Clusters inside the gray rectangle are the oropharyngeal clusters merged after cross-validation by random forest analysis. Bars below the dendrogram indicate clusters, samples in which pneumococcus was identified, and sampling site Fig. 2  Oropharynx and nasopharynx microbiota profiles. Dendrogram showing the clusters identified by hierarchical cluster analysis performed on the Euclidean distance of the centered log-ratio transformed data (Aitchison distance). The gray lines surrounding clades represent the clusters identified by the Calinski and Harabasz index. Clusters inside the gray rectangle are the oropharyngeal clusters merged after cross-validation by random forest analysis. Bars below the dendrogram indicate clusters, samples in which pneumococcus was identified, and sampling site Fig. 2  Oropharynx and nasopharynx microbiota profiles. Dendrogram showing the clusters identified by hierarchical cluster analysis performed on the Euclidean distance of the centered log-ratio transformed data (Aitchison distance). The gray lines surrounding clades represent the clusters identified by the Calinski and Harabasz index. Clusters inside the gray rectangle are the oropharyngeal clusters merged after cross-validation by random forest analysis. Bars below the dendrogram indicate clusters, samples in which pneumococcus was identified, and sampling site Paulo et al. Microbiome (2023) 11:199 Page 8 of 20 Paulo et al. Microbiome Fig. 3  Nasopharyngeal microbiota clusters. Taxonomic heat trees for the five clusters identified. A Bacillus-Streptococcus (48.2% of the total samples), B Corynebacterium-Moraxella in dark blue (16.3%), C Streptococcus-Acinetobacter (13.3%), D Streptococcus-Pseudomonas (12.0%), and E Pseudomonas-Corynebacterium (10.2%). The center of each tree represents the kingdom and has a relative abundance of 1. In the extremities, the relative abundances at the genus level are represented. From the center to the extremities, each taxonomic level from kingdom to genus is indicated. The gradient of colors represents relative abundance. Nasopharyngeal profiles of subpopulations of pneumococcal carriers, adults who have close contact with children, and smokersf The Bacillus-Streptococcus profile was the most frequent and thus was used as a reference against which the other profiles were compared ZIGM models were fitted. The nasopharyngeal micro- biota between individuals identified as pneumococcal carriers and noncarriers showed significant differences (PERMANOVA, P = 0.001). Among pneumococcal carri- ers, four ASVs were found to be overrepresented (Fig. 4A, Table S2). These were identified as presumptive H. influ- enzae (ASV23), Fusobacterium nucleatum (ASV87), Parvimonas micra or Dialister spp. (ASV116), and S. pneumoniae, S. pseudopneumoniae, or S. mitis (ASV5). In addition, eight ASVs were found to be underrepresented. These were identified as presumptive Haemophilus para- haemolyticus or Actinobacillus spp. (ASV32), Staphylo- coccus lugdunensis (ASV159), and Staphylococcus aureus (ASV2970).h sputorum (ASV41), Corynebacterium propinquum or Corynebacterium pseudodiphtericum (ASV46), and D. pigrum or uncultured Alloiococcus spp. (ASV72).f ZIGM models were fitted. The nasopharyngeal micro- biota between individuals identified as pneumococcal carriers and noncarriers showed significant differences (PERMANOVA, P = 0.001). Among pneumococcal carri- ers, four ASVs were found to be overrepresented (Fig. 4A, Table S2). These were identified as presumptive H. influ- enzae (ASV23), Fusobacterium nucleatum (ASV87), Parvimonas micra or Dialister spp. (ASV116), and S. pneumoniae, S. pseudopneumoniae, or S. mitis (ASV5). In addition, eight ASVs were found to be underrepresented. These were identified as presumptive Haemophilus para- haemolyticus or Actinobacillus spp. (ASV32), Staphylo- coccus lugdunensis (ASV159), and Staphylococcus aureus (ASV2970).h Finally, differences between the nasopharyngeal microbiota of adults who had regular contact with children compared to adults who did not have fre- quent contact with children were also significant (PERMANOVA, P = 0.04). Among adults who had contact with children, five ASVs were overrepresented (Fig. 4C, Table S4). Of these, ASV5 ­(log2FC = 2.57), ASV23 ­(log2FC = 3.61), and ASV40 ­(log2FC = 3.69) had the highest FCs. The latter was identified as presump- tive F. nucleatum or Fusobacterium naviforme. ASV159 ­(log2FC =  − 1.51), ASV307 ­(log2FC =  − 1.62), and ASV402 ­(log2FC =  − 1.94) were found to be underrep- resented. These were identified as presumptive S. lug- dunensis (ASV159), Sphingomonas spp. (ASV307), and Dermacoccus nishinomiyaensis (ASV402). The nasopharyngeal microbiota between individuals identified as smokers and nonsmokers also showed sig- nificant differences (PERMANOVA, P = 0.001). Among smokers, seven ASVs were found to be overrepresented (Fig. 4B, Table S3). Of these, the three with the highest FCs were ASV1 ­(log2FC = 4.19), ASV10 ­(log2FC = 3.85), and ASV63 ­(log2FC = 1.88). Nasopharyngeal profiles of subpopulations of pneumococcal carriers, adults who have close contact with children, and smokersf These were identified as presumptive Bacillus spp. (ASV1), Burkholderia spp. (ASV10), and Burkholderia spp. or Paraburkholderia spp. (ASV63). Ten ASVs were found to be underrep- resented. Of these, ASV41 ­(log2FC =  − 2.05), ASV46 ­(log2FC =  − 3.64), and ASV72 ­(log2FC =  − 2.46) showed the lowest FCs (Fig. 4B, Table S3). These were identified as presumptive H. parahaemolyticus or Haemophilus Nasopharyngeal profiles of subpopulations of pneumococcal carriers, adults who have close contact with children, and smokersf CI 1.8–376.1), whereas being a smoker decreased the likelihood of having that cluster (OR = 0.13, 95% CI 0.02–0.72). Of note, Pseudomonas-Corynebacterium included only one pneumococcal carrier  (Table  3), which may indicate that this microbiota profile has a protective role against pneumococcal carriage. To identify which ASVs differed between the naso- pharyngeal microbiota based on the pneumococcal car- rier state, smoking habits, and contact with children, Paulo et al. Microbiome (2023) 11:199 Page 9 of 20 Table 3  Association between nasopharyngeal microbiota profiles and variables under study Ref, variable used as reference. NA, nonadmissible as there are no data in the reference. Bold indicates statistically significant results. The Bacillus-Streptococcus profile was the most frequent and thus was used as a reference against which the other profiles were compared Characteristics Microbiota profiles Bacillus- Streptococcus N = 80 Streptococcus- Pseudomonas N = 20 Streptococcus- Acinetobacter N = 22 Corynebacterium- Moraxella N = 27 Pseudomonas- Corynebacterium N = 17 n (%) n (%) ORadj (95% CI) n (%) ORadj (95% CI) n (%) ORadj (95% CI) n (%) ORadj (95% CI) Pneumococcal carrier, yes 21 (26.3) 3 (18.7) 0.26 (0.04–1.21) 3 (18.7) 0.22 (0.03–1.18) 5 (18.5) 0.71 (0.04–10.59) 1 (5.8) NA Age, > 37 years 43 (53.7) 9 (20.0) 0.64 (0.14–2.62) 9 (20.0) 0.58 (0.15–2.06) 7 (25.9) 0.68 (0.10–3.89) 8 (47.1) 1.08 (0.12–10.15) Gender, male 22 (27.5) 6 (37.5) 1.19 (0.35–3.77) 6 (37.5) 0.74 (0.21–2.40) 26 (96.3) 226.97 (25.16– 679.59) 14 (82.6) 12.52 (1.51– 310.27) Children, yes 41 (52.2) 7 (43.7) 4.70 (1.04–25.82) 7 (43.7) 0.60 (0.15–2.39) 7 (25.9) 0.24 (0.02–1.99) 10 (58.8) 15.59 (1.83– 376.07) Smoker, yes 39 (48.7) 7 (43.7) 0.56 (0.16–1.81) 7 (43.7) 0.41 (0.12–1.23) 11 (40.7) 0.65 (0.12–3.26) 4 (23.5) 0.13 (0.02–0.72) Season   Spring 28 (35.0) 12 (75.0) Ref 12 (75.0) Ref 3 (11.1) Ref 0 (0.0) Ref   Summer 22 (27.5) 8 (50.0) 3.54 (0.94–15.86) 8 (50.0) 0.78 (0.23–2.53) 2 (7.4) 0.50 (0.04–4.75) 4 (23.5) NA   Autumn 11 (13.7) 0 (0.0) 2.55 (0.38–16.32) 0 (0.0) NA 2 (7.4) 0.71 (0.06–7.38) 12 (70.1) NA   Winter 19 (23.8) 2 (12.5) 1.11 (0.21–5.76) 2 (12.5) 0.18 (0.02–0.85) 20 (74.1) 28.1 (4.38– 307.16) 1 (5.9) NA Table 3  Association between nasopharyngeal microbiota profiles and variables under study Ref, variable used as reference. NA, nonadmissible as there are no data in the reference. Bold indicates statistically significant results. Diversity of nasopharyngeal profiles of subpopulations of pneumococcal carriers, adults who have close contact with children, and smokersi Diversity, at the taxonomic level of genus, was signifi- cantly lower in the nasopharynx of pneumococcal carri- ers (0D = 37.6, 1D = 4.3, 2D = 2.8) than in the nasopharynx of noncarriers (0D = 49.7, 1D = 7.1, 2D = 4.3) for each diversity number (P = 0.037, P = 0.003, and P = 0.006, respectively) (Fig. 5A and Fig. S5A). In addition, the Page 10 of 20 Paulo et al. Microbiome (2023) 11:199 Paulo et al. Microbiome Fig. 4  Volcano plots representing ASVs that showed differential abundance in the nasopharynx. A Effect of pneumococcal carrier status. B Effect of smoking status. C Effect of having contact with children. Bacterial taxa overrepresented among pneumococcal carriers, smokers, and adults who have regular contact with children are represented by red circles on the right side of each corresponding plot. Bacterial taxa underrepresented among pneumococcal carriers, smokers, and adults who have regular contact with children are represented by green circles on the left side of each corresponding plot. Gray circles indicate bacterial taxa that were not differentially abundant Fig. 4  Volcano plots representing ASVs that showed differential abundance in the nasopharynx. A Effect of pneumococcal carrier status. B Effect of smoking status. C Effect of having contact with children. Bacterial taxa overrepresented among pneumococcal carriers, smokers, and adults who have regular contact with children are represented by red circles on the right side of each corresponding plot. Bacterial taxa underrepresented among pneumococcal carriers, smokers, and adults who have regular contact with children are represented by green circles on the left side of each corresponding plot. Gray circles indicate bacterial taxa that were not differentially abundant Fig. 4  Volcano plots representing ASVs that showed differential abundance in the nasopharynx. A Effect of pneumococcal carrier status. B Effect of smoking status. C Effect of having contact with children. Bacterial taxa overrepresented among pneumococcal carriers, smokers, and adults who have regular contact with children are represented by red circles on the right side of each corresponding plot. Bacterial taxa underrepresented among pneumococcal carriers, smokers, and adults who have regular contact with children are represented by green circles on the left side of each corresponding plot. Gray circles indicate bacterial taxa that were not differentially abundant of all genera (summing up to more than 50% of all genera in only 3% of noncarriers) (Fig. 5D − E and Fig. Diversity of nasopharyngeal profiles of subpopulations of pneumococcal carriers, adults who have close contact with children, and smokersi S6).f of all genera (summing up to more than 50% of all genera in only 3% of noncarriers) (Fig. 5D − E and Fig. S6). Only marginal differences between the diversity exhib- ited by the nasopharynx of smokers (0D = 42.7, 1D = 5.4, 2D = 3.4) compared to nonsmokers (0D = 50.2, 1D = 7.3, 2D = 4.3) were found when comparing each diversity number (P = 0.063, P = 0.019, and P = 0.032, respectively) (Fig. 5B and Fig. S5B), and no significant differences were observed when the diversity exhibited by the micro- biota of the nasopharynx of individuals who had regular contact with children (0D = 45.4, 1D = 5.9, 2D = 3.6) was nasopharynx microbiota of pneumococcal carriers was less even than the nasopharynx microbiota of noncarri- ers, supporting the higher dominance of the most abun- dant species found in this niche (Fig. S5A).i Only marginal differences between the diversity exhib- ited by the nasopharynx of smokers (0D = 42.7, 1D = 5.4, 2D = 3.4) compared to nonsmokers (0D = 50.2, 1D = 7.3, 2D = 4.3) were found when comparing each diversity number (P = 0.063, P = 0.019, and P = 0.032, respectively) (Fig. 5B and Fig. S5B), and no significant differences were observed when the diversity exhibited by the micro- biota of the nasopharynx of individuals who had regular contact with children (0D = 45.4, 1D = 5.9, 2D = 3.6) was In parallel, the proportion of reads classified as Strep- tococcus in the nasopharynx of pneumococcal carri- ers compared to noncarriers was significantly higher (P < 0.001); among carriers, Streptococcus accounted for much as 30% on average of all genera (summing up to more than 50% of all genera in 29% of carriers); among noncarriers, Streptococcus accounted, on average, for 10% Paulo et al. Microbiome (2023) 11:199 Page 11 of 20 Paulo et al. Microbiome Microbiome (2023) 11:199 versity profiles. A Diversity of nasopharyngeal and oropharyngeal microbiota given by Hill numbers of order 0 to 4 in pneum nd noncarriers. B Diversity of nasopharyngeal and oropharyngeal microbiota given by the Hill numbers of order 0 to 4 of smo mokers. C Diversity of nasopharyngeal and oropharyngeal microbiota given by the Hill numbers of order 0 to 4 of individuals ontact with children and those who do not have. Characterization of oropharyngeal microbiota clustersh Characterization of oropharyngeal microbiota clusters The two oropharyngeal microbiota clusters were named Prevotella-Streptococcus and Neisseria-Fusobacterium. The Prevotella-Streptococcus cluster accounted for 83.9% (n = 151) of all samples from the oropharynx. The three most abundant genera in this cluster were Prevotella (20.2%), Streptococcus (16.3%), and Veillonella (10.3%) (Fig. 6A). The Neisseria-Fusobacterium cluster accounted for 16.1% (n = 29) of all samples, and the three most abundant genera were Neisseria (15.9%), Fusobacterium (12.1%), and Streptococcus (10.9%) (Fig. 6B). Both clus- ters had comparable diversity (Fig. S4B) and a high abun- dance of ASV2, classified as presumptive Streptococcus spp., which included, among other species, presumptive S. pneumoniae (supplementary information “Bacterial profiles in the oropharynx and nasopharynx”). Table 4  Association between oropharyngeal microbiota profiles and variables under study Table 4  Association between oropharyngeal microbiota profiles and variables under study Ref, variable used as reference. Bold indicates statistically significant results. The Prevotella-Streptococcus profile was the most frequent and thus was used as a reference against which the other profile was compared Characteristics Microbiota profiles Prevotella- Streptococcus N = 142 Neisseria-Fusobacterium N = 29 n (%) n (%) ORadj (95% CI) Pneumococcal carrier, yes 24 (16.9) 11 (37.9) 3.6 (1.3–12.1) Age, > 37 years 73 (51.4) 7 (24.1) 0.4 (0.1–1.1) Gender, male 66 (46.5) 14 (48.4) 2.1 (0.4–3.0) Children, yes 70 (49.3) 12 (41.4) 0.6 (0.2–1.9) Smoker, yes 67 (47.2) 4 (13.7) 0.14 (0.04–0.4) Season   Spring 40 (28.2) 6 (20.7) Ref   Summer 40 (28.2) 8 (27.6) 1.56 (0.5–5.6)   Autumn 23 (16.2) 5 (17.2) 1.93 (0.4–9.0)   Winter 39 (27.4) 10 (34.5) 1.23 (0.4–4.4) Diversity of nasopharyngeal profiles of subpopulations of pneumococcal carriers, adults who have close contact with children, and smokersi D Average relative abundance of the ten most frequent genera found in the opharynx represented by stacked bar plots. The remaining less abundant genera were grouped as a single bar (other). E Abu most abundant genera found in the nasopharynx of carriers and noncarriers. F Average relative abundance of the ten most und in the microbiota of the oropharynx represented by stacked bar plots. The remaining less abundant genera were groupe e bar (other). G Abundance of the ten most abundant genera found in the oropharynx of carriers and noncarriers. P-values de coxon rank-sum test Fig. 5  Diversity profiles. A Diversity of nasopharyngeal and oropharyngeal microbiota given by Hill numbers of order 0 to 4 in pneumococcal carriers and noncarriers. B Diversity of nasopharyngeal and oropharyngeal microbiota given by the Hill numbers of order 0 to 4 of smokers and nonsmokers. C Diversity of nasopharyngeal and oropharyngeal microbiota given by the Hill numbers of order 0 to 4 of individuals who have regular contact with children and those who do not have. D Average relative abundance of the ten most frequent genera found in the microbiota of the nasopharynx represented by stacked bar plots. The remaining less abundant genera were grouped as a single bar (other). E Abundance of the ten most abundant genera found in the nasopharynx of carriers and noncarriers. F Average relative abundance of the ten most frequent genera found in the microbiota of the oropharynx represented by stacked bar plots. The remaining less abundant genera were grouped as a single bar (other). G Abundance of the ten most abundant genera found in the oropharynx of carriers and noncarriers. P-values determined by the Wilcoxon rank-sum test Paulo et al. Microbiome (2023) 11:199 Page 12 of 20 compared with those without regular contact (0D = 48.4, 1D = 6.9, 2D = 4.1) (Fig. 5C and Fig. S5C). status of the oropharynx and sociodemographic and environmental characteristics. The cluster Prevotella- Streptococcus was used as a reference since this cluster accounted for the highest number of samples (Table 4). Association between oropharyngeal microbiota profiles and variables under study Mixed general linear models were used to investi- gate potential associations between the oropharyn- geal microbiota profiles and the pneumococcal carrier Ref, variable used as reference. Bold indicates statistically significant results. The Prevotella-Streptococcus profile was the most frequent and thus was used as a reference against which the other profile was compared Ref, variable used as reference. Bold indicates statistically significant results. The Prevotella-Streptococcus profile was the most frequent and thus was used as a reference against which the other profile was compared Fig. 6  Oropharyngeal microbiota clusters. Taxonomic heat trees for the two clusters identified. A Prevotella-Streptococcus (83.9% of the total samples) and B Neisseria-Fusobacterium (16.1%). The center of each tree represents the kingdom and has a relative abundance of 1. In the extremities, the relative abundances at the genus level are represented. From the center to the extremities, each taxonomic level from kingdom to genus is indicated. The gradient of colors represents relative abundance. In each cluster, the relative abundance of the three most common genera is specified Fig. 6  Oropharyngeal microbiota clusters. Taxonomic heat trees for the two clusters identified. A Prevotella-Streptococcus (83.9% of the total samples) and B Neisseria-Fusobacterium (16.1%). The center of each tree represents the kingdom and has a relative abundance of 1. In the extremities, the relative abundances at the genus level are represented. From the center to the extremities, each taxonomic level from kingdom to genus is indicated. The gradient of colors represents relative abundance. In each cluster, the relative abundance of the three most common genera is specified Fig. 6  Oropharyngeal microbiota clusters. Taxonomic heat trees for the two clusters identified. A Prevotella-Streptococcus (83.9% of the total samples) and B Neisseria-Fusobacterium (16.1%). The center of each tree represents the kingdom and has a relative abundance of 1. In the extremities, the relative abundances at the genus level are represented. From the center to the extremities, each taxonomic level from kingdom to genus is indicated. The gradient of colors represents relative abundance. In each cluster, the relative abundance of the three most common genera is specified Page 13 of 20 Paulo et al. Microbiome (2023) 11:199 carriers were found using PERMANOVA (P = 0.002). Eleven ASVs were overrepresented among pneumococ- cal carriers (Fig. 7A, Table S5), with ASV5 ­(log2FC = 4.31) and ASV281 ­(log2FC = 2.63) showing the highest FC. The latter was identified as Lachnospiraceae. Oropharyngeal profiles of subpopulations Oropharyngeal profiles of subpopulations Association between oropharyngeal microbiota profiles and variables under study On the other hand, ASV44 ­(log2FC =  − 1.85) and ASV86 ­(log2FC =  − 2.49) were underrepresented (Fig. 7A, Table S5). These were identified as presumptive Leptotrichia spp. and Alloprevotella tannerae, respectively. Pneumococcal carriers were 3.6-fold (95% CI 1.3–12.1) more likely to have their oropharyngeal microbiota described by cluster Neisseria-Fusobacterium com- pared to Prevotella-Streptococcus, whereas smokers were 86% less likely to have their oropharyngeal micro- biota described by cluster Neisseria-Fusobacterium (95% CI 0.04–0.4). Discussion Few metagenomic studies focusing on adults have been published, with the majority about microbiota dysbiosis in relation to disease [15, 35, 36]. Here, we took advan- tage of a longitudinal study conducted among immuno- competent healthy adults aged between 25 and 50 years old [9] to study the nasopharyngeal and oropharyngeal microbiota. In addition, we also compared the nasophar- ynx and oropharynx microbiota based on S. pneumoniae carrier status, smoking habits, and regular contact with children.f We found several differences between the microbiota of the nasopharynx vs. the oropharynx. We observed a higher bacterial load in oropharyngeal samples and a more homogeneous microbiota across individuals with just two clusters compared to five clusters identified in the nasopharynx. These observations are in line with a study that shows that the oropharynx has a high bacterial load, and that it varies little between individuals [37].h Diversity of oropharyngeal profiles of subpopulations of pneumococcal carriers, adults who have close contact with children, and smokersif Diversity at the genus level was not significantly differ- ent when the oropharyngeal microbiota of pneumococcal carriers (0D = 47.3, 1D = 11.7, 2D = 7.4) and noncarriers (0D = 44.9, 1D = 11.2, 2D = 7.1) were compared (Fig. 5A and Fig. S7A). There was also no difference (P = 0.129) between the average proportion of reads belonging to the genus Streptococcus found in the oropharynx of carriers (19.8%) vs. noncarriers (14.4%) (Fig. 5F − G and Fig. S6). In contrast, the oropharynx of smokers (0D = 43.5, 1D = 10.2, 2D = 6.3) was significantly less diverse than the oropharynx of nonsmokers (0D = 46.7, 1D = 12.2, 2D = 7.8) when comparing each diversity number (P = 0.095, P = 0.010, and P = 0.010, respectively) (Fig. 5B and Fig. S7B). Finally, no significant differences were observed between the oropharyngeal microbiota of indi- viduals who had regular contact with children (0D = 45.1, 1D = 11.3, 2D = 7.0) and those who did not (0D = 45.6, 1D = 11.3, 2D = 7.2) (Fig. 5C and Fig. S7C). Diversity at the genus level was not significantly differ- ent when the oropharyngeal microbiota of pneumococcal carriers (0D = 47.3, 1D = 11.7, 2D = 7.4) and noncarriers (0D = 44.9, 1D = 11.2, 2D = 7.1) were compared (Fig. 5A and Fig. S7A). There was also no difference (P = 0.129) between the average proportion of reads belonging to the genus Streptococcus found in the oropharynx of carriers (19.8%) vs. noncarriers (14.4%) (Fig. 5F − G and Fig. S6). The oropharyngeal microbiota and the nasopharynx revealed continuity and niche-specific characteristics: the bacteria thriving in the oropharynx were obliga- tory anaerobes (e.g., Prevotellaceae, Veillonellaceae, or Leptotrichiaceae), whereas the bacteria thriving in the nasopharynx were mostly facultative anaerobes (e.g., Moraxellaceae and Corynebacteriaceae). Streptococ- caceae, on the other hand, were common in both sites.i In contrast, the oropharynx of smokers (0D = 43.5, 1D = 10.2, 2D = 6.3) was significantly less diverse than the oropharynx of nonsmokers (0D = 46.7, 1D = 12.2, 2D = 7.8) when comparing each diversity number (P = 0.095, P = 0.010, and P = 0.010, respectively) (Fig. 5B and Fig. S7B). of pneumococcal carriers, adults who have close contact with children, and smokers Of these, ASV130 ­(log2FC =  − 3.06), ASV136 ­(log2FC =  − 3.30), and ASV269 ­(log2FC =  − 3.59) showed the lowest FCs and were identified as presumptive Campylobacter showae or Campylobacter rectus, Lep- totrichia spp., and Mollicutes, respectively. were ASV1 ­(log2FC = 4.08) and ASV10 ­(log2FC = 4.17). Fifteen ASVs were underrepresented among smok- ers. Of these, ASV130 ­(log2FC =  − 3.06), ASV136 ­(log2FC =  − 3.30), and ASV269 ­(log2FC =  − 3.59) showed the lowest FCs and were identified as presumptive Campylobacter showae or Campylobacter rectus, Lep- totrichia spp., and Mollicutes, respectively. carrying pneumococci (41.6% vs. 8.8%, chi-square test, P = 0.015), suggesting a higher stability in the former case (Fig. 8A). This result was supported by a lower volatility of the nasopharyngeal microbiota of pneumococcal car- riers vs. noncarriers (Wilcoxon rank-sum test, P < 0.001) (Fig. 8C). In the oropharynx, this was not observed (Fig. 8D). Other factors, such as having contact with chil- dren, being a smoker, gender, age, and season, did not impact the dynamics of carriage. pp p y Finally, differences between the oropharyngeal micro- biota of individuals who had regular contact with chil- dren compared to those who did not (P = 0.004) were observed. Ten ASVs were overrepresented in individu- als who had contact with children (Fig. 7C, Table S7), with ASV32 ­(log2FC = 2.62) and ASV40 ­(log2FC = 2.54) showing the highest FC. On the other hand, four ASVs were underrepresented (Fig. 7C, Table S7): ASV112 ­(log2FC =  − 2.32), ASV121 ­(log2FC =  − 1.87), ASV170 ­(log2FC =  − 1.60), and ASV283 ­(log2FC =  − 1.60). These were identified as presumptive Porphyromonas gingi- valis or Capnocytophaga spp., Alloprevotella rava or Prevotella spp., Prevotella melaninogenica, and Neisse- ria spp., respectively. Diversity of oropharyngeal profiles of subpopulations of pneumococcal carriers, adults who have close contact with children, and smokersif Finally, no significant differences were observed between the oropharyngeal microbiota of indi- viduals who had regular contact with children (0D = 45.1, 1D = 11.3, 2D = 7.0) and those who did not (0D = 45.6, 1D = 11.3, 2D = 7.2) (Fig. 5C and Fig. S7C). In the five nasopharyngeal microbiota clusters, the genus Streptococcus was one of the most abundant gen- era in three of these clusters, and the genus Corynebac- terium was the most abundant in the remaining two clusters. Of note, although these genera showed high abundances, they were never equally abundant in the same cluster. This result agrees with the observation of antagonistic relationships between species of these genera. For example, it has been shown that C. accolens is able to produce lipases and modify triacylglycerols present in the human skin, including the human nos- trils, into free fatty acids, thus inhibiting the growth of S. pneumoniae [38]. A healthy nasopharyngeal micro- biota has been frequently associated with Corynebac- terium, Dolosigranulum, and/or Moraxella-dominated profiles [39–41], which coincides with two of the of pneumococcal carriers, adults who have close contact with children, and smokers Differences between the oropharyngeal microbiota of smokers and nonsmokers were also observed (P = 0.001). Five ASVs were overrepresented among smokers (Fig. 7B, Table S6). Among these, the ones with the highest FC Significant differences between the oropharyngeal micro- biota of pneumococcal carriers and nonpneumococcal Fig. 7  Volcano plots representing ASVs that showed differential abundance in the oropharynx. A Effect of pneumococcal carrier status. B Effect of smoking status. C Effect of having contact with children. Bacterial taxa overrepresented among pneumococcal carriers, smokers, and adults who have regular contact with children are represented by red circles on the right side of each corresponding plot. Bacterial taxa underrepresented among pneumococcal carriers, smokers, and adults who have regular contact with children are represented by green circles on the left side of each corresponding plot. Gray circles indicate bacterial taxa that were not differentially abundant Fig. 7  Volcano plots representing ASVs that showed differential abundance in the oropharynx. A Effect of pneumococcal carrier status. B Effect of smoking status. C Effect of having contact with children. Bacterial taxa overrepresented among pneumococcal carriers, smokers, and adults who have regular contact with children are represented by red circles on the right side of each corresponding plot. Bacterial taxa underrepresented among pneumococcal carriers, smokers, and adults who have regular contact with children are represented by green circles on the left side of each corresponding plot. Gray circles indicate bacterial taxa that were not differentially abundant Fig. 7  Volcano plots representing ASVs that showed differential abundance in the oropharynx. A Effect of pneumococcal carrier status. B Effect of smoking status. C Effect of having contact with children. Bacterial taxa overrepresented among pneumococcal carriers, smokers, and adults who have regular contact with children are represented by red circles on the right side of each corresponding plot. Bacterial taxa underrepresented among pneumococcal carriers, smokers, and adults who have regular contact with children are represented by green circles on the left side of each corresponding plot. Gray circles indicate bacterial taxa that were not differentially abundant Paulo et al. Microbiome (2023) 11:199 Page 14 of 20 Page 14 of 20 were ASV1 ­(log2FC = 4.08) and ASV10 ­(log2FC = 4.17). Fifteen ASVs were underrepresented among smok- ers. Dynamics of microbiota carriage While 71.2% of the individuals maintained the same oropharyngeal cluster across the three time points, only 15.7% maintained the same nasopharyngeal cluster (chi- square test, P < 0.001) (Fig. 8A − B). On average, there was a higher volatility in the nasopharynx than in the oro- pharynx (Fig. 8C − D). In addition, in the nasopharynx, individuals carrying pneumococci were more likely to maintain the same nasopharyngeal cluster than those not Page 15 of 20 Paulo et al. Microbiome (2023) 11:199 Paulo et al. Microbiome Fig. 8  Dynamics of the nasopharyngeal and oropharyngeal microbiota. A Alluvial plot representing the change in individuals’ nasopharyngeal clusters, stratified by carriage of pneumococcus, at the three sampling times. B Alluvial plot representing the change in individuals’ oropharyngeal clusters, stratified by carriage of pneumococcus, at the three sampling times. C Volatility of the nasopharyngeal microbiota depending on pneumococcal carrier state. D Volatility of the oropharyngeal microbiota depending on pneumococcal carrier state. In C and D, left graphics show volatility per individual calculated as the Aitchison distance between microbiota at consecutive time points: first vs. second and second vs. third. Lines connect volatility values of each individual. In C and D, the graphics on the right show boxplots of aggregated volatility. Yellow indicates pneumococcal carriers; blue indicates pneumococcal noncarriers Fig. 8  Dynamics of the nasopharyngeal and oropharyngeal microbiota. A Alluvial plot representing the change in individuals’ nasopharyngeal clusters, stratified by carriage of pneumococcus, at the three sampling times. B Alluvial plot representing the change in individuals’ oropharyngeal clusters, stratified by carriage of pneumococcus, at the three sampling times. C Volatility of the nasopharyngeal microbiota depending on pneumococcal carrier state. D Volatility of the oropharyngeal microbiota depending on pneumococcal carrier state. In C and D, left graphics show volatility per individual calculated as the Aitchison distance between microbiota at consecutive time points: first vs. second and second vs. third. Lines connect volatility values of each individual. In C and D, the graphics on the right show boxplots of aggregated volatility. Yellow indicates pneumococcal carriers; blue indicates pneumococcal noncarriers identified clusters in this study: cluster Corynebacte- rium-Moraxella and cluster Pseudomonas-Corynebac- terium, which, together, were observed in 26.5% of the samples identified in the nasopharyngeal micro- biota clusters. Nonetheless, in this study, the majority (73.5%) of nasopharyngeal microbiota samples were represented by clusters codominated by Streptococcus. Dynamics of microbiota carriage This raises the possibility of a specialization of bacteria able to thrive in this niche. We have also looked for differences in the upper res- piratory tract microbiota based on two population char- acteristics that we know from our previous study [9] to be important for pneumococcal acquisition, namely, smok- ing habits and having contact with children. These charac- teristics were associated with differences in the microbiota in both niches. Although different, both the nasopharynx and oropharynx of smokers showed high abundances of Bacillus (ASV1 and ASV48) and Burkholderia (ASV10 and ASV63). These genera comprise a high range of human pathogenic species and have been frequently asso- ciated with environmental contamination. Nonetheless, in this study, they were spread across individuals and were most likely present due to the high proportion of indi- viduals with smoking habits in our sample. In fact, both genera have been reported as being part of the bacterial metagenome of cigarettes, providing evidence that the source of these pathogenic bacteria may be the cigarettes themselves [57]. Furthermore, we also found that Rothia dentocariosa (ASV73), Prevotella melaninogenica (ASV3 and ASV18), and Veillonella atypica (ASV4) were present in high abundance in the nasopharyngeal microbiota and Selenomonas sputigena (ASV348) in the oropharyngeal microbiota of smokers. These are all bacterial taxa usually found to be associated with oral diseases such as caries [58–61], which may be expected in smokers [62]. The presence of S. pneumoniae in the oropharyngeal niche seems not to disrupt the normal microbiota since the microbiota of both carriers and noncarriers are very similar. As reported, the oropharynx showed higher bac- terial diversity, and as an ecosystem, a higher diversity contributes to niche stability [45]. There are several reports of a synergistic relationship between pneumococcus and H. influenzae [46]. Both bacteria are part of the nasopharyngeal niche of healthy humans. However, these are also pathobionts that can cause several infections, such as bronchitis, pneumonia, otitis media, septicemia, and meningitis. To date, it is not yet known whether this interaction is strain and/or sero- type-specific or their molecular mechanisms [47]. Cope et  al. [48] showed that biofilms with both species had higher cell densities, and that these bacteria can modu- late each other’s virulence gene expression, leading to a persistent biofilm. Aside from this interaction, Horiuchi et al. [49] also reported a synergistic interaction between P. micra and F. nucleatum. Dynamics of microbiota carriage As the participants in the study were healthy individu- als, this suggests a broader range of clusters associated with a healthy state. Since season (winter or sum- mer) was associated with two clusters codominated by Corynebacterium and Streptococcus, we hypothesized that nasopharyngeal clusters may be very dynamic and may shift between clusters codominated by different genera. An alternation between different nasopharyn- geal microbiota profiles in children due to changes associated with seasonality was previously described for healthy youth and infants, which further supports our own observations [42, 43]. In the oropharynx, we found only two microbiota clus- ters. The most abundant genera comprised Prevotella, Streptococcus, Neisseria, and Fusobacterium, which have already been described in the healthy oropharyngeal microbiota of adults [1, 44]. We found that the microbiota composition of the naso- pharynx and oropharynx could depend on population demographic characteristics (i.e., age and gender) and/or environmental factors (i.e., smoking habits, contact with Paulo et al. Microbiome (2023) 11:199 Paulo et al. Microbiome (2023) 11:199 Paulo et al. Microbiome (2023) 11:199 Page 16 of 20 Page 16 of 20 Along with Streptococcus (ASV5), we found other bac- terial taxa associated with the oropharyngeal microbiota of pneumococcal carriers. These were either Lachno- spiraceae (ASV281) or Mollicutes (ASV269), which are frequently identified as being part of a healthy microbiota [53]. However, Capnocytophaga (ASV114 and ASV192), Oribacterium (ASV297), and Tannerella (ASV306) were also detected. These genera are frequently associated with diseases such as periodontitis [54]. children, and season). Indeed, we found that by compar- ing to a reference microbiota profile, there were two out of five nasopharyngeal microbiota profiles that could be associated with pneumococcal carrier state, smoking status, contact with children, sampling season, and gen- der. Regarding the oropharyngeal microbiota profiles, we found that the two clusters were associated with pneu- mococcal carrier status, smoking habits, and age. These results are in line with previous studies that also found that demographic characteristics and environmental fac- tors can affect the microbiota of the upper respiratory tract [1, 42]. The oropharyngeal microbiota of nonpneumococcal carriers comprised a higher abundance of genera such as Alloprevotella (ASV86) and Leptotrichia (ASV44). The first was found in the human oral cavity [55], whereas the latter was found to be present in the oropharynx of healthy adults [13, 56]. We observed that the nasopharyngeal microbiota among pneumococcal carriers had a lower evenness than that among nonpneumococcal carriers. Conclusions In conclusion, our study revealed notable differences between the nasopharyngeal and oropharyngeal micro- biota, with the nasopharyngeal niche exhibiting lower diversity. The presence of S. pneumoniae in the naso- pharyngeal niche led to a microbiota shift not observed at the genus level in the oropharyngeal niche. Moreo- ver, we identified various bacterial taxa that differ in prevalence between pneumococcal carriers and non- carriers, indicating potential interactions influencing the microbiota composition. Although some of these interactions are known, there may be additional uni- dentified factors playing a crucial role. For instance, P. micra was present in both the nasopharyngeal and oro- pharyngeal microbiota of pneumococcal carriers, hint- ing at intricate relationships yet to be fully elucidated. Additionally, our study highlighted differences in the upper respiratory tract microbiota based on smoking status and contact with children. Smokers’ microbiota exhibited an excess of pathogenic bacteria often found in cigarette metagenomes and are associated with peri- odontal diseases. Adults with contact with children showed higher abundances of pathobionts frequently found in children, such as Streptococcus, H. influenzae, and M. catarrhalis. In terms of dynamics, our results are in agreement with others that showed that the microbiota of the oro- pharynx is stable [37]. In addition, we observed that the nasopharyngeal microbiota of adults is much less stable. However, pneumococcal carriers tend to have a more stable nasopharyngeal microbiota than noncarriers. This may be the result of S. pneumoniae dominance, reflected by the lower evenness in the nasopharyngeal community of S. pneumoniae carriers. Our study has some limitations. First, the original study aimed to investigate the dynamics of carriage of S. pneu- moniae in immunocompetent healthy adults; therefore, STGG medium was used to store the samples. Although this may not be the ideal medium for such studies, it has been successfully used and validated previously by others [24]. Second, we were unable to use STGG as a negative control in our analyses, as no aliquots from the original study had been stored. Nevertheless, several unsupervised methods have been used to remove possible contami- nants. Also, the current comparison of the upper respira- tory microbiota based on the S. pneumoniae carrier state was performed exclusively based on the previous identifi- cation of this bacterium by culture methods and/or qPCR [1]. Dynamics of microbiota carriage This type of interaction may explain the increased abundance of these specific ASVs in pneumococcal carriers. On the other hand, the nasopharyngeal microbiota of the nonpneumococcal carriers also showed several bac- terial taxa that were overrepresented or even unique in the nasopharyngeal microbiota. Among these were Neisseria spp. (ASV24, ASV57, and ASV131), S. aureus (ASV2970), and S. lugdunensis (ASV159), for example. Several reports have observed a negative relationship between pneumococcus and S. aureus and identified mechanisms possibly associated with it [50, 51]. Addi- tionally, Brozyna et al. observed that S. aureus is able to enhance the growth of S. lugdunensis [52]. Finally, the upper respiratory tract microbiota of adults with contact with children was found to be different from that of adults without contact with children. For exam- ple, Streptococcus (ASV5), M. catarrhalis (ASV7), and H. influenzae (ASV23) were overrepresented in the naso- pharyngeal microbiota of individuals who have contact with children. Interestingly, these are the most common pathobionts known to colonize the upper respiratory tract of children [18, 46]. These bacteria are capable of Paulo et al. Microbiome (2023) 11:199 Page 17 of 20 respiratory tract regarding immunocompetent healthy adults. Second, it is the first study that aimed to under- stand the impact of S. pneumoniae colonization on the microbiota of both the nasopharyngeal and oropharyn- geal niches. causing infections such as bronchitis, otitis media, sinusi- tis, and pneumonia in both children and adults, although they are more frequent in the first age group [1]. Thus, colonization and, consequently, infection in young adults, albeit low, may be due to the transmission of these bacte- ria through contact with children. The oropharyngeal microbiota of adults with and with- out contact with children, as expected, was found to be mostly represented by genera already described as part of this niche [39]. Examples of these genera were Fuso- bacterium (ASV40), Leptotrichia (ASV418), Haemo- philus (ASV155 and ASV106), Veillonella (ASV275), Prevotella (ASV121, ASV170 and ASV163), and Neisseria (ASV283). Although we observed an increase in several pathobionts in the nasopharyngeal microbiota of indi- viduals who have regular contact with children, regard- ing the oropharyngeal microbiota, this increase was only noticeable for Streptococcus (ASV5). Received: 28 April 2023 Accepted: 4 August 2023 Received: 28 April 2023 Accepted: 4 August 2023 Received: 28 April 2023 Accepted: 4 August 2023 Funding h k g This work was supported by Fundação para a Ciência e a Tecnologia (FCT), I.P., through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020). JL and STA were supported by FCT grants UI/BD/153385/2022 and SFRH/BD/108380/2015, respectively. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. 9. Almeida ST, Paulo AC, Froes F, de Lencastre H, Sá-Leão R. Dynamics of pneumococcal carriage in adults: a new look at an old paradigm. J Infect Dis. 2021;223(9):1590–600. https://​doi.​org/​10.​1093/​infdis/​jiaa5​58. 10. Sá-Leão R, Nunes S, Brito-Avô A, Alves CR, Carriço JA, Saldanha J, et al. High rates of transmission of and colonization by Streptococcus pneumo- niae and Haemophilus influenzae within a day care center revealed in a longitudinal study. J Clin Microbiol. 2008;46(1):225–34. https://​doi.​org/​10.​ 1128/​JCM.​01551-​07. Availability of data and materials Metagenomic sequencing data as well as meta information were deposited at NCBI’s Sequence Read Archive under the BioProject accession number PRJNA962709. 11. Almeida ST, Nunes S, Santos Paulo AC, Valadares I, Martins S, Breia F, et al. Low prevalence of pneumococcal carriage and high serotype and geno‑ type diversity among adults over 60 years of age living in Portugal. PLoS One. 2014;9(3):90974. https://​doi.​org/​10.​1371/​journ​al.​pone.​00909​74. 12. Greenberg D, Givon-Lavi N, Broides A, Blancovich I, Peled N, Dagan R. The contribution of smoking and exposure to tobacco smoke to Streptococcus pneumoniae and Haemophilus influenzae carriage in children and their moth‑ ers. Clin Infect Dis. 2006;42(7):897–903. https://​doi.​org/​10.​1086/​500935. References 1. Man WH, de Steenhuijsen Piters WA, Bogaert D. The microbiota of the respiratory tract: gatekeeper to respiratory health. Nat Rev Microbiol. 2017;15(5):259–70. https://​doi.​org/​10.​1038/​nrmic​ro.​2017.​14. 1. Man WH, de Steenhuijsen Piters WA, Bogaert D. The microbiota of the respiratory tract: gatekeeper to respiratory health. Nat Rev Microbiol. 2017;15(5):259–70. https://​doi.​org/​10.​1038/​nrmic​ro.​2017.​14. 2. Henriques-Normark B, Tuomanen EI. The pneumococcus: epidemiol‑ ogy, microbiology, and pathogenesis. Cold Spring Harb Perspect Med. 2013;3(7):a010215. https://​doi.​org/​10.​1101/​cshpe​rspect.​a0102​15. 3. Brooks LRK, Mias GI. Virulence and host immunity: aging, diagnostics, and prevention. Front Immunol. 2018;9:1366. https://​doi.​org/​10.​3389/​fimmu.​ 2018.​01366. 4. Siegel SJ, Weiser JN. Mechanisms of bacterial colonization of the respira‑ tory tract. Annu Rev Microbiol. 2015;69:425–44. https://​doi.​org/​10.​1146/​ annur​ev-​micro​091014-​104209. 5. Nunes S, Félix S, Valente C, Simões AS, Tavares DA, Almeida ST, et al. The impact of private use of PCV7 in 2009 and 2010 on serotypes and antimi‑ crobial resistance of Streptococcus pneumoniae carried by young children in Portugal: comparison with data obtained since 1996 generating a 15-year study prior to PCV13 introduction. Vaccine. 2016;34(14):1648–56. https://​doi.​org/​10.​1016/j.​vacci​ne.​2016.​02.​045. 6. Félix S, Handem S, Nunes S, Paulo AC, Candeias C, Valente C, et al. Impact of private use of the 13-valent pneumococcal conjugate vaccine (PCV13) on pneumococcal carriage among Portuguese children living in urban and rural regions. Vaccine. 2021;39(32):4524–33. https://​doi.​org/​10.​1016/j.​ vacci​ne.​2021.​06.​035. 7. Wyllie AL, Rümke LW, Arp K, Bosch AATM, Bruin JP, Rots NY, et al. Molecu‑ lar surveillance on Streptococcus pneumoniae carriage in nonelderly adults; little evidence for pneumococcal circulation independent from the reservoir in children. Sci Rep. 2016;6:34888. https://​doi.​org/​10.​1038/​ srep3​4888. Competing interests The authors declare no competing interests. Additional file 1: Fig. S1. Quality plot of the reverse and forward read sequences resulting from FastQC. Forward sequences were trimmed at position (cycle) 240 and reverse sequences were trimmed at position 230 to reach a quality score (QS) of 30 at the ­25th percentile. Fig. S2. Oro‑ pharynx and nasopharynx microbiota profiles. Barplot representing the relative abundances of the taxonomic level Class in the oropharynx and nasopharynx and of samples misclassified in each site. Fig. S3. Identifica‑ tion of balances between groups of taxa associated with discrimination of the oropharynx and nasopharynx. The components defining the selected balance are specified on top of the boxplot that represents the distribution of the balance score for each of the groups. On the right the Receiver Operator Curve (ROC) with its AUC value and the density curve for each group is shown. TPR indicates true positive rate and FPR indicates false positive rate. Fig. S4. Diversity profiles of nasopharyngeal and oropharyngeal clusters. Fig. S5. Diversity of nasopharyngeal microbiota, for each Hill number. A. Comparison between pneumococcal carriers and non-carriers. B. Comparison between smokers and non-smokers. C. Com‑ parison between individuals who have contact with children with adults who do not have contact. Fig. S6. Ten most abundant genera in the nasopharynx and oropharynx depending on pneumococcal carrier state. Fig. S7. Alpha diversity for the oropharyngeal microbiota calculated with the Hill numbers. A according to their pneumococcal carriage status. B. smoking and C contact with children. Table S1. Differences in abundance at the taxonomic level of family between nasopharynx and oropharynx. Table S2. Bacteria (ASV) differentially present in the nasopharyngeal microbiota of pneumococcal carriers and non-carriers. Table S3. Bacteria (ASV) differentially present in the nasopharyngeal microbiota of smokers and non-smokers. Table S4. Bacteria (ASV) differentially present in the nasopharyngeal microbiota of individuals that have and do not have regu‑ lar contact with children. Table S5. Bacteria (ASV) differentially present in the oropharyngeal microbiota of pneumococcal carriers and non-carriers. Table S6. Bacteria (ASV) differentially present in the oropharyngeal micro‑ biota of smokers and non-smokers. Table S7. Bacteria (ASV) differentially present in the oropharyngeal microbiota of individuals that have and do not have regular contact with children. Supplementary Information The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s40168-​023-​01640-9. The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s40168-​023-​01640-9. Authors’ contributions ACP, JL, STA, MH and RSL conceived the study. STA and JL did DNA extraction and quantification. ACP and JL did metagenomic and data analysis. ACP, JL and RSL interpreted the results. ACP, JL and RSL wrote the manuscript. All authors read and critically revised the manuscript. ACP, JL, STA, MH and RSL conceived the study. STA and JL did DNA extraction and quantification. ACP and JL did metagenomic and data analysis. ACP, JL and RSL interpreted the results. ACP, JL and RSL wrote the manuscript. All authors read and critically revised the manuscript. 8. Trzciński K, Bogaert D, Wyllie A, Chu ML, van der Ende A, Bruin JP, et al. Superiority of trans-oral over trans-nasal sampling in detecting Strepto- coccus pneumoniae colonization in adults. PLoS One. 2013;8(3):e60520. https://​doi.​org/​10.​1371/​journ​al.​pone.​00605​20. Author details 1 1 Instituto de Tecnologia Química E Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal. 2 Faculty of Medicine, Institute for Infectious Diseases, University of Bern, Bern, Switzerland. Conclusions Finally, to meet our established criteria for the selec- tion of individuals, we only used 12 out of 25 pneumococ- cal carrier individuals identified in a previous study [9] (leading to 72 paired samples out of 224 nasopharyngeal samples and 240 oropharyngeal samples). However, when taking into account the total number of individuals and samples included in this study, it is currently one of the largest studies performed in healthy adults. In summary, our findings contribute to increase our understanding of how different factors shape the upper respiratory tract microbiota of adults opening the pos- sibility of using such information to design strategies aimed to promote a healthy respiratory microbiota. Abbreviations ASV Amplicon sequence variant AUC​ Area under the ROC curve CI Confidence interval CLR Centered ­log10 ratio Ct Cycle threshold SD Standard deviation FC Fold change FPR False-positive rate OR Odds ratio PERMANOVA Permutational analysis of variance qPCR Real-time polymerase chain reaction ROC Receiver operating characteristic STGG​ Skim milk, tryptone, glucose, and glycerin TPR True-positive rate URT​ Upper respiratory tract WHO World Health Organization ZIGMM Zero-inflated Gaussian mixed model Our study also has some strengths. First, to the best of our knowledge, it is a microbiota study with one of the largest number of samples from the upper Page 18 of 20 Paulo et al. 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Anaphylaxis to supplemental oral lactase enzyme
Allergy, asthma & clinical immunology/Allergy, asthma, and clinical immunology
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Allergy, Asthma & Clinical Immunology Allergy, Asthma & Clinical Immunology Voisin and Borici‑Mazi  Allergy Asthma Clin Immunol (2016) 12:66 DOI 10.1186/s13223-016-0171-8 Open Access CASE REPORT © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background:  Anaphylactic reactions involving IgE mediated hypersensitivity have been frequently reported for a number of uncommon foods. However, cases of anaphylaxis to over the counter vitamins and oral supplements have been rarely published. Lactose intolerance affects approximately 20% of Canadians and roughly 70% of the world’s population of any age. Lactose intolerance develops primarily due to the absence of the enzyme lactase and treat‑ ment involves avoidance of lactose-containing foods or ingestion of commercially available lactase enzyme prepa‑ rations prior to their consumption. This case report represents the first documented evidence of anaphylaxis after exposure to supplemental lactase enzyme preparation. Case presentation:  A 38 years old Caucasian female presented with a history of self-diagnosed adult-onset lactose intolerance and a suspected allergy to lactase containing tablets. She reported an episode of bilateral orbital swell‑ ing, shortness of breath, and throat constriction after oral ingestion of a supplemental lactase enzyme tablet. Her symptoms slowly resolved with the administration of inhaled salbutamol and oral diphenhydramine. She handled lactase tablets for years to her children who were lactose intolerant, but had never ingested the tablets herself prior to the reported episode. In clinic, physical examination was benign, and skin prick testing to a slurry of the lactase tablet revealed a strongly positive reaction wheal size of 10 mm and flare of 60 mm with normal controls. The patient reported throat tightness and constriction after skin prick testing and required cetirizine treatment and observation in clinic. Subsequent skin testing was performed with individual ingredients of the lactase tablet provided by the manu‑ facturer and Aspergillus niger, a common bacteria used in lactase preparations. Only concentrated lactase enzyme elic‑ ited a positive response. The patient was diagnosed with lactase tablet induced anaphylaxis due to synthetic lactase enzyme IgE mediated allergy, and was advised to avoid all products containing lactase enzymes as an ingredient and to carry an epinephrine auto-injector. Conclusion:  This is the first documented case report of an anaphylactic reaction to supplemental lactase enzyme. This case report reinforces the importance of thorough allergy assessment, education on avoidance of triggers, in particular with uncommon allergens. Keywords:  Anaphylaxis, Lactase allergy, Aspergillus, Lactose intolerance, IgE mediated Anaphylaxis to supplemental oral lactase enzyme M. R. Voisin1 and R. Borici‑Mazi2* © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. *Correspondence: rb62@queensu.ca 2 Division of Allergy and Immunology, Department of Medicine, Queen’s University, 166 Brock Street, Kingston, ON K7L 5G2, Canada Full list of author information is available at the end of the article Case presentation A previously healthy 38 years old Caucasian female pre- sented to the outpatient allergy clinic in the fall of 2014 with self-diagnosed adult-onset lactose intolerance and a suspected allergy to a commercial available lactase enzyme tablets. She reported sudden onset of bilateral orbital swelling and facial urticaria, and rapidly worsen- ing throat constriction and shortness of breath after her first oral ingestion of a supplemental lactase tablet. She took two tablets of diphenhydramine and 4 puffs of a salbutamol puffer which relieved her throat constric- tion and shortness of breath within 20–30  min. The bilateral orbital swelling slowly decreased within next 24 h with additional doses of oral diphenhydramine. She had avoided ingestion or handling of the supplemental lactase tablets since this reaction. Upon careful review of the history, patient had experienced recurrent itchiness and urticarial lesions in her hands and face after having been in contact with the supplemental lactase tablets she handed to her kids. She had no history of prior inges- tion of any commercial supplemental lactase-containing foods. Patient was diagnosed with anaphylaxis caused by sup- plemental lactase tablet due to synthetic lactase enzyme IgE mediated allergy. She was advised to avoid handling and ingestion of all products containing lactase enzymes as an ingredient and to carry an epinephrine auto-injec- tor. She has had no further accidental exposures. Subse- quently, patient was gradually introduced to regular dairy products and has remained asymptomatic, without the need for further investigations. Fig. 1  Skin test response to a slurry of lactase enzyme tablet applied to patient’s forearm Patient’s past medical history included well-controlled mild intermittent asthma, oral allergy syndrome to raw fruits and vegetables, allergic rhinitis, GERD, and hyper- tension. She reported a self-diagnosis of lactose intoler- ance approximately twelve months prior to her reported episode of anaphylaxis. Her medication list included ran- itidine, fluticasone nasal spray, and salbutamol as needed. She reported that her children were diagnosed with lac- tose intolerance and she was handling supplemental lactase tablets for years with no reactions, prior to the initiation of her skin symptoms a few months earlier. Upon assessment in clinic, physical examination was unremarkable. Skin prick testing (SPT) to environmental and food allergens was performed using allergens from Omega Laboratories, Montreal Quebec. Background of the enzyme lactase, leading to bloating, flatulence and diarrhea after the ingestion of lactose-containing foods. The prevalence of lactose intolerance varies considerably depending upon ethnic background, with overall rates approaching 20% [1]. Lactose intolerance can develop in two main patterns; primary or secondary, with congenital lactase deficiency being a third extremely rare cause [2, 3]. Management of lactose intolerance focuses primar- ily on avoidance of lactose-containing foods or ingestion of commercially prepared lactase enzyme preparations, Anaphylactic reactions involving IgE mediated hyper- sensitivity have been frequently reported for a number of uncommon foods; however, cases of anaphylaxis to over the counter oral supplements and vitamins have been rarely published. Lactose intolerance results from lack *Correspondence: rb62@queensu.ca 2 Division of Allergy and Immunology, Department of Medicine, Queen’s University, 166 Brock Street, Kingston, ON K7L 5G2, Canada Full list of author information is available at the end of the article Voisin and Borici‑Mazi Allergy Asthma Clin Immunol (2016) 12:66 Page 2 of 4 respectively, with adequate controls (Fig. 1). After skin testing was completed, patient reported sudden onset of throat tightness which resolved after treatment with oral cetirizine 20 mg and 2 puffs of inhaled salbutamol. During a subsequent office visit, the patient underwent skin testing to ingredients of the lactase tablet provided by the manufacturer. Skin testing was strongly positive for a slurry of approximately 10% weight/volume of the lactase enzyme (4000 FCC lactase units) and negative for the other non-enzyme ingredients of the tablet including dextrose, microcrystalline cellulose, calcium stearate and natural mint flavour. Skin testing to the crushed lactase tablet and its ingredients was performed in similar fash- ion in two healthy volunteers and it was entirely negative with appropriate controls. often produced by subspecies of Aspergillus [4]. Although previous reports of allergic reactions to supplemental lactase either via ingestion or occupational exposure have been published, this is the first case report of a systemic, anaphylactic reaction in a patient after first ingestion of a supplemental lactase enzyme preparation [5–9]. Discussionh This report describes the case of a 38 years old female who reported systemic anaphylactic symptoms after her first oral ingestion of supplemental lactase enzyme- containing tablet. Enzymes are known to be high molecular weight sensitizers, and evidence of aller- gic symptoms has been previously reported most commonly as respiratory allergic symptoms includ- ing asthma and/or rhinitis at variable levels of expo- sure [7]. Previous studies have described occupational rhino conjunctivitis, asthma, and contact dermatitis in pharmaceutical workers exposed to lactase powder for commercial use [4, 8, 9]. The earliest study reported lactase enzyme sensitization in 31% or 65 of 207 work- ers involved in the handling of lactase containing prod- ucts [9]. Another study elaborated on a cross-sectional survey of 94 pharmaceutical workers exposed to lactase, of which 29% or 27 had positive skin testing [4]. Finally, a study involving 13 employees at a lactase tablet man- ufacturing plant demonstrated lactase sensitization in 69% or 9 employees [8]. Unlike these reports, our patient developed solitary skin involvement, but denied respiratory symptoms of rhino conjunctivitis or asthma after repeated dermal exposures that occurred during handling of lactase enzyme tablet. Instead, she experi- enced involvement of respiratory tract as part of sys- temic anaphylactic reaction after her first oral ingestion of supplemental lactase enzyme. gy p Pharmaceutical molds are widely used in industry as a source for producing supplemental lactase enzyme prep- aration. In our case report, the manufacturer confirmed that the lactase enzyme concentrate used to make the lactase tablet was derived from cultures of Aspergillus oryzae. Aspergillus oryzae is an aerobic, filamentous fun- gus with many applications in the food industry includ- ing its traditional use in China and Japan to produce koji, a complex enzyme preparation used in the production of soy sauce, miso, and sake [10, 11]. Its ability to secrete large amounts of proteins has facilitated its use in mod- ern biotechnology including large scale production of enzymes including lactase [12]. Antigenic determinants identified from Aspergillus include α-amylase—the major cause of Baker’s asthma—and lipase, and previ- ous reports of sensitization were thought to be due to repeated inhalational exposure to the enzyme [4, 5, 11]. Case presentation SPT was posi- tive for dust mites, trees, grasses, ragweed, and molds of Alternaria and Cladosporium, but it was negative for cow’s milk, egg, wheat, soy, sesame, inhalant Aspergillus species and subspecies of Aspergillus niger. SPT to sub- species of Aspergillus oryzae was not performed due to lack of allergenic extract. SPT to a saline slurry of the crushed lactase tablet (approximately 10%  weight/vol- ume) revealed a positive wheal and flare reaction, long- est diameters of wheal and flare were 10 and 60  mm, Fig. 1  Skin test response to a slurry of lactase enzyme tablet applied to patient’s forearm Voisin and Borici‑Mazi Allergy Asthma Clin Immunol (2016) 12:66 Page 3 of 4 Voisin and Borici‑Mazi Allergy Asthma Clin Immunol (2016) 12:66 Page 3 of 4 The sensitization pattern of our patient was similar to the case report published in 2007 by Laukkanen et al. [6]. He described the presence of serum lactase specific IgE anti- bodies in a pharmaceutical worker exposed to powdered form of lactase enzyme who suffered from contact der- matitis and allergic rhino conjunctivitis. However, in our case report, confirmation of IgE mediated sensitization was obtained via positive SPT to the slurry of crushed lactase tablet and concentrated lactase enzyme, as well as lack of sensitivity to other non-lactase ingredients of the lactase tablet. The negative SPT responses in two healthy volunteers and the appropriate controls we obtained dur- ing the SPT procedure confirmed that positive skin tests were specifically attributed to IgE mediated sensitivity to lactase enzyme and not due to skin irritation. In addition, although to a milder degree, patient experienced similar symptoms in clinic after the positive skin testing with the crushed lactase tablet confirming that lactase enzyme allergy was the culprit. Availability of data and materials Data sharing not applicable to this article as no datasets were generated or analysed during the current study. Data sharing not applicable to this article as no datasets were generated or analysed during the current study. • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Competing interests The authors declare that they have no competing interests. References 1. Vesa T, et al. Lactose intolerance. J Am Coll Nutr. 2000;19(2):165–75. 1. Vesa T, et al. Lactose intolerance. J Am Coll Nutr. 2000;19(2):165–75. 2. Swagerty D, et al. Lactose intolerance. Am Fam Phys. 2002;65(9):1845–50. 1. Vesa T, et al. Lactose intolerance. J Am Coll Nutr. 2000;19(2):165 75. 2. Swagerty D, et al. Lactose intolerance. Am Fam Phys. 2002;65(9):1845–50. 3. Heyman M. Lactose intolerance in infants, children, and adolescents. Am Acad Pediatr. 2006;118(3):1279–86. 4. Bernstein J, et al. A cross-sectional survey of sensitization to Aspergillus oryzae-derived lactase in pharmaceutical workers. J Allergy Clin Immunol. 1999;103:1153–7. Author details 1 8. Stocker B, et al. Occupational sensitization to lactase in the dietary sup‑ plement industry. Arch Environ Occup Health. 2015. doi:10.180/1933824 4.2015.1066294. 1 School of Medicine, Faculty of Health Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada. 2 Division of Allergy and Immunology, Department of Medicine, Queen’s University, 166 Brock Street, Kingston, ON K7L 5G2, Canada. 9. Muir D, et al. Occupational sensitization to lactase. Am J Ind Med. 1997;31:570–1. Conclusionh Written informed consent was obtained from the patient for the publication of this report and the accompanying image. This case represents the first report of a systemic ana- phylactic reaction in a patient with self-diagnosed lactose intolerance after oral ingestion of a supplemental lactase tablet, resulting from an IgE mediated allergy to lactase enzyme. This study highlights the potential for serious and potentially life-threatening anaphylactic reactions to commonly used over the counter supplements and a need for patients and healthcare providers to be aware of rare but significant allergies to a variety of uncom- mon allergens. Patients presenting with a history of aller- gic and/or anaphylactic symptoms should be screened by both a thorough history and adequate allergy testing and be educated about the risks of future exposures and treatment of potential anaphylactic reactions. Received: 2 September 2016 Accepted: 25 November 2016 Received: 2 September 2016 Accepted: 25 November 2016 Received: 2 September 2016 Accepted: 25 November 2016 Authors’ contributions 5. Binkley K. Allergy to supplemental lactase enzyme. J Allergy Clin Immu‑ nol. 1996;97:1414–6. MRV assessed the patient in clinic under the supervision of RBM, and RBM performed all of the allergy investigations. MRV drafted the abstract and the first draft of the manuscript, and both authors were involved in revising the manuscript for final approval. Both authors read and approved the final manuscript. 6. Laukkanen A, et al. Lactase-induced occupational protein contact derma‑ titis and allergic rhino conjunctivitis. Contact Dermat. 2007;57:89–93. g j 7. Larsen AI, et al. Incidence of respiratory sensitization and allergy to enzymes among employees in an enzyme producing plant and the rela‑ tion to exposure and host factors. Occup Environ Med. 2007;64:763–8. Acknowledgements 10. Barbesgaard P, et al. On the safety of Aspergillus oryzae: a review. Appl Microbiol Biotechnol. 1992;36:569–72. g We would also like to thank the manufacturers of the oral supplemental lactase tablet for providing information and individual ingredient samples for allergy testing. 11. Van Kampen V, et al. Occupational airway sensitizers: an overview of the respective literature. Am J Ind Med. 2000;38:164–218. 12. Machida M, et al. Genome sequencing and analysis of Aspergillus oryzae. Nature. 2005;438:22–9. Consent to participate Written informed consent was obtained from the patient regarding participat‑ ing in this case report and collecting individual clinical data. Discussionh Since our patient did not demonstrate IgE mediated sensitivity to inhalant Aspergillus species upon SPT, but reacted to the lactase ingredient derived from Aspergil- lus oryzae, the likely mechanism of sensitization remains repeated dermal exposure to the same lactase tablet, but possible prior oral consumption of foods that may have contained lactase enzyme derived from Aspergillus ory- zae cannot be excluded. The patient was educated about the risk of future accidental exposures with consump- tion of supplemental lactase-containing products and the need to carry an epinephrine auto injector, although she denied any allergic-like symptoms outside of the context of the above mentioned episodes. In addition to studies describing occupational expo- sure to lactase enzyme preparations, there have been two case reports in the literature documenting more specific allergic reactions to lactase enzyme [5, 6]. The earliest case report by Binkley [5], described an IgE mediated allergic reaction to Aspergillus oryzae derived lactase, with the patient experiencing allergic symptoms con- fined to the oropharynx after oral ingestion of lactase supplements. The author suggested that the sensitiza- tion of this patient occurred either due to prior expo- sure to cross-reacting Saccharomyces fragilis found in the specific brand of lactase tablet, or as a result of the patient’s inhalant allergy to Aspergillus species, with a similar mechanism to an “oral allergy syndrome”. Unlike this case report, our patient did not demonstrate an IgE mediated sensitivity to inhalant Aspergillus species and she also denied known previous consumption of other supplemental lactase products to account for the sensiti- zation. Therefore, to the best of our knowledge, the most likely route of sensitization to synthetic lactase enzyme was with repeated prior dermal exposure when handling lactase tablets to her children. Eventually she experienced contact urticaria when handling the lactase tablets and later on suffered a systemic reaction compatible with anaphylaxis upon first oral ingestion of the lactase tablet. Finally, this patient was self-diagnosed with lac- tose intolerance. Subsequently to our assessment, she was able to re-introduce the dairy products in her diet. Therefore, there was no need for further investigation of Page 4 of 4 Voisin and Borici‑Mazi Allergy Asthma Clin Immunol (2016) 12:66 lactose intolerance and future consumption of alternate supplemental lactase enzyme. Funding The design of the paper, literature review and writing of the manuscript were self funded. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: • We accept pre-submission inquiries
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Mediadores e Desafios Hodiernos: Regenerar Diversidades Através da Confiança
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Iva Maria da Costa Fernandes Centro de Investigação em Estudos da Criança, Instituto de Educação, Universidade do Minho, Portugal https://orcid.org/0000-0003-1322-3708 ivafernandes2000@gmail.com Resumo A mediação é perspetivada como uma área contemporânea que paira sobre um pre­ sente demarcado pela abundância de significações, interpretações, singularidades e divergências. Hoje, mais do que nunca, o mediador assume a responsabilidade de facilitar a comunicação entre aqueles que persistem, firmemente, à mudança e à diferença. Constroem contextos dialógicos que valorizam a palavra do “próximo” e acolhem o sentimento escondido daquele que se sente enfraquecido. Entre as várias sessões e processos de mediação, a confiança é a principal sensação que permite a cada mediado transpor as suas histórias de vida, os seus olhares sobre o mundo e as emoções que denotam a sua individualidade. Em base de uma reflexão crítica, apelo pela promoção de novas competências profissionais, construindo uma perspetiva que ultrapassa a teoria e atinge o meio que circunscreve qualquer processo media­ tivo: espaço, objetos, dimensões, aparências e sensações. O principal objetivo deste trabalho é compreender o efeito que a confiança acarreta no desenvolvimento das sessões de mediação, desmistificando os obstáculos que impedem que a confiança seja uma realidade familiar e inquestionável na mediação. Mediadores e Desafios Hodiernos: Regenerar Diversidades Através da Confiança https://doi.org/10.21814/uminho.ed.78.11 Introdução A proposta “Mediadores e Desafios Hodiernos: Regenerar Diversidades Através da Confiança” corresponde, originalmente, a um trabalho académico desenvolvido no âmbito da unidade curricular Fundamentos e Modelos de Mediação, do mestrado em mediação educacional da Universidade do Minho. Esta temática surge pela minha curiosidade pessoal em explorar, de um outro ângulo, as sessões de mediação, um olhar que paira sobre pormenores que circunscrevem cada contexto mediativo. Desta forma, esta proposta permite explorar as minhas curiosidades e fazer com que os meus valores, enquanto estudante e futura mediadora, se tornem num alvo de apreciação crítica. É com estas palavras que quero evidenciar a minha pessoa, alguém que estima e identifica a mediação como um espaço dialógico, um contexto que valoriza a palavra do próximo e acolhe o sentimento escondido daquele que se sente enfraquecido. Porém, não podemos deixar de questionar qual a base essencial para que haja, efetivamente, esta abertura por parte dos mediados. Tal como acon­ tece no quotidiano, só nos sentimos confortáveis a partilhar as nossas vivências ou ideologias quando o ambiente torna as nossas palavras leves, livres de preconceito e, sobretudo, quando o meio ao nosso redor nos permite experienciar a sensação de pertença e confiança para sermos nós mesmos. É a partir desta visão que pretendo desenvolver a seguinte reflexão, edificando a confiança como o conceito que se alia, indispensavelmente, a este mundo mediador e de paz. Assim, o seguinte trabalho contempla um foco dirigido aos espaços físicos nos quais ocorrem os processos de mediação — o conforto espacial: as paredes que acolhem conflitos, acreditando que estes são contextos inundados de significações e interpre­ tações que poderão promover uma maior sensação de segurança. Posteriormente, enfatizo um pequeno olhar sobre a aplicabilidade do princípio da imparcialidade e as competências que este subtende. Numa abordagem mais teórica, é evidenciada a perspetiva de alguns autores que enfatizam a confiança como uma conquista pro­ gressiva e um ciclo vicioso. A reflexão terminará com as minhas considerações finais, elevando a mediação como uma área naturalmente confiável. MEDIADORES E DESAFIOS HODIERNOS MEDIADORES E DESAFIOS HODIERNOS 152 Palavras-Chave confiança, imparcialidade, mediação de conflitos Conforto Espacial: As Paredes que Acolhem Conflitos Acredito que tudo aquilo que nos envolve apresenta uma função, comunica um sig­ nificado. Cada recinto é caracterizado pela sua organização, dimensão ou, até mesmo, pelos elementos que o constituem, fazendo com que o seu aspeto imediato provo­ que um impacto sobre a nossa pessoa que, (in)conscientemente, interpreta aqui­ lo que observa, provocando a vivência de um conjunto de sensações familiares ou desconhecidas. O espaço no qual ocorre o processo de mediação é, igualmente, um contexto repleto de mensagens sensoriais, disfarçadas através da disposição do local e dos objetos. Os autores Diez e Tapia (2006) chamam à atenção sobre a caracteri­ zação do espaço envolvente num processo de mediação, pois este é um contexto que abraçará um conflito, uma situação delicada que impossibilitou a adaptação e a transformação dos sujeitos, devido a uma vivência menos positiva. Não podemos esquecer qual a “receita mágica” que fundamenta a nossa sobrevi­ vência que, a meu ver, é a interação humana. Todos os dias comunicamos: compar­ tilhamos os nossos sentimentos íntimos a quem mais confiamos; transmitimos per­ ceções distintas sobre o mundo para com aqueles que se escondem na sombra do senso-comum; mantemo-nos em silêncio quando sentimos que o ambiente se torna constrangedor. Entre as várias formas de comunicação e entre as inúmeras pessoas com quem dialogamos, o conflito emerge como manifesto das divergências que dis­ tinguem a nossa bagagem individual. O conflito é fruto da nossa essência, representa parte da nossa história de vida e transmite aqueles que são os nossos sentimen­ tos, perceções e interesses. Isto para dizer que o espaço onde decorrerá o processo é um ambiente que acolherá um caso particular e, acima de tudo, seres humanos insubstituíveis (Mangini, 2020). É a partir deste olhar que, juntamente com outros olhares, enfatizo a necessidade de construirmos um espaço físico e intangível, um contexto que permita que os mediados se sintam confortáveis para dialogar. Tal como a essência da mediação, no que diz respeito ao princípio da voluntariedade e confidencialidade, o seu contexto é pensado com o intuito de promover o conforto dos mediados para que se sintam à vontade para compartilhar as suas vivências. (RE)PENSAR A FORMAÇÃO EM MEDIAÇÃO (RE)PENSAR A FORMAÇÃO EM MEDIAÇÃO partilha profunda do nosso ser, num contexto dialógico, de interação e transforma­ ção. Porém, a segurança que conduz todo o processo implica a atenção e o controlo sobre vários pormenores que nos rodeiam que, por vezes, são invisivelmente impac­ tantes. Mas como é que isto se aplica na mediação? Quais os aspetos que devemos de ter em conta? A Confiança na Mediação Observo que a confiança é definida como um sentimento de segurança relativamen­ te à nossa pessoa, uma crença firme de lealdade para com o “outro” ou um sentimen­ to de familiaridade (Infopédia, s.d.). Pessoalmente, perspetivo-a como um sentimento complexo, uma sensação que comporta a influência de vários fatores explícitos e implícitos, variáveis que poderão abrigar os nossos medos ou impedir que sejamos genuínos. É desta forma que pretendo analisar o conceito de confiança, ao longo de todo o processo de mediação, assimilando o impacto do papel do mediador, a importância do reconhecimento da mediação e a caracterização do espaço físico que circunscreve as sessões mediativas. A confiança é como uma sensação mútua entre todos, mediador e mediados, um sentimento capaz de nos conduzir para uma 153 MEDIADORES E DESAFIOS HODIERNOS 154 equilíbrio do espaço, isto é, desde a sua organização à dimensão espacial. Digamos que o espaço é como um “recurso” que tenta alcançar não só o reconforto dos me­ diados como também o do mediador, aquele que permitirá criar uma conexão entre todos os envolvidos para que haja, efetivamente, um ambiente de confiança (Diez & Tapia, 2006). equilíbrio do espaço, isto é, desde a sua organização à dimensão espacial. Digamos que o espaço é como um “recurso” que tenta alcançar não só o reconforto dos me­ diados como também o do mediador, aquele que permitirá criar uma conexão entre todos os envolvidos para que haja, efetivamente, um ambiente de confiança (Diez & Tapia, 2006). A verdade é que, na maioria dos casos, as pessoas desconhecem a realidade da me­ diação, sentindo que caminham por um percurso estranho até a uma pessoa nunca antes vista, o mediador. É por esta razão que pretendo enfatizar a caracterização do espaço, visto que o contexto acaba por ser um reflexo do profissional, uma evidência da pessoa que ele constitui: “as minhas roupas e os meus modos, a disposição física dos espaços, os ruídos, as cores e os cheiros do lugar geram um contexto repleto de significados.” (Diez & Tapia, 2006, p. 34). Isto não significa que existe um design ou uma estrutura rígida a ser seguida porque, na verdade, cada profissional é distinguido pela sua personalidade, crenças e missões, não deixando de referir que, na maioria dos casos, o profissional não desempenha as suas funções no contexto que idealiza. Quando se trata do sistema público de mediação, como por exemplo o Sistema de Mediação Familiar, os facilitadores de comunicação são notificados para conduzir um dado processo que, caso seja aceite, existirá um espaço concebido para o efeito. Estes ambientes, que acolherão os obstáculos, dilemas e preocupações dos mediados, são meios camuflados de outras finalidades. O que pretendo referir é que o espaço não é pensado ou construído em função do acolhimento de um conflito. Pessoalmente, va­ lorizo a adjetivação daquilo que me rodeia, seja em situações quotidianas ou profis­ sionais, porque o meio acaba por ser um “bónus” no âmbito da promoção do conforto da minha pessoa e do outro. Em base de relatos reais, denoto que alguns mediadores partilham este mesmo olhar atento sobre o espaço: no dia e hora combinados, lá estava, numa sala protocolada para o efeito. Conforto Espacial: As Paredes que Acolhem Conflitos O facto de, habitualmente, existirem mesas redondas ao invés de uma secretária e poltronas, revela que este processo é um espaço dialógico e cooperativo, sem hie­ rarquias, que acolhe, abertamente e sem preconceito, as dificuldades dos mediados: “o elemento mais importante a ter em conta quando preparamos a mediação é que o espaço concede significados aos sucessos que nele ocorrem, porque faz parte do contexto comunicacional” (Diez & Tapia, 2006, p. 33). Efetivamente, o conforto mútuo é como um requisito fundamental para que, de facto, se consiga usufruir da melhor forma possível da sessão, mas repito que este bem-estar só é conseguido a partir do MEDIADORES E DESAFIOS HODIERNOS (RE)PENSAR A FORMAÇÃO EM MEDIAÇÃO (RE)PENSAR A FORMAÇÃO EM MEDIAÇÃO distância física (e afetiva) entre os mediados e o mediador. É curioso como a ausência de uma mesa redonda ou oval pode impossibilitar que o profissional observe todo o cenário numa amplitude de cento e oitenta graus, isto é, atender todas as locuções que são partilhadas por um mediado em questão e, simultaneamente, escoltar os gestos e expressões daquele que o escuta. Acredito que a presença de uma mesa retangular leva a que o nosso contacto ocular seja um pouco mais limitado e rigida­ mente distribuído pelos sujeitos presentes, podendo influenciar a conexão pessoal entre mediador-mediado e a análise que se realiza sobre o meio. Na verdade, os contextos nos quais se desenvolvem os processos transformam-se em “ferramentas” de trabalho (Diez & Tapia, 2006). Mesmo que a caracterização dos objetos e do espaço não favoreçam a essência da mediação, o profissional, através das suas competências interrelacionais e comunicacionais, criará um contexto neu­ tro e imparcial, sustentado no valor da colaboração, para que os mediados se sintam indispensáveis ao longo de todo o processo. Para além de “ampliar os canais de comunicação entre o mediador e as partes” (Diez & Tapia, 2006, p. 36), a relação de proximidade é, progressivamente, (co)construída. Porém, questiono-me sobre a credibilidade das nossas práticas em espaços que, apa­ rentemente, desafiam a nossa capacidade de saber ser mediador, de saber estar como pessoa e de saber fazer o conforto chegar, mesmo quando parece ser impossível: quando chegámos, deparámo-nos com um espaço da câmara municipal onde se encontravam a funcionar vários serviços da área social e que tinha uma sala com uma mesa oval (mais próxima de uma sala de reuniões do que de uma sala de mediação). Era um local sem aquecimento, num edifício em pedra, em que o frio era de tal forma insuportável que eu e a minha colega (e, posterior­ mente, os próprios mediados) tivemos de estar de casaco (e mesmo assim a tremer) durante todas as sessões. (Reis, 2021, pp. 25–26) Tento imaginar este momento inicial da sessão de pré-mediação, mas um pensa­ mento insiste e persiste dentro de mim: “e se o ambiente fosse diferente?”. As me­ diadoras foram desafiadas, pois, para além da imparcialidade a adotar em relação às posições dos mediados, presumo que tiveram que demonstrar uma dada neutra­ lidade em relação ao ambiente ao seu redor. MEDIADORES E DESAFIOS HODIERNOS Uma sala vazia de aconchego e cheia de tudo o que não faz sentido para os fins pretendidos. Uma mesa retangular a espraiar-se pelo espaço de mosaico, papéis e dossiês empilhados e, entre tantos outros artefactos administrativos, uma bandeira, num canto, a olhar-nos pelos castelos e quinas da nossa portu­ galidade. (Quintanilha, 2021, p. 2) Espontaneamente, o contexto supramencionado reclama em mim a necessidade de erguer o valor da mediação. Imagino um ambiente agitado que, ao ser facultado para o processo, entrou numa pausa temporária de silêncio e de período de substituição. A adaptação existiu, mas por parte do mediador que, dadas as circunstâncias, esta­ beleceu um espaço intocável em base da comunicação interpessoal, a linguagem corporal e o à-vontade oferecido para o momento da partilha. A confiança foi cons­ truída através das competências profissionais, de forma a atenuar aquelas paredes que realçavam um ambiente burocrático e solene. Provavelmente, aquela mesa re­ tangular não transmitiu o verdadeiro significado da mediação, visto que, ao invés de revelar que se tratava de um espaço construtivo e colaborativo, no âmbito da pro­ cura de novas soluções consensuais e benéficas para os envolvidos, aparentava uma 155 MEDIADORES E DESAFIOS HODIERNOS 156 extremidades, nas quais se encontram os réus. A distância física entre as partes transparece a inexistência de uma tentativa de conjugar os seus interesses, não dei­ xando de referir que a posição do juiz comunica aquele que será o decisor final, independente das vontades, necessidades e interesses manifestados pelos argui­ dos. (In)Conscientemente a nossa pessoa começa a absorver a informação que o espaço comunica, seja a partir das suas dimensões ou dos objetos distribuídos pelo contexto, fazendo com que naveguemos entre sensações familiares ou misteriosas. Como será que as pessoas se sentem ao entrar, pela primeira vez, num gabinete de mediação ou em outro contexto no qual se desenvolverá o processo? A resposta para esta questão levaria para a profundidade de um estudo qualitativo. Mas, recorrendo agora ao pensamento crítico, penso que esta interrogativa nos eleva para um mo­ mento de reflexão: os tribunais, por exemplo, são contextos institucionalizados (se assim os posso designar), mas a mediação é uma realidade ainda por descobrir. O que difere? Será que é o reconhecimento que cada uma das áreas agrega? Será o estado de consolidação científica? Bem, a verdade é que os espaços também nos revelam os seus estados de desenvolvimento: entramos numa pequena mercearia em que os produtos estão distribuídos em prateleiras altas de madeira e caso desejarmos algo que se encontra no cimo dessas mesmas, precisamos de chamar o colaborador para alcançar o que desejamos com um escadote. No momento de pagar, reparamos que o senhor tem dificuldade em realizar a leitura do código de barras e de inserir o nosso contribuinte no sistema, aludindo que tem saudades da sua antiga máqui­ na registadora. Apesar de estarmos num ambiente antiquado e com dificuldades de adaptação, a doçura do senhor transmite-nos uma sensação de bem-estar, devido à sua sabedoria e humildade. O que quero dizer com esta viagem pelo imaginário é que, apesar de não conse­ guirem encontrar, para já, o espaço ideal para o desenvolvimento das suas práticas, os mediadores são sujeitos capazes de transformar os ambientes solenes e sem aquecimento, em espaços seguros para o acolhimento dos conflitos, vulnerabilida­ des e indecisões. MEDIADORES E DESAFIOS HODIERNOS O nosso olhar, o rasgar de um simples sorriso, o tom da nossa voz e a postura corporal, inundam os espaços de novas significações e interpretações, fazendo com que as paredes que causam desconforto ou desconfiança se transfor­ mem em paredes íntimas que acolherão, seguramente, as inquietações dos media­ dos que nos procuram. (RE)PENSAR A FORMAÇÃO EM MEDIAÇÃO Caso contrário, iriam ser manipuladas pela baixa temperatura e, consequentemente, persuadidas pela distração. Com isto, pergunto-me se “nós”, mediadores, estamos preparados para antever o contexto e se somos capacitados para nos adaptar às circunstâncias espaciais. Acredito que são os pormenores que potenciam a verdadeira diferença. Um simples saber estar num espaço desajustado às nossas necessidades, transparecerá que, independentemente do ambiente, o mediador atenta as histórias de vida de cada mediado. Não há nada mais reconfortante do que sentirmos que alguém está ali para nos escutar, orientar e auxiliar num momento em que acudimos por uma melhor solução. Os espaços geram significados, assim como promovem a sensação de conforto e bem-estar. Basta transpormo-nos para os contextos e ambientes dos tribunais: deparamo-nos com um lugar centrado (e, por vezes, mais elevado) entre duas MEDIADORES E DESAFIOS HODIERNOS Imparcialidade: Um Princípio e uma Competência Somos movidos pelas nossas crenças, missões e valores. A forma como observamos o mundo, transparece, inevitavelmente, parte daquilo que somos. Os fenómenos que ocorrem no nosso quotidiano despertam um conjunto de emoções, espelhadas pela comunicação: os olhos observam, a mente processa, a alma converte e o corpo rea­ ge através de comportamentos. Por outras palavras, as emoções e a capacidade de comunicá-las reivindicam a nossa essência enquanto seres humanos: somos o único ser vivo capaz de sentir, raciocinar e de comunicar. Partindo do princípio destas qua­ lidades, questiono-me se somos seres capazes de dominar, em particular, o nosso lado emocional, pois “não somos criaturas de lógica: somos criaturas de emoções” 157 (RE)PENSAR A FORMAÇÃO EM MEDIAÇÃO (Carnegie, 2021, p. 60). Atualmente, o autoconhecimento e a inteligência emocional são temas que têm ganho um grande destaque no mercado de trabalho. Mas será que todos os profissionais apresentam estas capacidades? E os mediadores? Será um fator inerente para a prática das suas ações? (Carnegie, 2021, p. 60). Atualmente, o autoconhecimento e a inteligência emocional são temas que têm ganho um grande destaque no mercado de trabalho. Mas será que todos os profissionais apresentam estas capacidades? E os mediadores? Será um fator inerente para a prática das suas ações? Vejamos que existe um conjunto de princípios aplicáveis à mediação: voluntarie­ dade, confidencialidade, igualdade e imparcialidade, independência, competência e responsabilidade, executoriedade (Lei n.º 29/2013, 2013). Tendo uma especial aten­ ção sobre a aplicabilidade do princípio da (igualdade e) imparcialidade, constata-se que “o mediador de conflitos não é parte interessada no litígio, devendo tratar de forma equitativa e imparcial as partes ( ... ) podendo frequentar ações de formação para melhorar as suas competências” (Pinto & Mendes, 2013, p. 143). Este princípio vigora a isenção, exige a ocultação das nossas perceções, valores e emoções. A nossa função não é determinar qual é o caminho que os mediados devem de percorrer, mas acompanhar os desejos dos mesmos. Entre decisões e vontades, neutralizamos a nossa pessoa. Porém, nunca deixamos de ser humanos, nunca deixamos de sentir, de pensar e, sobretudo, de reagir: ora, se o mediador é um humano, reage a estímulos e padece de fragilidade – também sente “impactos”. Sente emoções como qualquer outro ser humano. Sente esperança, contentamento, felicidade, irritação, preocupação, alívio, tris­ teza, entusiasmo, medo, entre tantas outras emoções. Na verdade, o mediador vai sentir tudo. (Sousa, 2020, p. MEDIADORES E DESAFIOS HODIERNOS MEDIADORES E DESAFIOS HODIERNOS 158 Aliás, a competência da imparcialidade reporta, nesta dimensão da promoção da confiança, uma grande influência na relação entre mediador-mediado. Nunca existe só e apenas uma verdade, isto é, o conflito provoca diferentes sensações e impactos nas pessoas, levando a que as suas posições e desejos se dividam entre diferentes interpretações da situação. Cada parte representa uma história e o mediador, neutro e imparcial, é o responsável por recontar o sucedido. Contudo, a arte de recontar, tal como já foi referido, exige o controlo sobre o nosso eu interior para que os sujeitos envolvidos não sintam que, entre si, existem personagens principais e secundárias. Por outras palavras, os mediadores detêm a responsabilidade de equiparar as dife­ rentes interpretações da história, no sentido em que nenhum mediado se deva sentir identificado como a parte que (não) tem razão. A sensação de desvantagem ou de julgamento poderá, sem dúvida, comprometer a relação entre mediador-mediado e também, o desenvolvimento de todo o processo. Porém, à priori de uma tentativa de fortalecimento da relação mediador-mediado, o profissional deve sentir-se confiante sobre si mesmo e as suas competências, habilidades, conquistas e percurso(s). Imparcialidade: Um Princípio e uma Competência 6) Se, inevitavelmente, existe a impossibilidade de deixarmos de sentir, como será que o princípio da imparcialidade é aplicado, sem colocar em causa a confiança dos media­ dos? O autoconhecimento será, provavelmente, o melhor escudo de autodefesa de qualquer mediador. Saber aquilo que somos, o que nos incomoda, as vias de escape quando não conseguimos controlar as nossas emoções, a capacidade de refletir na e para a ação, permite-nos conter a nossa essência e obter um maior controlo sobre o processo. Na verdade, o autoconhecimento transparece o estado de consciencializa­ ção que nós detemos da nossa posição em relação ao meio que nos rodeia, incluindo os momentos em que as nossas ações poderão comprometer o direito à imparcia­ lidade dos mediados (Aragão, 2016). Nestas situações, a reflexividade contínua das nossas práticas permitirá que, ao longo do processo, consigamos regular a nossa imparcialidade e reconstruir a confiança: “é necessário sermos conscientes da nossa incompetência de imparcialidade robótica para chegarmos à melhor imparcialidade possível que consigamos alcançar como ser humano” (Sousa, 2020, p. 7). A confiança poderá ser vista como um ciclo: ao sermos conscientes da nossa in­ dividualidade e profissionalismo tornamo-nos, consequentemente, mais confiantes sobre as nossas práticas. Posteriormente, esta alegoria da nossa pessoa confiante é transparecida para o meio ao nosso redor, seja através da postura que adotamos ou da nossa linguagem não-verbal e para-verbal. A aplicabilidade ponderada da im­ parcialidade permite que os mediados não se sintam categorizados ou julgados e, sobretudo, que se sintam confiantes e confortáveis ao longo de todo o processo. (RE)PENSAR A FORMAÇÃO EM MEDIAÇÃO (RE)PENSAR A FORMAÇÃO EM MEDIAÇÃO Por último, o vértice referente ao “outro” é alcançado através da empatia, uma compe­ tência social que permite perspetivar a contraparte em base do respeito e considera­ ção, independentemente da sua posição, das necessidades e interesses que defende. Porém, isto só é concretizável quando o mediador edifica um clima de segurança entre os mediados, pois é entre si que será possível encontrar uma solução conjunta e consensual. Caso contrário, como é que os mediados procuram uma resposta se não conseguem confiar no “outro”? Apesar de a confiança estar representada em pormenores e de ser alcançada gradual­ mente, a essência do trabalho do mediador é como um fator contínuo, isto é, um fator indispensável para apaziguar o conflito, transformar os indivíduos e para promover reconhecimento da mediação: “a geração de confiança é essencial para o desenvolvi­ mento do nosso trabalho e é foco permanente de atenção do mediador ao longo de todo o processo de mediação” (Diez & Tapia, 2006, p. 51). Digamos que a confiança é como um ciclo que não pode ser corrompido, aliás, é referenciado a necessidade de trabalharmos aprofundadamente a geração de confiança passo a passo, apelando por evidências positivas que nos permitam dar continuidade ao processo: “se perce­ bemos que não existe um nível mínimo de confiança para com o outro quanto a ele, isso indica-nos que temos que continuar a trabalhar mais um pouco” (Diez & Tapia, 2006, p. 51). É importante visualizar toda esta conceção de geração confiança como um processo sequencial que se inicia por uma base consolidada, um espaço seguro e reconfortante para desenvolver o processo de mediação. E isto porquê? Segundo Diez e Tapia (2006), o conflito, dependendo das conjunturas em que se origina, poderá ser o reflexo da impossibilidade de as pessoas resolverem autono­ mamente uma dada situação conflituosa, reconhecendo “isto” como um obstáculo complexo e difícil de superar, pois exige confiança e segurança para ultrapassar (no­ vamente) a situação. Por outro lado, Mangini (2020) perspetiva o conflito como uma constante que constitui uma bagagem pessoal — “cada conflito tem a sua dinâmica, sua intensidade, sua História, seu objeto e seus personagens próprios” (para.7), na qual o mediado carrega parte da sua história de vida, tendo que confiá-la a alguém. A Confiança Como uma Conquista Cíclica e Progressiva Diez e Tapia (2006) defendem que a conquista da confiança é conseguida através de uma estrutura sequencial assegurada em base de quatro “vértices”: o mediador; a mediação; o eu mesmo e o outro. À imagem desta convicção, a estratégia em questão é uma ferramenta indispensável para compreender qual é a intervenção mais ade­ quada para cada situação, tendo em conta o que é manifestado ao longo das sessões de mediação. De uma forma geral, o mediador é visto como um criador de con­ fiança, porém recomenda-se que o próprio profissional se sinta capaz e confortável em realizar o processo em questão, pois a sua (in)segurança será espontaneamente manifestada através das suas ações (Diez & Tapia, 2006). Por outro lado, os autores evidenciam a importância do profissional ser claro face ao(s) objetivo(s) e às regras que subentendem o processo de mediação, quer para si mesmo ou para os mediados. Tendo em conta a necessidade de promover um clima de confiança, a técnica da es­ cuta ativa é vista como uma ferramenta essencial para que o mediado se sinta não só escutado como também compreendido, existindo assim a possibilidade de criar uma maior fluidez dialógica, facilitada pelo mediador ao longo de todas as sessões. Efetivamente, o processo de mediação não é possível de ser concebido sem o envol­ vimento dos mediados, logo a necessidade de estes confiarem na mediação. Para que este “vértice” seja concretizável, é necessário darmos vida à mediação, demonstrando que esta funciona, por exemplo, através da exibição de resultados conseguidos pela metodologia mediativa. Para além da necessidade de se confiar na mediação, torna­ -se fulcral que os próprios mediados confiem em si mesmos para que haja, efetiva­ mente, o seu empoderamento. Isto é conseguido através da ajuda do mediador (Diez & Tapia, 2006), que deteta “todos aqueles relatos, episódios e sinais que revelam in­ dícios certos de capacidade” para que, posteriormente, possa fundamentar questões específicas que possibilitem uma reflexão por parte dos mediados, de forma a que “as partes possam vê-los de um novo ângulo, mais positivo para si mesmas” (p. 48). 159 MEDIADORES E DESAFIOS HODIERNOS 160 e Tapia (2006) enfatizam a necessidade de criarmos um contexto adequado e apto para receber e analisar os conflitos. Todavia, existem inúmeras estratégias e visões distintas no que diz respeito à promoção da confiança, relativamente ao processo e ao mediador. Aliás, a possibilidade de um mediado não confiar no mediador, levará a que este não volte a reconhecer a mediação como uma opção alternativa. É desta forma que vários autores alertam os profissionais sobre a forma como se envolvem nos processos de mediação. O reconhecido mediador Mangini (2020) revela que o conhecimento e a experiência são alguns dos conceitos-chave que nos possibilitam ser agentes confiáveis perante o mediado. Porém, destaca que a confiança não basta partir apenas do conhecimento do mediador, mas também de um saber que é par­ tilhado por todos, isto é, para que se confie na mediação necessita-se que esta seja validada socialmente (Mangini, 2020): e Tapia (2006) enfatizam a necessidade de criarmos um contexto adequado e apto para receber e analisar os conflitos. Todavia, existem inúmeras estratégias e visões distintas no que diz respeito à promoção da confiança, relativamente ao processo e ao mediador. Aliás, a possibilidade de um mediado não confiar no mediador, levará a que este não volte a reconhecer a mediação como uma opção alternativa. É desta forma que vários autores alertam os profissionais sobre a forma como se envolvem nos processos de mediação. O reconhecido mediador Mangini (2020) revela que o conhecimento e a experiência são alguns dos conceitos-chave que nos possibilitam ser agentes confiáveis perante o mediado. Porém, destaca que a confiança não basta partir apenas do conhecimento do mediador, mas também de um saber que é par­ tilhado por todos, isto é, para que se confie na mediação necessita-se que esta seja validada socialmente (Mangini, 2020): por mais que a Mediação esteja prevista nos ordenamentos jurídicos e seja, de fato, uma prática, é necessário também que ela esteja inserida dentro dos códigos da sociedade, ancorada naquela cultura e mais ainda, vinda de um conhecimento construído por todos os agentes envolvidos. (para.18) Considerações Finais: A Mediação Como uma Área Natu­ ralmente Confiável Termino e começo esta dimensão final, com a alusão a uma reflexão que obtive ao longo do desenvolvimento desta proposta: será que a mediação, já por natureza, é edificada em base da confiança? (RE)PENSAR A FORMAÇÃO EM MEDIAÇÃO Apesar de serem visões um pouco distintas, os autores supramencionados conectam o termo confiança com o processo de mediação, um contexto que deverá assegurar o à-vontade do mediado para partilhar as suas posições e necessidades. Uma outra opinião partilhada entre estes autores é a sensação de desconfiança que ainda existe sobre a realidade da mediação, devido à falta de conhecimento sobre esta metodologia alternativa de resolução de conflitos. Neste aspeto, Mangini (2020) evidencia que este desconhecimento parte da responsabilidade da sociedade, ca­ bendo a esta e aos profissionais envolvidos na área proclamarem a luz da mediação, tentando que esta cresça connosco e que fique enraizada na próxima geração, pois é muito mais fácil confiarmos em algo que já conhecemos, visto que a segurança já é manifestada naturalmente. Efetivamente, quando existe esta incerteza em relação à mediação, a promoção da confiança terá que ser fomentada a partir do primeiro instante que os mediados entram no espaço no qual decorrerá o processo. Tal como já foi mencionado, Diez MEDIADORES E DESAFIOS HODIERNOS (RE)PENSAR A FORMAÇÃO EM MEDIAÇÃO (RE)PENSAR A FORMAÇÃO EM MEDIAÇÃO causa os seus interesses, fazendo com que haja uma desconfiança, principalmente, face ao princípio da cooperação, porque a ideia de nos unirmos com a contraparte leva-nos à falsa ideia de que poderemos sair lesados. Mas como é que podemos re­ verter esta realidade? Não diria que o primeiro passo é (re)formatar a sociedade, mas sim os mediadores. Digo isto, porque acredito que não é qualquer indivíduo que possa ser um verda­ deiro mediador. É preciso acreditar, mais do que ninguém, na força que a mediação sustenta para a transformação das pessoas e do meio. Como será uma sessão de mediação em que o mediador desconfia sobre a possibilidade de as pessoas con­ seguirem, autonomamente, encontrar uma melhor solução? Provavelmente, as suas intervenções iriam transparecer isto mesmo, gerando um ambiente contrário àquilo que se espera, um espaço repleto de dúvidas e inseguranças, podendo até levar os mediados a idealizar uma falsa imagem da mediação. Contrariamente, um mediador otimista em que a sua crença se baseia na união e cooperação, levará a que as pes­ soas envolvidas vivenciem uma experiência completamente diferente, confiando a mediação como um lugar seguro para partilhar as suas vivências. Sem dúvida que, para sermos mediadores, não podemos descartar a nossa pessoa: mediar implica que sejamos humanos, sujeitos empáticos e profissionais que acreditam no seu valor para o auxílio da transformação do mundo. Teria muito mais para dizer, mas o que quero demonstrar é que, por detrás da palavra confiança existem muitos desafios que têm de ser alcançados, acreditando que a for­ mação de mediadores é um dos primeiros passos a serem dados para que a confiança deixe de ser uma possível sensação, mas uma realidade inquestionável na mediação — não podemos ser só e apenas capacitados para dominar a teoria, para redigir acor­ dos ou para ser um expert a reformular questões. É urgente sermos humanos, sermos profissionais com a capacidade de nos envolvermos, neutralmente, com o próximo. É necessário, saber ser sensível e empático no momento certo. É necessário formarmos bons mediadores em prol da construção de espaços seguros. Atenção, a mediação nunca deixou de ser um processo confiável, o meio ao seu redor é que esconde a sua verdadeira essência. MEDIADORES E DESAFIOS HODIERNOS Vejamos que estamos perante um processo que só se desenvolve a partir da voluntariedade, confidencialidade, igualdade e imparcia­ lidade, isto é, princípios que asseguram a singularidade dos sujeitos, no sentido em que estes não terão de sentir o receio de perder a sua essência ou aceitar a derrota de um conflito, pois aqui todos são vencedores. É neste sentido que defendo que a própria forma como a mediação é executada é já, intencionalmente, construída para que o mediado se sinta seguro ao longo de todo o processo, sendo que a questão do espaço e o profissionalismo do mediador são fatores que poderão exibir, ainda mais, esta confiança sobre a mediação. Todavia, o problema é que a mediação ainda não é uma realidade bem consolidada: ainda é uma área desconhecida pela maior parte dos cidadãos; muitos são os profissionais que alegam ser mediadores e, na verdade, não possuem qualquer tipo de formação ou acreditação; o individualismo e a competitividade são ainda características que nos impedem de ser um mundo cooperante na conquista da inclusão e equidade. Acredito vivamente que o meio que nos envolve é uma grande influência na pro­ clamação da mediação. O facto de ainda existirem contextos, sejam eles profis­ sionais ou formativos, em que preservam somente o valor do individualismo, leva a que a sociedade sofra uma formatação egocêntrica em que a hipótese de atuar em conformidade com o outro é desprezada, e que o conflito seja visto como algo comprometedor para o sucesso e o bem-estar individual. Isto pode levar a que algumas pessoas perspetivem a mediação como uma opção que poderá pôr em 161 MEDIADORES E DESAFIOS HODIERNOS MEDIADORES E DESAFIOS HODIERNOS 162 (RE)PENSAR A FORMAÇÃO EM MEDIAÇÃO “Nós”, mediadores, temos que alumiar esta força para que a sombra seja movida, temos que revelar a verdadeira natureza da mediação às pessoas, fazer com que esta faça parte da nossa cultura. A culpa não é daqueles que nunca ouviram falar sobre a mediação, mas sim dos profissionais envolvidos que não a dão a conhecer esta realidade à população. Como é que vão confiar na mediação se desconhecem a sua existência? Hoje existe um maior acesso à informação, é só tirarmos proveito disso. Podemos escrever artigos para jornais e revistas, criar eventos que permitam às pessoas (re)conhecer esta metodologia, partilhar informação nas redes sociais, entre outros. A confiança é acumulativa e não podemos esperar que nasça apenas quando um mediado entra pelo nosso gabinete. A confiança nasce em pequenas “coisas”: dar a conhecer a mediação à sociedade é e será um grande impacto. Referências Aragão, S. (2016, 1 de janeiro). Decifra-me ou te devoro: A importância do autoconhecimento em nosso percurso existencial. Psicologia.pt. https://www.psicologia.pt/artigos/ver_opiniao.php?codigo=AOP0384 Carnegie, D. (2021). Como superar as preocupações e lidar com o stress. Penguin Random House Grupo Editorial Aragão, S. (2016, 1 de janeiro). Decifra-me ou te devoro: A importância do autoconhecimento em nosso percurso existencial. Psicologia.pt. https://www.psicologia.pt/artigos/ver_opiniao.php?codigo=AOP0384 Aragão, S. (2016, 1 de janeiro). Decifra-me ou te devoro: A importância do autoconhecimento em nosso percurso existencial. Psicologia.pt. https://www.psicologia.pt/artigos/ver_opiniao.php?codigo=AOP0384 Carnegie, D. (2021). Como superar as preocupações e lidar com o stress. Penguin Random House Grupo Editorial Aragão, S. (2016, 1 de janeiro). Decifra-me ou te devoro: A importância do autoconhecimento em nosso percurso existencial. Psicologia.pt. https://www.psicologia.pt/artigos/ver_opiniao.php?codigo=AOP0384 percurso existencial. Psicologia.pt. https://www.psicologia.pt/artigos/ver_opiniao.php?codigo=AOP0384 Carnegie, D. (2021). Como superar as preocupações e lidar com o stress. Penguin Random House Grupo Editorial. Carnegie, D. (2021). Como superar as preocupações e lidar com o stress. Penguin Random House Grupo Editorial. Diez, F., & Tapia, G. (2006). Herramientas para trabajar en mediación (1.ª ed). Paidós. Infopédia. Dicionários da Porto Editora. (s.d.). Confiança. https://www.infopedia.pt/dicionarios/lingua­ -portuguesa/confiança Lei n.º 29/2013, de 19 de abril, Diário da República n.º 77, Série I de 2013-04-19 (2013). https://files.dre. pt/1s/2013/04/07700/0227802284.pdf Mangini, Z. (2020, 7 de maio). A confiança na mediação. Algi Mediação. https://algimediacao.com. br/2021/a-confianca-na-mediacao/ Pinto, A., & Mendes, J. (2013). Os princípios gerais aplicáveis à mediação e o regime da mediação civil e comercial em Portugal. Actualidad Jurídica Uría Menéndez, 35, 143–145. Quintanilha, A. (2021). Caso 1. In Federação Nacional de Mediação de Conflitos (Ed.). Casos práticos de me­ diação de conflitos: Relatos reais (pp.1–12). Pactor — Edições de Ciências Sociais, Forenses e da Educação. Reis, C. (2021). Caso 3. In Federação Nacional de Mediação de Conflitos (Ed.). Casos práticos de mediação de conflitos: Relatos reais (pp.25–36). Pactor — Edições de Ciências Sociais, Forenses e da Educação. Sousa, M. R. (2020). Gestão de emoções na mediação. Jornal Jurídico, 2(2), 3–16. https://doi.org/10.29073/ j2.v2i2.217
https://openalex.org/W3129582904
https://scholar.ummetro.ac.id/index.php/poace/article/download/610/320
Indonesian
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EVALUASI SISTEM PENJAMINAN MUTU INTERNAL (SPMI) PADA SEKOLAH MODEL LAMPUNG TIMUR
POACE
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ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) POACE: Jurnal Program Studi Administrasi Pendidikan Volume 1, No. 1, Februari 2021, 20-32 Abstrak Lembaga Penjaminan Mutu Pendidikan (LPMP) Propinsi Lampung mengembangkan satuan pendidikan yang dipilih untuk menjadi sekolah model didalam menjalankan Sistem Penjaminan Mutu Internal (SPMI) yang harapannya dapat dijadikan contoh oleh satuan pendidikan yang lain didalam menerapkan penjaminan mutu pendidikan secara mandiri. Fokus dari penelitian kualitatif ini adalah: (a) Bagaimana sosialisasi Permendikbud No. 28 tahun 2016 tentang Sistem Penjaminan Mutu Pendidikan Dasar dan Menengah kepada warga sekolah model dan sekolah imbas, (b) Bagaimana implementasi (SPMI) di Sekolah Model oleh Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS), (c) Bagaimana peran kepala sekolah memfasilitasi TPMPS didalam mengimplementasikan SPMI, (d) Bagaimana tingkat efektifitas kegiatan pendampingan yang dilakukan oleh Fasda di sekolah model, (e) Bagaimana peran dan tanggungjawab Dinas Pendidikan Kabupaten melalui Tim Penjaminan Mutu Pendidikan Daerah (TPMPD) di dalam menjalankan fungsi pembinaan, pembimbingan, pendampingan serta supervisi terhadap pelaksanaan SPMI di sekolah model. Berdasarkan hasil analisis dalam penelitian ini diperoleh kesimpulan bahwa: (a) Kegiatan sosialisasi oleh TPMPS disetiap sekolah model terhadap warga sekolah sudah berjalan dengan baik, terjadwal dan melibatkan seluruh anggota TPMPS, (b) Implementasi SPMI oleh TPMPS yang meliputi tahap pemetaan mutu, perencanaan program pemenuhan mutu, pelaksanaan program pemenuhan mutu, evaluasi dan monitoring serta penetapan standar baru sebagai bentuk tahap tindak lanjut dari pelaksanaan program secara umum sudah dapat berjalan sesuai dengan aturan dari Permendikbud No.28 Tahun 2016, jika mungkin ada kendala dan kelemahan itu karena program perencanaan pemenuhan mutu yang dibuat oleh TPMPS belum dapat masuk pada RKAS sehingga berdampak belum maksimalnya pelaksanaan program pemenuhan mutu, (c) peran kepala sekolah model dalam memfasilitasi TPMPS melaksanakan implementasi SPMI sudah berjalan baik, hanya perlu ditingkatkan dalam hal melibatkan pihak dinas pendidikan dan unsur masyarakat melalui komite sekolah, (d) peran Fasda sudah cukup efektif didalam menjalankan kegiatan pendampingan di sekolah model dan (e) peran serta tanggungjawab dinas pendidikan kabupaten melalui Tim Penjaminan Mutu pendidikan Daerah (TPMPD) sampai dengan selesainya penelitian ini belum begitu kelihatan peran sertanya dalam menjalankan Sistem penjaminan Mutu Eksternal (SPME). Kata Kunci: Evaluasi, Sistem Penjaminan Mutu Internal (SPMI), Sekolah Model. ata Kunci: Evaluasi, Sistem Penjaminan Mutu Internal (SPMI), Sekolah Model. EVALUASI SISTEM PENJAMINAN MUTU INTERNAL (SPMI) PADA SEKOLAH MODEL LAMPUNG TIMUR Elvina Maya Puspa1, Agus Sutanto2*, Bambang Suhada3 1,2*,3 Universitas Muhammadiyah Metro, Lampung, Indonesia E-mail: elvina.taher15@gmail.com1) Abstract The Lampung Province Education Quality Assurance Institution (LPMP) has developed an educational unit chosen to become a model school in implementing the Internal Quality Assurance System (SPMI) which is hoped to be used as an example by other educational units in implementing education quality assurance independently. The focus of this qualitative research is: (a) How does the socialization of Permendikbud No. 28 of 2016 concerning the Quality Assurance System for Primary and Secondary Education to members of model schools and impact schools, (b) How is the implementation (SPMI) in Model Schools by the School Education Quality Assurance Team (TPMPS), (c) How is the role of the principal in facilitating TPMPS in implementing SPMI, (d) What is the level of effectiveness of assistance activities carried out by Fasda in model schools, (e) What are the roles and responsibilities of the District Education Office through the Regional Education Quality Assurance Team (TPMPD) in carrying out the functions of guidance, mentoring, mentoring and supervision of implementation of SPMI in model schools. Based on the results of the analysis in this study, it is concluded that: (a) The socialization activities by TPMPS in each model school to school members have been going well, are scheduled and involve all TPMPS members, (b) Implementation of SPMI by TPMPS which includes the quality mapping 20 POACE: Jurnal Program Studi Administrasi Pendidikan Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) PENDAHULUAN Penjaminan Mutu pendidikan adalah suatu mekanisme yang sistematis, terintegrasi, dan berkelanjutan untuk memastikan bahwa seluruh proses penyelenggaraan pendidikan telah sesuai dengan standar mutu. Terbitnya Permendikbud No. 28 Tahun 2016 tentang Sistem Penjaminan Mutu Pendidikan Dasar dan Menengah (SPMPDM) memberikan penjelasan teknis tentang penerapan sistem penjaminan mutu pendidikan yang memuat ketentuan umum, fungsi dan tujuan SPMPDM, pembagian tugas dan wewenang, pemantauan dan evaluasi, sanksi dan ketentuan penutup. Dalam rangka mendorong tersosialisasinya Permendikbud No. 28 tahun 2016 serta ikut mengawal agar sistem penjaminan mutu internal dapat diimplementasikan di setiap satuan pendidikan khususnya di daerah Propinsi Lampung, maka sesuai dengan tugas pokok dan fungsinya LPMP Lampung menyelenggarakan pembinaan terhadap beberapa sekolah terpilih untuk dijadikan sebagai sekolah model yang mampu menjalankan Sistem Penjaminan Mutu Internal (SPMI) dan harapannya setiap sekolah model yang telah dibina dan sekaligus dibiayai melalui dana bantuan pemerintah dapat mengimbaskan penerapan sistem penjaminan mutu internal ke satuan pendidikan yang lainnya. Sekolah model adalah sekolah yang ditetapkan dan dibina oleh Lembaga Penjamin Mutu Pendidikan (LPMP) untuk menjadi sekolah acuan bagi sekolah lain di sekitarnya dalam penerapan Sistem Penjaminan Mutu Internal (SPMI). POACE: Jurnal Program Studi Administrasi Pendidikan Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) stage, program planning fulfillment of quality, implementation of quality compliance programs, evaluation and monitoring as well as setting new standards as a form of follow-up phase of program implementation in general can run according to the regulations of Permendikbud No.28 of 2016, if possible there are obstacles and weaknesses because of the compliance planning program. the quality made by TPMPS has not been able to enter the RKAS so that the impact on the implementation of the quality compliance program has not been maximized, (c) the role of the model school principal in facilitating TPMPS in implementing the SPMI has been going well, it only needs to be improved in terms of involving the education office and community elements through the committeeschools, (d) the role of Fasda has been quite effective in carrying out assistance activities in model schools and (e) the role and responsibilities of the district education office through the Regional Education Quality Assurance Team (TPMPD) until the completion of this research has not been so visible that its participation in implementing the guarantee system External Quality (SPME). Keywords: Evaluation, Internal Quality Assurance System (SPMI), Model School. Keywords: Evaluation, Internal Quality Assurance System (SPMI), Model School. POACE: Jurnal Program Studi Administrasi Pendidikan Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) e. Bagaimana proses pembinaan, pembimbingan, pendampingan supervisi dan evaluasi dari Tim penjaminan Mutu Daerah (TPMPD), terhadap pelaksanaan implementasi Sistem Penjaminan Mutu Pendidikan Internal (SPMI) di sekolah model? e. Bagaimana proses pembinaan, pembimbingan, pendampingan supervisi dan evaluasi dari Tim penjaminan Mutu Daerah (TPMPD), terhadap pelaksanaan implementasi Sistem Penjaminan Mutu Pendidikan Internal (SPMI) di sekolah model? Fokus Penelitian a. Bagaimana bentuk-bentuk kegiatan sosialisasi Permendikbud No. 28 tahun 2016, tentang Sistem Penjaminan Mutu Pendidikan Dasar dan Menengah, kepada warga sekolah model? b. Bagaimana upaya yang dilakukan Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS) didalam melaksanakan keseluruhan tahapan Sistem Penjaminan Mutu Internal (SPMI)? c. Bagaimana peran yang seharusnya dilakukan oleh Kepala Se kolahterhadap TPMPS dalam mengimplementasikan sistem penjaminan mutu internal sesuai dengan tugasnya? d. Bagaimana tingkat efektifitas kegiatan pendampingan yang dilakukan oleh fasilitator daerah (Fasda) di sekolah model? 21 POACE: Jurnal Program Studi Administrasi Pendidikan ISSN 2775-6564 (Print) Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-7048 (Online) POACE: Jurnal Program Studi Administrasi Pendidikan Volume 1, No. 1, Februari 2021, 20-32 Tinjauan Pustaka 1, Februari 2021, 20-32 ISSN 2775-7048 (Online) POACE: Jurnal Program Studi Administrasi Pendidikan Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) dengan pendapat M. Ihsan Dacholfany (2017: 6) bahwa “Dalam sebuah organisasi, peranan sumber daya manusia sangat urgent dan penting. Peran sumber daya manusia ini akan maksimal jika dikelola dengan baik. Pimpinan lembaga pendidikan sebagai top leader dalam lembaga sekolah mempunyai peran sentral dalam pengelolaan personalia sehingga sangat penting bagi pimpinan lembaga pendidikan untuk memahami dan menerapkan pengelolaan personalia dengan baik dan benar” dengan pendapat M. Ihsan Dacholfany (2017: 6) bahwa “Dalam sebuah organisasi, peranan sumber daya manusia sangat urgent dan penting. Peran sumber daya manusia ini akan maksimal jika dikelola dengan baik. Pimpinan lembaga pendidikan sebagai top leader dalam lembaga sekolah mempunyai peran sentral dalam pengelolaan personalia sehingga sangat penting bagi pimpinan lembaga pendidikan untuk memahami dan menerapkan pengelolaan personalia dengan baik dan benar” Berdasarkan Peraturan Menteri Pendidikan Dan Kebudayaan Republik Indonesia Nomor 28 tahun 2016, Pasal 11 Ayat (4), tugas dari tim penjaminan mutu satuan pendidikan (TPMPS) adalah: Berdasarkan Peraturan Menteri Pendidikan Dan Kebudayaan Republik Indonesia Nomor 28 tahun 2016, Pasal 11 Ayat (4), tugas dari tim penjaminan mutu satuan pendidikan (TPMPS) adalah: p a. Mengorganisasikan pelaksanaan penjaminan mutu di tingkat satuan pendidikan; b. Melakukan pembinaan, pengembangan, pendampingan, dan supervisi terhadap pelaku pendidikan di satuan pendidikan dalam pengembangan dan penjaminan mutu pendidikan; c. Melaksanakan pemetaan mutu pendidikan berdasarkan data mutu pendidikan di satuan pendidikan d. Melakukan monitoring dan evaluasi proses pelaksanaan pemenuhan mutu yang telah dilakukan ; dan e. Memberikan rekomendasi strategi peningkatan mutu baerdasarkan hasil monitong dan evaluasi kepada kepala setuan pendidikan. Sekolah model adalah sekolah yang ditetapkan dan dibina oleh Lembaga Penjamin Mutu Pendidikan (LPMP) untuk menjadi sekolah acuan bagi sekolah lain di sekitarnya dalam menerapkan Sistem Penjaminan Mutu Internal (SPMI). Sekolah model menerapkan seluruh siklus penjaminan mutu pendidikan secara sistematik, holistic, dan berkelanjutan, sehingga budaya mutu tumbuh dan berkembang secara mandiri pada sekolah tersebut. Keberhasilan sekolah memberikan layanan pendidikan yang berkualitas bagi seluruh peserta didik melalui penerapan SPMI sangat tergantung pada komitmen dan kesungguhan kepala sekolah dan warga sekolah terutama dari peran serta dari Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS) dalam menerapkan Sistem Penjaminan Mutu Internal (SPMI). Tinjauan Pustaka Melalui program sekolah model ini sesungguhnya Kementrian Pendidikan dan kebudayaan telah menunjuk sekolah-sekolah yang dijadikan piloting dalam menerapkan sistem penjaminan mutu internal yang selanjutnya pembinaan sekolah-sekolah model tersebut diamanatkan kepada Lembaga Penjaminan Mutu Pendidikan (LPMP). Harapannya sekolah yang sudah dibina ini mampu mengimbaskan kepada sekolah lain bagaimana mengimplementasikan sistem penjaminan mutu internal. Tinjauan Pustaka Permendiknas No. 63 Tahun 2009 tentang Sistem Penjaminan Mutu Pendidikan yang ditetapkan pada tanggal 25 September 2009, menyebutkan bahwa “Penjaminan mutu pendidikan adalah kegiatan sistemik dan terpadu oleh satuan atau program pendidikan, penyelenggara satuan atau program pendidikan, pemerintah daerah, pemerintah, dan masyarakat untuk menaikkan tingkat kecerdasan kehidupan bangsa melalui pendidikan” Menurut Barnawi, M.Arifin (2017: 25) mengatakan bahwa Sistem Penjaminan Mutu Pendidikan (SPMP) merupakan bagian penting yang tidak dapat dipisahkan dari sistem peningkatan mutu, yang mencakup dua kegiatan besar yaitu: pertama, peningkatan mutu yang dilandasi dengan target mutu yang sekolah harapkan. Kedua, mengukur mutu pencapaian kinerja untuk mengetahui tingkat pemenuhan standar berdasarkan target program yang telah ditetapkan. Apabila sistem ini dijalankan dengan baik, lembaga pendidikan akan terbiasa dengan budaya peningkatan mutu. Dari penjelasan diatas, dari pendapat diatas dapat disimpulkan bahwa Pengertian Penjaminan Mutu Pendidikan adalah suatu mekanisme yang sistematis, terintegrasi, dan berkelanjutan untuk memastikan bahwa seluruh proses penyelenggaraan pendidikan telah sesuai dengan standar mutu. Sesuai dengan isi Peraturan Menteri Pendidikan dan kebudayaan No. 28 Tahun 2016, Sistem Penjaminan Mutu Internal (SPMI) adalah sistem penjaminan mutu yang dilakukan oleh seluruh komponen dalam satuan pendidikan. Pelaksanaan SPMI dimaksudkan agar pemenuhan mutu dapat direncanakan, dan dievaluasi secara internal oleh satuan pendidikan. Agar budaya mutu dapat diciptakan disetiap satuan pendidikan, maka dibutuhkan sebuah proses penjaminan mutu secara internal yang digerakkan oleh orang atau sekelompok orang yang memilki tugas dan tanggung jawab untuk melakukan proses kegiatan penjaminan mutu tersebut, hal ini sejalan dengan pendapat yang dikemukakan oleh Ridwan A. Sani Dkk (2018: 9) menyatakan bahwa “Upaya menjamin mutu sebuah satuan pendidikan harus dilakukan dengan menerpakan manajemen mutu atau sistem penjaminan mutu. Kepala sekolah harus membentuk sebuah tim yang membantunya dalam melakukan penjaminan mutu, karena sebuah proses pendidikan merupakan proses yang komplek” Tim Penjaminan Mutu Pendidkan Sekolah (TPMPS) adalah “Sekelompok orang yang ditugaskan oleh kepala sekolah untuk melakukan tugas pelaksanaan proses penjaminan mutu pendidikan secara internal di tingkat sekolah”. Tim penjaminan mutu pendidikan sekolah tersebut paling sedikit terdiri atas: (1) perwakilan pimpinan sekolah; (2) perwakilan guru; (3) perwakilan tenaga kependidikan; dan (4) perwakilan komite sekolah. TPMPS akan dapat bekerja secara maksimal jika kepala sekolah selaku top manajer disebuah lembaga pendidikan mampu mengorganisir tim tersebut, hal ini sesuai 22 POACE: Jurnal Program Studi Administrasi Pendidikan ISSN 2775-6564 (Print) Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-7048 (Online) POACE: Jurnal Program Studi Administrasi Pendidikan ISSN 2775-6564 (Print) Volume 1, No. a. Untuk mendeskripsikan upaya Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS) Model didalam melakukan sosialisasi Permendikbud No. 28 Tahun 2016 kepada warga sekolah model dan sekolah imbas. Deskripsi Penelitian Selanjutnya untuk menggambarkan upaya dari TPMPS dalam menerapkan Sistem Penjaminan Mutu Internal di Sekolah Model jenjang SMP kabupaten Lampung Timur, dapat dirumuskan tujuan secara rinci, sebagai berikut: a. Untuk mendeskripsikan upaya Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS) Model didalam melakukan sosialisasi Permendikbud No. 28 Tahun 2016 kepada warga sekolah model dan sekolah imbas. a. Untuk mendeskripsikan upaya Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS) Model didalam melakukan sosialisasi Permendikbud No. 28 Tahun 2016 kepada warga sekolah model dan sekolah imbas. 23 POACE: Jurnal Program Studi Administrasi Pendidikan ISSN 2775-6564 (Print) Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-7048 (Online) POACE: Jurnal Program Studi Administrasi Pendidikan Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) Untuk mendeskripsikan upaya Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS) b. Untuk mendeskripsikan upaya Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS) Model didalam menerapkan siklus Sistem Penjaminan Mutu Internal (SPMI). b. Untuk mendeskripsikan upaya Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS Model didalam menerapkan siklus Sistem Penjaminan Mutu Internal (SPMI). p p y j Model didalam menerapkan siklus Sistem Penjaminan Mutu Internal (SPMI). c. Untuk mendeskripsikan peran kepala sekolah model terhadap TPMPS dalam mengimplementasikan SPMI. c. Untuk mendeskripsikan peran kepala sekolah model terhadap TPMPS dalam mengimplementasikan SPMI. d. Untuk mendeskripsikan apakah proses pendampingan yang dilakukan oleh Fasilitator daerah (Fasda) di sekolah model sudah sesuai dengan juknis dari LPMP Lampung. d. Untuk mendeskripsikan apakah proses pendampingan yang dilakukan oleh Fasilitator daerah (Fasda) di sekolah model sudah sesuai dengan juknis dari LPMP Lampung. e. Untuk mendeskripsikan upaya dan peran Tim Penjaminan Mutu Pendidikan Daerah (TPMPD) didalam melaksanakan Sistem Penjaminan Mutu Eksternal (SPME) sesuai dengan Permendikbud No. 28 tahun 2016. e. Untuk mendeskripsikan upaya dan peran Tim Penjaminan Mutu Pendidikan Daerah (TPMPD) didalam melaksanakan Sistem Penjaminan Mutu Eksternal (SPME) sesuai dengan Permendikbud No. 28 tahun 2016. METODE PENELITIAN (15%) akan diteliti terhadap 5 (lima) sekolah model, dengan demikian terdapat 5 (lima) Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS) di jenjang SMP. HASIL DAN PEMBAHASAN (70%) Pembahasan hasil penelitian difokuskan dalam 5 (lima) hal, yaitu; (1) upaya yang dilakukan oleh TPMPS didalam melaksanakan sosialisasi Permendikbud No. 28 tahun 2016 (2) Upaya yang dilakukan oleh TPMPS didalam menajalankan siklus Sistem Penjaminan Mutu Internal (SPMI) (3) Peran kepala sekolah dalam memfasilitasi TPMPS didalam mengimplementasikan siklus Sistem Penjaminan Mutu Internal (SPMI) (4) Untuk mengetahui efektifitas pendampingan yang dilakukan oleh Fasilitator Daerah (Fasda) didalam melakukan pendampingan di sekolah model (5) Untuk mengetahui peran Tim Penjaminan Mutu Pendidikan Daerah (TPMPD) didalam menjalankan tupoksinya sesuai Permendikbud No. 28 Tahun 2016. METODE PENELITIAN (15%) Menurut Yanuar Ikbar (2014: 105) Metode penelitian adalah suatu pembelajaran tentang metode ilmiah yang meliputi penetapan masalah, premis, hipotesis, tujuan, kegunaan, tinjauan pustaka, metode penelitian, pembahasan hasil penelitian, dan cara menarik kesimpulan yang bertujuan memperbaiiki prosedur dan kriteria baku dalam penelitian ilmiah, selanjutnya menurut Sugiyono (2018: 210) metode penelitian kualitatif adalah metode penelitian yang digunakan untuk meneliti pada kondisi obyek yang alamiah dimana peneliti adalah sebagai instrumen kunci, teknik pengumpulan data dilakukan secara triangulasi (gabungan), analisis data bersifat induktif, dan hasil penelitian, kualitatif lebih menekankan makna dan keunikan daripada generalisasi. sementara menurut Suharsimi Arikunto (2007: 222) penelitian evaluasi dapat diartikan suatu proses yang dilakukan dalam rangka menentukan kebijakan dengan terlebih dahulu mempertimbangkan nilai-nilai positif dan keuntungan suatu program, serta mempertimbangkan proses serta teknik yang telah digunakan untuk melakukan suatu penelitian. Sugiyono (2018: 1) penelitian evaluasi digunakan untuk mengetahui apakah suatu program atau aktivitas telah mencapai tujuan yang ditetapkan, dengan alasan antara lain: 1. Penelitian ini bersifat deskriptif analitis, hal ini dapat dilihat dari cara mengumpulkan dan merekap data dengan membuat penjelasan sejelas-jelasnya. dengan pendekatan induktif, yaitu penelitian yang dimulai dari data atau gejala yang ada dilapangan yang kemudian memunculkan teori. 2. Penelitian ini berfokus pada makna yang terdapat dalam suatu fenomena yang teliti, yang dapat digali dari persepsi objek penelitian tentang upaya yang dilakukan oleh Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS)di dalam melaksanakan Sistem Penjaminan Mutu Internal (SPMI) di sekolah model dan mengutamakan akan pentingnya proses penelitian yang berjalan. Bukan hanya mengacu pada hasil yang ingin dicapai. Dalam penelitian ini, peneliti memperhatikan dan menelaah fokus fenomena yang akan diteliti, dengan melihat berbagai aspek subjektif dari perilaku objek. Selanjutnya peneliti akan melakukan penggalian data berupa bagaimana pemaknaan objek dalam memberikan arti terhadap fenomena terkait. Subjek dalam penelitian ini adalah Tim Penjaminan Mutu Pendidikan Sekolah Model (TPMPS), sedangkan fokus yang diteliti adalah upaya Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS) dalam menerapkan Sistem Penjaminan Mutu Internal (SPMI) di sekolah model. Dengan jumlah sekolah model jenjang SMP di kabupaten Lampung Timur berjumlah 7 sekolah, karena terbatasnya kondisi dan kemampuan yang ada pada peneliti, maka dalam penelitian ini hanya 24 POACE: Jurnal Program Studi Administrasi Pendidikan Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) akan diteliti terhadap 5 (lima) sekolah model, dengan demikian terdapat 5 (lima) Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS) di jenjang SMP. POACE: Jurnal Program Studi Administrasi Pendidikan Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) Tetapi pada indikator adanya anggaran khusus untuk biaya operasional TPMPS dalam RKAS nya belum ada yang mengalokasikan untuk biaya operasional. Hal lain yang menjadi temuan dalam penelitian ini adalah banyaknya tim-tim lain disekolah yang belum terintegrasi kedalam wadah TPMPS, sehingga terjadinya beban kerja yang besar dan proses pengelolaan sekolah menjadi tumpang tindih. Hal ini terlihat di hasil observasi pada indikator “terintegrasi dengan tim-tim lain” p g g Sosialisasi Permendikbud No. 28 tahun 2016 secara umum telah dilakukan oleh sekolah model dengan melibatkan seluruh guru dan perwakilan sekolah imbas sesuai ketentuan. Hal-hal yang disampaikan dalam kegiatan sosialisasi terdiri dari (1) tentang isi Permendikbud No. 28 Tahun 2016, (2) tentang pengertian Sistem Penjaminan Mutu Internal (SPMI) dan Sistem Penjaminan Mutu Eksternal (SPME), (3) Struktur Organisasi TPMPS), (4) Tugas TPMPS, (5) Siklus atau tahapan SPMI, (6) Membaca Rapor Mutu Sekolah dan melakukan pemetaan mutu sekolah. Kegiatan Sosialisasi Permendikbud No. 28 tahun 2016 yang dilakukan oleh TPMPS. Berdasarkan hasil wawancara terhadap seluruh responden di 5 (lima) sekolah model tentang bentuk-bentuk kegiatan sosialisasi Permendikbud No. 28 tahun 2016, diperoleh gambaran hasil penelitian sebagai berikut Tabel 1. Hasil kegiatan Sosialisasi Nama Sekmod Bentuk – Bentuk Sosialisasi Keterangan Sistem Penjaminan Mutu (SPMI dan SPME) SK. Dan Struktur TPMPS Rapor Mutu dan Siklus SPMI Standar Nasional Pendidikan (SNP) S B S B S B S B SMPN 1 Sekampung Udik     Ke 5 sekolah model sudah melakukan sosialisasi Permendikbud No.28 Tahun 2016. Indikatornya sudah memiliki SK TPMPS dan Struktur Organisasi. SMPN 1 Marga Sekampung     SMPN 2 Batanghari     SMPN 1 Bumi Agung     SMPN 1 Way Bungur     Keterangan: S= Sudah, B= Belum Dari tabel diatas terlihat bahwa sosialisasi telah dilakukan oleh seluruh sekolah model, yang berkaitan dengan (1) Sistem penjaminan Mutu Internal, (2) Pemetaan mutu melalui pembacaan rapor mutu sekolah dan siklus SPMI, (3) Surat Keputusan Kepala Sekolah tentang TPMPS serta Struktur Organisasi TPMPS sudah dapat berjalan dengan baik , semua sekolah model telah memiliki SK. Kepala Sekolah tentang TPMPS beserta struktur organisasinya, hal ini sesuai dengan hasil observasi yang dilakukan dengan kode Ob. K1,2,3,4,5/I. Dari tabel diatas terlihat bahwa sosialisasi telah dilakukan oleh seluruh sekolah model, yang berkaitan dengan (1) Sistem penjaminan Mutu Internal, (2) Pemetaan mutu melalui pembacaan rapor mutu sekolah dan siklus SPMI, (3) Surat Keputusan Kepala Sekolah tentang TPMPS serta Struktur Organisasi TPMPS sudah dapat berjalan dengan baik , semua sekolah model telah memiliki SK. Kepala Sekolah tentang TPMPS beserta struktur organisasinya, hal ini sesuai dengan hasil observasi yang dilakukan dengan kode Ob. K1,2,3,4,5/I. Dari tabel diatas terlihat bahwa sosialisasi telah dilakukan oleh seluruh sekolah model, yang berkaitan dengan (1) Sistem penjaminan Mutu Internal, (2) Pemetaan mutu melalui pembacaan rapor mutu sekolah dan siklus SPMI, (3) Surat Keputusan Kepala Sekolah tentang TPMPS serta Struktur Organisasi TPMPS sudah dapat berjalan dengan baik , semua sekolah model telah memiliki SK. Kepala Sekolah tentang TPMPS beserta struktur organisasinya, hal ini sesuai dengan hasil observasi yang dilakukan dengan kode Ob. K1,2,3,4,5/I. 25 POACE: Jurnal Program Studi Administrasi Pendidikan Volume 1, No. 1, Februari 2021, 20-32 Peran Kepala Sekolah Model terhadap Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS) dalam mengimplementasikan SPMI. g p Dari data yang diperoleh, maka dapat digambarkan peran kepala sekolah sesuai dengan tupoksinya sebagai berikut: Tabel 3. Peran Kepala Sekolah dalam mengimplementasikan SPMI Tabel 3. Peran Kepala Sekolah dalam mengimplementasikan SPMI Nama Sekmod Peran Kepala Sekolah Keterangan Membuat SK. TPMPS Memberikan Fasilitas kepada TPMPS Dalam melakukan SPMI Memiliki anggaran untuk operasioal TPMPS di RKAS Melibatkan pemangku kepentingan terhadap pelaksanaan siklus SPMI S B Y T Y T Y T SMPN 1 Sekampung Udik     Terdapat 2 sekmod peran kepala sekolah belum maksimal dalam hal melibatkan pemangku kepentingan SMPN 1 Marga Sekampung     SMPN 2 Batanghari     SMPN 1 Bumi Agung     SMPN 1 Way Bungur     Keterangan: S= Sudah, B= Belum, Y= Ya, T=Tidak Sesuai dengan peran dan tanggungjawabnya sebagai manajer, maka sudah barang tentu keterlibatan kepala sekolah sangat menentukan berhasil tidaknya program pendampingan sekolah model. Sesuai dengan hasil penelitian yang telah dilakukan diperoleh gambaran bahwa dari 5(lima) sekolah model terdapat 3 sekolah model yang kepala sekolahnya telah melakukan kegiatan dalam bentuk (1) Membuat SK. TPMPS, (2) memberikan fasilitas kepada TPMPS untuk melaksanakan siklus SPMI, (3) menyiapkan anggaran diluar yang diberikan oleh pihak LPMP dari dana sekolah (BOS), (4) melibatkan pemangku kepentingan dalam hal ini Dinas Pendidikan dan komite sekolah. Tetapi ada 2 (dua) sekolah model yang kepala sekolahnya belum mampu melaksanakan peran nya dalam hal Menyiapkan (1) anggaran khusus dari dana BOS untuk kegiatan pendampingan sekolah model dan (2) belum melibatkan pemangku kepentingan yaitu pihak dinas pendidikan dan komite sekolah, yaitu SMPN 2 Batanghari dan SMPN 1 Way Bungur. Upaya Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS) dalam melaksanakan siklus SPMI. Upaya yang dilakukan TPMPS dalam rangka mengimplementasikan Sistem Penjaminan Mutu Internal (SPMI), dapat dijelaskan sebagai berikut: Tabel 2. Kegiatan yang dilakukan TPMPS Nama Sekmod Kegiatan yang dilakukan TPMPS Keterangan Semua anggota TPMPS terlibat dalam proses pemetaan mutu Semua nggota TPMPS terlibat dalam proses perencanaan mutu Semua nggota TPMPS terlibat dalam proses pelaksanaan pemenuhan mutu Seluruh anggota Tim Auditor terlibat dalam proses moneva S B S B S B S B SMPN 1 Sekampung Udik     Upaya TPMPS dalam meng - implementasikan SPMI sudah berjalan baik. Ada 2 sekmod yang belum maksimal SMPN 1 Marga Sekampung     SMPN 2 Batanghari     SMPN 1 Bumi Agung     SMPN 1 Way Bungur     Keterangan: S= Sudah, B= Belum Tabel 2. Kegiatan yang dilakukan TPMPS Berdasarkan tumuan data hasil penelitian tentang bagaimana upaya TPMPS dalam rangka mengimplementasikan tahapan/siklus SPMI secara garis besar dapat berjalan dengan baik, terdapat 2 (dua) sekolah yaitu SMPN 1 Sekampung Udik dan SMPN 2 Batanghari yang pada siklus kegiatan proses pemetaan mutu , proses perencanaan pemenuhan mutu dan proses kegiatan pemenuhan mutu tidak semua anggota tim terlibat, hanya ada beberapa anggota yang hadir, hal ini sesuai dengan hasil observasi yang dilakukan dengan kode Ob.T1,3 / II. Hal lainnya yang dapat disampaikan sesuai data yang diperoleh adalah Tim Auditor Internal dari ke 5 (lima) sekolah model tersebut belum melaksanakan tugas monitoring dan dan evaluasi (moneva) setelah selesainya kegiatan pemenuhan mutu, hal ini terlihat pada hasil observasi dengan kode Ob. T 1,2,3,4,5/II pada indikator “seluruh anggota tim audit terlibat dalam proses monitoring dan evaluasi”. Hal ini terjadi 26 POACE: Jurnal Program Studi Administrasi Pendidikan ISSN 2775-6564 (Print) Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-7048 (Online) POACE: Jurnal Program Studi Administrasi Pendidikan Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) disebabkan kurangnya pemahaman tim audit internal tersebut terhadap tugas pokok dan fungsi sebagai anggota tim audit internal yang merupakan bagian yang tidak terpisahkan dari keberadaan TPMPS. disebabkan kurangnya pemahaman tim audit internal tersebut terhadap tugas pokok dan fungsi sebagai anggota tim audit internal yang merupakan bagian yang tidak terpisahkan dari keberadaan TPMPS. Peran Kepala Sekolah Model terhadap Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS) dalam mengimplementasikan SPMI. terhadap sekolah model. Berdasarkan data yang diperoleh, ditemukan hal-hal sebagai berikut: 27 POACE: Jurnal Program Studi Administrasi Pendidikan ISSN 2775-6564 (Print) Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-7048 (Online) POACE: Jurnal Program Studi Administrasi Pendidikan Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) Tabel 4. Kegiatan Fasilitator Daerah terhadap Sekolah Model Nama Sekmod Kegiatan yang dilakukan Fasilitator Daerah (Fasda) Keterangan Bimtek SPMI Menyampaikan Materi (Jelas, bisa dipahami, jelas) Waktu pelaksanaan kegiatan pendampingan Sekmod Efektifitas kegiatan pendampingan S B J TJ sesuai tidak efektif tidak SMPN 1 Sekampung Udik     Ada 2 sekmod yang menyatakan bahwa waktu proses pendamping-an tidak tepat pelaksanaan nya SMPN 1 Marga Sekampung     SMPN 2 Batanghari     SMPN 1 Bumi Agung     SMPN 1 Way Bungur     Keterangan: S= Sudah, B= Belum, J= Jelas, TJ= Tidak Jelas Tabel 4. Kegiatan Fasilitator Daerah terhadap Sekolah Model Tabel 4. Kegiatan Fasilitator Daerah terhadap Sekolah Model Nama Sekmod Kegiatan yang dilakukan Fasilitator Daerah (Fasda) Keterangan Bimtek SPMI Menyampaikan Materi (Jelas, bisa dipahami, jelas) Waktu pelaksanaan kegiatan pendampingan Sekmod Efektifitas kegiatan pendampingan S B J TJ sesuai tidak efektif tidak SMPN 1 Sekampung Udik     Ada 2 sekmod yang menyatakan bahwa waktu proses pendamping-an tidak tepat pelaksanaan nya SMPN 1 Marga Sekampung     SMPN 2 Batanghari     SMPN 1 Bumi Agung     SMPN 1 Way Bungur     Keterangan: S= Sudah, B= Belum, J= Jelas, TJ= Tidak Jelas Dari hasil kegiatan wawancara dan observasi langsung ke 5 (lima) sekolah model secara garis besar dapat digambarkan sebagai berikut : bahwa pelaksanaan pendampingan sekolah model oleh Fasilitator daerah (Fasda) sudah berjalan dengan baik sesuai ketentuan dan sudah cukup efektif hal ini terlihat dari capaian kegiatan yang dapat selesai tepat waktu sesuai dengan yang disediakan oleh pihak LPMP. Ada 2 (dua) sekolah yang menjawab berbeda dengan 3 (tiga) sekolah model yang lainnya yaitu masalah waktu pelaksanaan pendampingan yang menurut mereka kurang tepat karena diadakan di pertengahan tahun, seharusnya pelaksanaan pendampingan dilaksanakan di awal tahun sehingga semua program dapat diamasukkan kedalam anggaran sekolah (RKAS). Proses pembinaan, pembimbingan, pendampingan supervisi dan evaluasi dari Tim Penjaminan Mutu Pendidikan daerah (TPMPD), terhadap pelaksanan Sistem Penjaminan Mutu Pendidikan Internal (SPMI) di sekolah model. Proses pembinaan, pembimbingan, pendampingan supervisi dan evaluasi dari Tim Penjaminan Mutu Pendidikan daerah (TPMPD), terhadap pelaksanan Sistem Penjaminan Mutu Pendidikan Internal (SPMI) di sekolah model. Dari hasil wawancara dan observasi yang telah dilaksanakan di 5 (lima) sekolah model terhadap peran TPMPD terhadap pelaksanaan implementasi SPMI di sekolah model oleh TPMPS, dapat digambarkan sebagai berikut: Tabel 5. Peran TPMPD Nama Sekmod Peran TPMPD Keterangan Melakukan sosialisasi tentang SPME Melakukan Pembinaan, pembimbing- an dan pen- dampingan Melakukan supervisi dan moneva Melakukan program tindak lanjut terhadap sekmod Y T Y T Y T Y T SMPN 1 Sekampung Udik     Secara garis besar peran TPMPD belum dapat menjalankan fungsinya didalam melakukan SMPN 1 Marga Sekampung     SMPN 2 Batanghari     SMPN 1 Bumi Agung     SMPN 1 Way Bungur     28 POACE: Jurnal Program Studi Administrasi Pendidikan ISSN 2775-6564 (Print) Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-7048 (Online) pendampingan di lima sekmod Keterangan: Y= Ya, T= Tidak POACE: Jurnal Program Studi Administrasi Pendidikan Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) Tim Penjaminan Mutu Pendidikan Daerah (TPMPD) yang memiliki kewenangan dan bertanggungjawab (a) melakukan pembinaan (b) pembimbingan (c) pendampingan (d) pengawasan, dan (e) pengendalian satuan pendidikan dalam pengembangan Sistem Penjaminan Mutu Internal (SPMI) khususnya di Dinas Pendidikan dan Kebudayaan Kabupaten Lampung Timur, berdasarkan data yang diperoleh dari hasil wawancara, observasi dan studi dokumentasi ternyata sampai dengan dilakukan penelitian ini belum terlihat peran TPMPD dalam menjalankan tugas dan tanggung jawab berkaitan dengan implementasi sistem penjaminan mutu internal di satuan pendidikan. Dari ke 5(lima) sekolah model yang diteliti belum ada satu dokumenpun yang menunjukkan peran serta keterlibatan TPMPD, hal ini sesuai dengan hasil wawancara, observasi serta studi dokumentasi yang dilakukan dengan kode W.T.1.2.3.4.5/VII dan Ob.T.1.2.3.4.5/VII juga berdasarkan studi dokumentasi dengan kode Dk. T.1.2.3.4.5/VII. KESIMPULAN DAN SARAN (5%) Berdasarkan data dan pembahasan hasil penelitian yang telah dilakukan, maka dapat disimpulkan hal- hal sebagai berikut : Berdasarkan data dan pembahasan hasil penelitian yang telah dilakukan, maka dapat disimpulkan hal- hal sebagai berikut : 1. Sosialisasi Permendikbud No. 28 Tahun 2016 sudah dilaksanakan oleh TPMPS terhadap anggota tim penjaminan mutu sekolah model dengan baik dalam bentuk sosialisasi dan bimtek secara terjadwal. 2. Kegiatan Implementasi Sistem penjaminan Mutu Internal (SPMI), Siklus atau tahapan sistem penjaminan mutu, yang terdiri dari (a) Pemetaan mutu, (b) Perencanaan program pemenuhan mutu, (c) Pelaksanaan kegiatan pemenuhan mutu, (d) Kegiatan Evaluasi dan Monitoring, (e) Penetapan standar baru. Berdasarkan hasil penelitian dan pembahasan maka dapat disimpulkan bahwa kegiatan implementasi SPMI oleh TPMPS sudah dapat berjalan dengan baik meskipun ada 2 (dua) sekolah model yang belum maksimal, hal ini semata-mata disebabkan tidak adanya dana khusus yang disiapkan oleh sekolah. Biaya operasional kegiatan sepenuhnya hanya bersumber dari dana bantuan pemerintah pusat melalui LPMP Lampung. p g 3. Peran kepala sekolah terhadap TPMPS dalam mengimplementasikan SPMI. Dari hasil pembahasan dapat disimpulkan bahwa kepala sekolah model sudah dapat memberikan kontribusi yang baik kepada TPMPS sehingga tim dapat melaksanakan semua tahapan penjaminan mutu disekolahnya, kelemahan yang masih ada adalah kurangnya kemampuan kepala sekolah melibatkan pemangku kepentingan untuk terlibat aktif dalam kegiatan penjaminan mutu di sekolahnya. Pihak dinas pendidikan kabupaten dan ketua komite sekolah hanya hadir pada saat acara pembukaan kegiatan pendampingan setelah itu pada kegiatan selanjutnya mereka tidak pernah bisa ikut terlibat dalam setiap kegiatan yang dilakukan oleh TPMPS. 4. Kegiatan pendampingan yang dilakukan oleh Fasilitator Daerah (Fasda) berdasarkan hasil wawancara, observasi dan pembahasan dapat disimpulkan bahwa proses pendampingan sudah berjalan dengan baik sesuai juknis yang sudah ditetapkan oleh pihak LPMP Lampung. Jika ada kendala adalah waktu pendampingan yang kurang tepat, karena dibulan Juli , Agustus sekolah sedang banyak menghadapi kegiatan 29 POACE: Jurnal Program Studi Administrasi Pendidikan Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) awal tahun pelajaran, sehingga dapat menyebabkan kurang efektifnya kegiatan pendampingan. 5. Peran Penjaminan Mutu Daerah (TPMPD) terhadap Implementasi Sistem Penjaminan Mutu Internal (SPMI) di satuan pendidikan. Berdasarkan pembahasan dan hasil penelitian dapat disimpulkan bahwa secara umum Tim Penjaminan Mutu Pendidikan Daerah (TPMPD) Kabupaten lampung Timur sampai dengan selesainya pelaksanaan program kegiatan sekolah model ternyata belum dapat memberikan kontribusi yang nyata terhadap pelaksanaan Implementasi Sistem Penjaminan Mutu Internal (SPMI) di 5 (lima) sekolah model. REKOMENDASI 1. Sebaiknya seluruh anggota Tim Penjaminan Mutu Pendidikan Sekolah (TPMPS) terlibat aktif didalam implementasi siklus penjaminan mutu sesuai dengan tugas yang ada di SK. Kepala Sekolah tentang TPMPS. y g p g 2. Sebaiknya setiap sekolah memasukkan semua program perencanaan pemenuhan mutu dapat dianggarkan melalui RKAS, sehingga pelaksanaan program pemenuhan mutu dapat berjalan sesuai dengan rencana. 3. Waktu kegiatan pendampingan sekolah model dalam melaksanakan Sistem Penjaminan Mutu internal (SPMI) diupayakan dilaksanakan diawal tahun, sehingga sekolah dapat menganggarkan kegiatan operasional TPMPS melalui dana BOS dalam RKAS. 4. Sebaiknya sekolah menganggarkan biaya operasional Tim Penjaminan Mutu pendidikan sekolah (TPMPS) melalui RKAS, sehingga TPMPS dapat bekerja secara maksimal karena didukung dengan biaya operasional yang diperlukan. 5. Pemerintah daerah melalui Tim Penjaminan Mutu Pendidikan Daerah (TPMPD) melakukan sosialisasi Permendikbud No. 28 tahun 2016 tentang Sistem Penjaminan Mutu pendidikan terhadap satuan pendidikan yang belum mendapatkan pengimbasan dari sekolah model yang ada. 5. Pemerintah daerah melalui Tim Penjaminan Mutu Pendidikan Daerah (TPMPD) melakukan sosialisasi Permendikbud No. 28 tahun 2016 tentang Sistem Penjaminan Mutu pendidikan terhadap satuan pendidikan yang belum mendapatkan pengimbasan dari sekolah model yang ada. KESIMPULAN DAN SARAN (5%) Sesuai dengan tugas yaitu : melakukan pembinaan, pembimbingan, pendampingan, dan supervisi terhadap satuan pendidikan dalam pengembangan SPMI Dikdasmen pada pendidikan dasar. POACE: Jurnal Program Studi Administrasi Pendidikan Volume 1, No. 1, Februari 2021, 20-32 DAFTAR PUSTAKA Arikunto Suharsimi. (1989). Manajemen Penelitian. Jakarta: Proyek Pengembangan LPTK. A Sani, Ridwan, dkk . (2018). Sistem Penjaminan Mutu Internal. 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Implementasi Sistem Penjaminan Mutu Internal Sebagai Upaya Meningkatkan Mutu Pendidikan di Universitas Gajah Mada. Diakses tanggal 16 Januari 2019, dari Jurnal Akuntabilitas Manajemen Pendidikan Volume 4 No 1 http://journal.uny.ac.id Sugiyono, (2017). Metode Penelitian Pendidikan. Cetakan ke-25: Penerbit Alfabeta Bandung. Sugiyono, (2018). Metode Penelitian Evaluasi. Cetakan ke-1: Penerbit Alfabeta Bandung. Yanuar Ikbar, (2014). Metode Penelitian Sosial Kualitatif. Penerbit Refika Aditama Bandung. POACE: Jurnal Program Studi Administrasi Pendidikan ISSN 2775-6564 (Print) Volume 1, No. 1, Februari 2021, 20-32 ISSN 2775-7048 (Online) ISSN 2775-6564 (Print) ISSN 2775-7048 (Online) Sulaiman, Ahmad, Udik Budi Wibowo, 2016. Implementasi Sistem Penjaminan Mutu Internal Sebagai Upaya Meningkatkan Mutu Pendidikan di Universitas Gajah Mada. Diakses tanggal 16 Januari 2019, dari Jurnal Akuntabilitas Manajemen Pendidikan Volume 4 No 1 http://journal.uny.ac.id Sugiyono, (2017). Metode Penelitian Pendidikan. Cetakan ke-25: Penerbit Alfabeta Bandung. Sugiyono, (2018). Metode Penelitian Evaluasi. Cetakan ke-1: Penerbit Alfabeta Bandung. Yanuar Ikbar, (2014). Metode Penelitian Sosial Kualitatif. Penerbit Refika Aditama Bandung. 32
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The evolution of Runx genes II. The C-terminal Groucho recruitment motif is present in both eumetazoans and homoscleromorphs but absent in a haplosclerid demosponge
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BioMed Central BioMed Central BioMed Central Short Report Address: 1Mount Desert Island Biological Laboratory, Salisbury Cove, Maine 04672, USA and 2School of Biological Sciences, University o Queensland, St Lucia, 4072 QLD, Australia Email: Anthony J Robertson - tony@mdibl.org; Claire Larroux - c.larroux1@uq.edu.au; Bernard M Degnan - b.degnan@uq.edu.au; James A Coffman* - jcoffman@mdibl.org * Corresponding author * Corresponding author Received: 24 December 2008 Accepted: 17 April 2009 Received: 24 December 2008 Accepted: 17 April 2009 BMC Research Notes 2009, 2:59 doi:10.1186/1756-0500-2-59 This article is available from: http://www.biomedcentral.com/1756-0500/2/59 © ; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BMC Research Notes Ope Short Report The evolution of Runx genes II. The C-terminal Groucho recruitment motif is present in both eumetazoans and homoscleromorphs but absent in a haplosclerid demosponge Anthony J Robertson1, Claire Larroux2, Bernard M Degnan2 and James A Coffman*1 Open Access Findings Nematostella vectensis and the placozoan Trichoplax adher- ens each have a single Runx gene, as do several triploblast species, including the lancelet Branchiostoma floridae and the sea squirt Ciona intestinalis among deuterostomes; the nematode Caenorhabditis elegans among ecdysozoans; and the polychaete Capitella sp.I and the mollusk Lottia gigantea among lophotrochozoans. In contrast, verte- brates, sea urchins (Strongylocentrotus purpuratus), dipteran insects (Drosophila melanogaster), clitellate annelids (Helobdella robusta), and planarians (Schmidtea mediterra- nea) each have two or more Runx genes. g The Runt domain (Runx) is a highly conserved 128 amino acid sequence motif that defines a metazoan family of sequence-specific DNA binding proteins required for the ontogeny of each of the animal species in which it has been functionally studied, as well as for the regulation of somatic stem cells and development of the lineages to which they give rise [1-4]. Runx genes facilitate develop- mental coordination of cell proliferation and differentia- tion [1], integrating the transduction of multiple signalling pathways [2] by nucleating the assembly of sig- nal-responsive cis-regulatory modules [5]. Runx genes have only been found in animals [6,7], suggesting that they may have evolved in concert with metazoan systems for developmental signalling. Comparison of the gene architectures suggests that the primordial Runx gene contained three introns, the first of which interrupts the coding sequence of the Runt domain (found in every representative except for the insect runt orthologues), the second of which lies at the C-terminal end of the Runt domain (found in all of the representa- tives except two, HrRunx2 and LgRunx, both from lopho- trochozoans), and the third lying between the two exons that encode the poorly conserved C-terminal sequence of the protein (missing in three of the insect genes and one of the leech genes; Fig. 1). This basic four-exon architec- ture is displayed by the demosponge, placozoan and anthozoan Runx genes, and among the known triploblast Runx genes, by the two sea urchin paralogues, the single lancelet orthologue, and the two planarian paralogues. Except for the additional intron within the sequence that encodes the N-terminal half of the Runt domain in all the vertebrate paralogues (Fig. 1), the basal architecture is conserved in vertebrate Runx3, which supports previous propositions for that gene being the most ancient of the vertebrate paralogues [17]. Abstract Background: The Runt DNA binding domain (Runx) defines a metazoan family of sequence- specific transcription factors with essential roles in animal ontogeny and stem cell based development. Depending on cis-regulatory context, Runx proteins mediate either transcriptional activation or repression. In many contexts Runx-mediated repression is carried out by Groucho/ TLE, recruited to the transcriptional complex via a C-terminal WRPY sequence motif that is found encoded in all heretofore known Runx genes. Findings: Full-length Runx genes were identified in the recently sequenced genomes of phylogenetically diverse metazoans, including placozoans and sponges, the most basally branching members of that clade. No sequences with significant similarity to the Runt domain were found in the genome of the choanoflagellate Monosiga brevicollis, confirming that Runx is a metazoan apomorphy. A contig assembled from genomic sequences of the haplosclerid demosponge Amphimedon queenslandica was used to construct a model of the single Runx gene from that species, AmqRunx, the veracity of which was confirmed by expressed sequences. The encoded sequence of the Runx protein OscRunx from the homoscleromorph sponge Oscarella carmella was also obtained from assembled ESTs. Remarkably, a syntenic linkage between Runx and Supt3h, previously reported in vertebrates, is conserved in A. queenslandica. Whereas OscRunx encodes a C-terminal Groucho-recruitment motif, AmqRunx does not, although a Groucho homologue is found in the A. queenslandica genome. Conclusion: Our results are consistent with the hypothesis that sponges are paraphyletic, and suggest that Runx-WRPY mediated recruitment of Groucho to cis-regulatory sequences originated in the ancestors of eumetazoans following their divergence from demosponges. Page 1 of 9 (page number not for citation purposes) Page 1 of 9 (page number not for citation purposes) BMC Research Notes 2009, 2:59 http://www.biomedcentral.com/1756-0500/2/59 http://www.biomedcentral.com/1756-0500/2/59 http://www.biomedcentral.com/1756-0500/2/59 Page 2 of 9 (page number not for citation purposes) Findings The additional N-terminal intron in Runx3, which is also found in each of the other vertebrate Runx paralogues, is also found in the C. intesti- nalis orthologue (but not in the cephalochordate B. flori- dae), consistent with recent phylogenies that place cephalochordates basal to {urochordates+vertebrates} in the chordate lineage [18]. All heretofore known Runx genes encode proteins that bear at their C-terminus a WRPY sequence motif (or a close variant thereof), which functions to recruit the Groucho/TLE corepressor to the cis-regulatory system [8- 12]. Runx-WRPY mediated recruitment of Groucho is rel- atively weak and controlled by cis-regulatory sequence context [12,13]. Depending on such context, Runx pro- teins can also function as Groucho-independent repres- sors, as well as activators [8,14]. The purpose of this study was to extend our previous investigation of the evolution of Runx genes [6] by analyz- ing and comparing several new Runx gene sequences col- lected from recently sequenced genomes of lophotrochozoans and basally branching metazoans (see Additional File 1 for detailed methods). Although cnidar- ian and sponge Runx genes were described in a recent report [7], that study left open the question of whether the sponge Runx proteins bear a C-terminal Groucho recruit- ment motif. To address that question we examined Runx- encoding genomic and cDNA sequences from two sponges (Amphimedon queenslandica and Oscarella car- mela), and compared these to Runx sequences collected from a phylogenetically broad sampling of other meta- zoan genomes, including that of the placozoan Trichoplax adhaerens [15]. To confirm and extend previous analyses of Runx family relations [6,7], we used our expanded Runx sequence dataset to calculate trees by Bayesian, distance neighbor- joining (NJ), and maximum likelihood (ML) methods. The three trees have slightly different topologies; the Baye- sian tree is shown in Figure 2A. All three analyses confi- dently support the branch separating the two sponge Runx genes from eumetazoan genes. Additionally, the protostome and chordate clades are recovered in all three trees but the positions of cnidarian, placozoan, and echi- noderm genes differ between analyses. While only the NJ tree places echinoderms correctly inside a deuterostome clade, this clade also erroneously includes cnidarian and placozoan genes. Bayesian and ML analyses correctly place the latter two genes at the base of the bilaterian clade Figure 1 (see legend on next page) Runx is a metazoan synapomorphy that has undergone independent duplications in a subset of triploblast lineages Runx is a metazoan synapomorphy that has undergone independent duplications in a subset of triploblast lineages Figure 1 depicts several representative examples of previ- ously known [6,7] or newly revealed (Table 1) Runx genes from across metazoan phylogeny, clustered according to the phylogenetic topology obtained by Sperling et al. [16]. As recently shown by Sullivan et al. [7], Runx-encoding sequences extend to the base of the metazoan family tree, with single orthologues encoded in the genome of the haplosclerid demosponge A. queenslandica and in expressed sequence tags from the homoscleromorph sponge O. carmela. Similarly, the anthozoan cnidarian Page 2 of 9 (page number not for citation purposes) Page 2 of 9 (page number not for citation purposes) BMC Research Notes 2009, 2:59 http://www.biomedcentral.com/1756-0500/2/59 re 1 (see legend on next page) Figure 1 (see legend on next page) Page 3 of 9 (page number not for citation purposes) BMC Research Notes 2009, 2:59 http://www.biomedcentral.com/1756-0500/2/59 Schematic structure of Runx genes from the major metazoan clades Figure 1 (see previous page) Schematic structure of Runx genes from the major metazoan clades. Scale models of Runx genes described previ- ously [6,7] from mouse (Mus musculus, Mm), sea squirt (Ciona intestinalis, Ci), fruit fly (Drosophila melanogaster, Dm), nematode worm (Caenorhabditis elegans, Ce), and sea anemone (Nematostella vectensis, Nv) are shown in comparison to new models obtained from various recent genome projects (Table 1). The latter include Runx genes from lancelet (Branchiostoma floridae, Bf), sea urchin (Strongylocentrotus purpuratus, Sp, corrected; the arrow points to an intron that was previously missed [6,21]), leech (Helobdella robusta, Hr), polychaete (Capitella sp. I, CspI), snail (Lottia gigantea) planarian (Schmidtea mediterranea, Sm), pla- cozoan (Trichoplax adherens, Ta), and demosponge (Amphimedon queesnslandica, Amq). The Runt domain is shaded grey and black, with the black box denoting the highly conserved exon encoding its C-terminal end. The C-terminal WRPY Groucho- recruitment motif is shaded Red. A hypothetical model of the homoscleromorph sponge Runx gene (Oscarella carmela, Osc) is shown; although as yet there is no genomic sequence from which exon-intron structure of this gene can be inferred (as indi- cated by question marks), the predicted exonic coding sequences containing the Runt domain and C-terminal LWRPY are rep- resented in assembled ESTs. but wrongly group echinoderm genes with protostome genes. Relationships within the protostomes are unclear and none of the three analyses separates these genes into lophotrochozoan and ecdysozoan clades. Runx is a metazoan synapomorphy that has undergone independent duplications in a subset of triploblast lineages This may be due to long-branch attraction between the Runx genes from S. mediterranea, H. robusta, and C. elegans. Thus, these genes were removed in a second set of analyses (Fig. 2B), where a lophotrochozoan clade and a clade compris- ing the four D. melanogaster genes are recovered in all three trees. These analyses suggest that there was only one Runx gene in the lineage between the metazoan and the lophotrochozoan-ecdysozoan last common ancestors. Hence, the multiple Runx genes present in some of the animals in this study are most likely the products of inde- pendent duplications within each of the lineages [6] (Fig. 1, colored boxes; note that a second sea urchin Runx gene, SpRunt-2, was recently found to be encoded in the sea urchin genome [19,20], in contradiction to several previ- ous reports [1,6,7,21]). but wrongly group echinoderm genes with protostome genes. Relationships within the protostomes are unclear and none of the three analyses separates these genes into lophotrochozoan and ecdysozoan clades. This may be due to long-branch attraction between the Runx genes from S. mediterranea, H. robusta, and C. elegans. Thus, these genes were removed in a second set of analyses (Fig. 2B), where a lophotrochozoan clade and a clade compris- ing the four D. melanogaster genes are recovered in all three trees. These analyses suggest that there was only one Runx gene in the lineage between the metazoan and the lophotrochozoan-ecdysozoan last common ancestors. Hence, the multiple Runx genes present in some of the animals in this study are most likely the products of inde- pendent duplications within each of the lineages [6] (Fig. 1, colored boxes; note that a second sea urchin Runx gene, SpRunt-2, was recently found to be encoded in the sea urchin genome [19,20], in contradiction to several previ- ous reports [1,6,7,21]). sequence motif in the M. brevicolis genome using tBLASTn searches. Thus, the Runt domain appears to have evolved in concert with complex multicellularity in the animal clade. Furthermore, unlike many other metazoan-specific transcription factor classes [23], the Runx gene did not duplicate in early animals, or even within some of the bilaterian lineages. AmqRunx lacks a Groucho recruitment motif q f As reported previously [7], Runx genes are found in both the haplosclerid demosponge A. queenslandica and the homoscleromorph sponge O. carmela. Although genome sequence is not yet available for the latter, a sequence encoding a Runx protein was recovered from an assembly of available ESTs. The predicted OscRunx protein termi- nates with the amino acid sequence WRPY (Fig. 3) [see Additional File 2], the C-terminal Groucho-recruitment motif found encoded in all heretofore known Runx genes (Fig. 1). Note that there are vertebrate splice variants that lack a C-terminal WRPY [24-26], and that one each of the two leech and two planarian paralogues do not appear to terminate in WRPY (Fig. 1) [see Additional File 2]. Thus, some contexts have functional requirements for Runx pro- tein isoforms lacking a C-terminal WRPY. Nevertheless, all of the eumetazoan species depicted in Fig. 1 (as well as Previous reports have noted the absence of any Runx homologues in sequenced genomes of unicellular organ- isms [6,7], including the choanoflagellate M. brevicolis [22], a member of the Holozoa taxon that is most closely related to Metazoa. We confirmed the absence of a Runx Table 1: Sources of sequences used in this analysis Table 1: Sources of sequences used in this analysis Table 1: Sources of sequences used in this analysis Species Genome Database (URL) Version NCBI Acc. No. S. purpuratus http://sugp.caltech.edu/SpBase 2.1 NW_001330224 B. floridae http://genome.jgi-psf.org 1.0 N.A. H. robusta http://genome.jgi-psf.org 1.0 N.A. Capitella sp. I* http://genome.jgi-psf.org 1.0 N.A. L. gigantean http://genome.jgi-psf.org 1.0 N.A. T. adhaerens http://genome.jgi-psf.org 1.0 N.A. S. mediterranea http://smedgd.neuro.utah.edu/index.html 1.3.14 N.A. A. queenslandica http://compagen.zoologie.uni-kiel.de/index.html N.A. N.A. O. carmela** http://compagen.zoologie.uni-kiel.de/index.html N.A. N.A. The table identifies the genome project and assembly version from which each of the new sequences described here was obtained, as well as available NCBI genomic contig reference assemblies. The sequences and links to each locus on the respective genome browsers are provided in Additional File 2. N.A., not available. *Complete gene model obtained from raw contig sequence using GeneScan. **ESTs only. The table identifies the genome project and assembly version from which each of the new sequences described here was obtained, as well as available NCBI genomic contig reference assemblies. The sequences and links to each locus on the respective genome browsers are provided in Additional File 2. N.A., not available. *Complete gene model obtained from raw contig sequence using GeneScan. **ESTs only. AmqRunx lacks a Groucho recruitment motif http://www.biomedcentral.com/1756-0500/2/59 BMC Research Notes 2009, 2:59 Bayesian trees of Runx sequences Figure 2 Bayesian trees of Runx sequences. In a first analysis (A), all the genes from Figure 1 were included and, in a second a (B), long-branched taxa (Runx genes from S. mediterranea, H. robusta, and C. elegans) were excluded from the dataset. Th were calculated using a multiple sequence alignment of amino acid sequences corresponding to the Runt domain of eac cies. Percentages of bootstrap support greater than or equal to 50% are indicated above the node for the distance anal (Phylip 3.6; 1000 replicates) and below the node for the maximum likelihood analysis (Phylip 3.6; 100 replicates). An ast under the node indicates a Bayesian posterior probability greater than or equal to 95%. Abbreviations as in Figure 1.                        ! " # $ ! ! $ $                                !  ! "  " $  ! "! $"  $ # #                                     ! " # $ ! ! $ $                                 !  ! "  " $  ! "! $"  $ # #                Page 5 of 9 (page number not for citation purposes) Bayesian tr Figure 2 Bayesian trees of Runx sequences Figure 2 Bayesian trees of Runx sequences. In a first analysis (A), all the genes from Figure 1 were included and, in a second analysis (B), long-branched taxa (Runx genes from S. mediterranea, H. robusta, and C. elegans) were excluded from the dataset. The trees were calculated using a multiple sequence alignment of amino acid sequences corresponding to the Runt domain of each spe- cies. Percentages of bootstrap support greater than or equal to 50% are indicated above the node for the distance analysis (Phylip 3.6; 1000 replicates) and below the node for the maximum likelihood analysis (Phylip 3.6; 100 replicates). An asterisk under the node indicates a Bayesian posterior probability greater than or equal to 95%. Abbreviations as in Figure 1. y q g Bayesian trees of Runx sequences. In a first analysis (A), all the genes from Figure 1 were included and, in a second analysis (B), long-branched taxa (Runx genes from S. mediterranea, H. robusta, and C. elegans) were excluded from the dataset. The trees were calculated using a multiple sequence alignment of amino acid sequences corresponding to the Runt domain of each spe- cies. Percentages of bootstrap support greater than or equal to 50% are indicated above the node for the distance analysis (Phylip 3.6; 1000 replicates) and below the node for the maximum likelihood analysis (Phylip 3.6; 100 replicates). An asterisk under the node indicates a Bayesian posterior probability greater than or equal to 95%. Abbreviations as in Figure 1. Page 5 of 9 (page number not for citation purposes) Page 5 of 9 (page number not for citation purposes) Page 5 of 9 (page number not for citation purposes) BMC Research Notes 2009, 2:59 http://www.biomedcentral.com/1756-0500/2/59 gnment of AmqRunx and OscRunx amino acid sequences gure 3 ignment of AmqRunx and OscRunx amino acid sequences. Identities are marked with asterisks, whereas conserv e changes are marked with dots. The Runt domains are highlighted in grey and black, for reference to the scheme depicte Figure 1. The arrows indicate predicted intron positions with respect to the coding sequence of AmqRunx. Proline residue the C-terminal domains are highlighted in green, whereas serines and threonines are highlighted in yellow. The C-termina RPY motif in OscRunx is highlighted in red. AmqRunx MPLIMSSESIPPPDGPLPPPSSKRYRGERT-FSELLAEYPGELVTTDSPNFVCTILPSHW OscRunx MRLMMEREPAPKRSKDSLELSSSM--GTLSASASAAAEHQGDLVKTDNPNFVCTILPSHW * *:*. *. * . **. * : :. **: *:**.**.************ AmqRunx RCNKTLPVPFKVLSLSDIT--DGTKVILTAGNDENSAAELRNAIATFKNQVARFNDLRFV OscRunx RVNKTLPVPFRVLAVGDISVPDGVKVTLKAFNEETVSGELRNATAIFRNNVARFNDLRFV * ********:**::.**: **.** *.* *:*. Bayesian tr Figure 2 :.***** * *:*:********** AmqRunx GRSGRGKMLTVTITIVTEPVQYATYSHAIKVTVDGPREPRRNRASTRSDDHPYL-RPNPF OscRunx GRSGRGKYFDVLITVQTDTVQKAIYKKAIKVTVDGPREPRRHKVKERQLLAAHQHHHSPY ******* : * **: *:.** * *.:**************::.. *. .: : .*: AmqRunx MGHLSPAGAGQVPPLIKDT--------------------------RCPPSGSIDLEGAMA OscRunx HGY--PNRQHVLPPDFMPLSSAAASSLSSSSSSLGSAGCETPQLQRAIHRDSLSAFSTIA *: * :** : *. .*:. .::* AmqRunx TDPSCRPPSMSDVFPAGIRSPVWQYPGVITSQSLIPSQLDTSSTNSVPQTSADSLSNGSS OscRunx TEPIMRRPAFS-------------QP-MVTMNHVHYTQEPTTMSTQMEQSHCPSIPPSIS *:* * *::* * ::* : : :* *: :..: *: . *:. . * AmqRunx TPPNVTQNGDHAQIN--------NSTGNVNDSKFLFPSGSAIPLSPGLFNAQSFFNPGGS OscRunx MPPTFTSEASLSMLAGPAFAPRQSVPGHI-EQGFVFPP--PFPLRSP-TSAAGFAFPGGP **..*.:.. : : . .*:: :. *:**. .:** . .* .* ***. AmqRunx N-NIPITPTLLAPSVSFSESYIRPGQIYSPFSFTPHGSLHGN------------------ OscRunx PGLVPLSPIPGDSASPFPHRNYRP--IACQVSISSHGRSSGSGYDAPAPAMPGLVSTSDL :*::* .: .*.. ** * . .*::.** *. AmqRunx -----LPRTPTL---PPPSPHA------IVSCSSFPALTAIAHPSFSTSSLQFQKGGSFL OscRunx FSLPVTPRTPITPTVRYAQMHAAQAAGQLMATSSSDAL-GYGHLTAAIGSFPFDQYATHL **** .. ** ::: ** ** . .* : : .*: *:: .:.* AmqRunx DDITKIGSISPFIVSPSLSPNRRPNGTTIFFPTTITAQGEAKFATIGEVGMAGSVGGVER OscRunx QDIQHMQQHSASMTSLNGEPSS--SGVTITLSP---PPNKAKPTLSRSSSFTSGAGNGKM :** :: . *. :.* . .*. .*.** :.. . .:** : . .::...*. : AmqRunx LSGTPDDGSIHSTDSPLIKREVSSPQHCYIDQEAA OscRunx DSGETDEKE-----------G---------LWRPY ** .*: . .. gnment of AmqRunx and OscRunx amino acid sequences gure 3 ignment of AmqRunx and OscRunx amino acid sequences. Identities are marked with asterisks, whereas conserv e changes are marked with dots. The Runt domains are highlighted in grey and black, for reference to the scheme depict Figure 1. The arrows indicate predicted intron positions with respect to the coding sequence of AmqRunx. Proline residu the C-terminal domains are highlighted in green, whereas serines and threonines are highlighted in yellow. The C-termin RPY motif in OscRunx is highlighted in red. AmqRunx MPLIMSSESIPPPDGPLPPPSSKRYRGERT-FSELLAEYPGELVTTDSPNFVCTILPSHW OscRunx MRLMMEREPAPKRSKDSLELSSSM--GTLSASASAAAEHQGDLVKTDNPNFVCTILPSHW * *:*. *. * . **. * : :. **: *:**.**.************ AmqRunx RCNKTLPVPFKVLSLSDIT--DGTKVILTAGNDENSAAELRNAIATFKNQVARFNDLRFV OscRunx RVNKTLPVPFRVLAVGDISVPDGVKVTLKAFNEETVSGELRNATAIFRNNVARFNDLRFV * ********:**::.**: **.** *.* *:*. :.***** * *:*:********** AmqRunx GRSGRGKMLTVTITIVTEPVQYATYSHAIKVTVDGPREPRRNRASTRSDDHPYL-RPNPF OscRunx GRSGRGKYFDVLITVQTDTVQKAIYKKAIKVTVDGPREPRRHKVKERQLLAAHQHHHSPY ******* : * **: *:.** * *.:**************::.. *. .: : .*: AmqRunx MGHLSPAGAGQVPPLIKDT--------------------------RCPPSGSIDLEGAMA OscRunx HGY--PNRQHVLPPDFMPLSSAAASSLSSSSSSLGSAGCETPQLQRAIHRDSLSAFSTIA *: * :** : *. .*:. .::* AmqRunx TDPSCRPPSMSDVFPAGIRSPVWQYPGVITSQSLIPSQLDTSSTNSVPQTSADSLSNGSS OscRunx TEPIMRRPAFS-------------QP-MVTMNHVHYTQEPTTMSTQMEQSHCPSIPPSIS *:* * *::* * ::* : : :* *: :..: *: . *:. . * AmqRunx TPPNVTQNGDHAQIN--------NSTGNVNDSKFLFPSGSAIPLSPGLFNAQSFFNPGGS OscRunx MPPTFTSEASLSMLAGPAFAPRQSVPGHI-EQGFVFPP--PFPLRSP-TSAAGFAFPGGP **..*.:.. : : . .*:: :. *:**. .:** . .* .* ***. AmqRunx N-NIPITPTLLAPSVSFSESYIRPGQIYSPFSFTPHGSLHGN------------------ OscRunx PGLVPLSPIPGDSASPFPHRNYRP--IACQVSISSHGRSSGSGYDAPAPAMPGLVSTSDL :*::* .: .*.. ** * . .*::.** *. AmqRunx -----LPRTPTL---PPPSPHA------IVSCSSFPALTAIAHPSFSTSSLQFQKGGSFL OscRunx FSLPVTPRTPITPTVRYAQMHAAQAAGQLMATSSSDAL-GYGHLTAAIGSFPFDQYATHL **** .. ** ::: ** ** . .* : : .*: *:: .:.* AmqRunx DDITKIGSISPFIVSPSLSPNRRPNGTTIFFPTTITAQGEAKFATIGEVGMAGSVGGVER OscRunx QDIQHMQQHSASMTSLNGEPSS--SGVTITLSP---PPNKAKPTLSRSSSFTSGAGNGKM :** :: . *. :.* . .*. .*.** :.. . .:** : . .::...*. : AmqRunx LSGTPDDGSIHSTDSPLIKREVSSPQHCYIDQEAA OscRunx DSGETDEKE-----------G---------LWRPY ** .*: . .. Page 6 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1756-0500/2/59 BMC Research Notes 2009, 2:59 http://www.biomedcentral.com/1756-0500/2/59 the homoscleromorph sponge) encode at least one Runx protein that terminates in WRPY or a close variant thereof. the EST-encoded 3' UTR region – were used to amplify the sequence both from A. queenslandica adult and embryonic RNA. An amplicon of the correct size and sequence was obtained (Additional File 4), thus confirming the veracity of the AmqRunx gene prediction. A genomic sequence contig from A. queenslandica was pre- dicted to encode a Runx gene with four exons, displaying an architecture very similar to that of the placozoan and cnidarian genes (Fig. 1) [7]. The predicted coding sequence of AmqRunx is 1,566 bp with the Runt domain contained within the first 474 bp. As is typical for Runx proteins, the predicted C-terminal domain of AmqRunx (amino acid residues 159–479) is enriched for proline (12%), serine (16%), and threonine (7%) residues, a PST enrichment similar to that previously reported for the C- terminal domain of NvRunx [7] and that displayed by the C-terminal domain of OscRunx (Fig. 3). Surprisingly however, the C-terminus of AmqRunx does not bear the WRPY motif or any variant thereof (Fig. 3). Furthermore, no open reading frames encoding WRPY were found along the genomic contig in which AmqRunx is found. The A. queenslandica genome does however encode a bona fide Groucho homologue (Additional File 3 and unpublished data), as well as several transcription factors that are pre- dicted to interact with Groucho [12], including a hairy/ Hey homologue with a FRPW motif and a number of NK class genes with an engrailed homology 1 (EH-1) motif ([27,28]; BMD, unpublished data). The contig bearing AmqRunx contains sequences predic- tive of additional genes flanking the Runx gene (Fig. 4), which argues against the possibility that the AmqRunx gene model is missing a C-terminal exon that might pro- duce alternative splice variants. Moreover, the veracity of the contig assembly is further supported by the remarka- ble fact that a syntenic relationship between Runx and Supt3h, previously reported to exist in vertebrates [29] and which we found also to exist in cnidarians (N. vectensis), lancelets (B. floridae), and polychaetes (Capitella sp. I), is conserved in the demosponge (Fig. 4). Although homoscleromorph sponges are still commonly grouped with demosponges in the phylum Porifera (Fig. 5A), this classification has been called into question, as has the monophyly of sponges (and hence 'Porifera' as a true phylum) [16]. Alignment Figure 3 Alignment of AmqRunx and OscRunx amino acid sequences Figure 3 Alignment of AmqRunx and OscRunx amino acid sequences. Identities are marked with asterisks, whereas conserva- tive changes are marked with dots. The Runt domains are highlighted in grey and black, for reference to the scheme depicted in Figure 1. The arrows indicate predicted intron positions with respect to the coding sequence of AmqRunx. Proline residues in the C-terminal domains are highlighted in green, whereas serines and threonines are highlighted in yellow. The C-terminal WRPY motif in OscRunx is highlighted in red. Alignment of AmqRunx and OscRunx amino acid sequences Figure 3 Alignment of AmqRunx and OscRunx amino acid sequences. Identities are marked with asterisks, whereas conserva- tive changes are marked with dots. The Runt domains are highlighted in grey and black, for reference to the scheme depicted in Figure 1. The arrows indicate predicted intron positions with respect to the coding sequence of AmqRunx. Proline residues in the C-terminal domains are highlighted in green, whereas serines and threonines are highlighted in yellow. The C-terminal WRPY motif in OscRunx is highlighted in red. Page 6 of 9 (page number not for citation purposes) Page 6 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1756-0500/2/59 http://www.biomedcentral.com/1756-0500/2/59 The fact that AmqRunx lacks a C-termi- nal WRPY motif is consistent with the more recent propo- sition that sponges are paraphyletic [16,30], with calcisponges and homoscleromorphs branching after demosponges along the lineage leading to eumetazoans (Fig. 5B). The conventional scenario, which holds that sponges are monophyletic (Fig. 5A), would require that several characters held in common between eumetazoans and homoscleromorph sponges (i.e., acrosomes, true epi- thelia, and a C-terminal WRPY motif linked to Runx) be either convergent homoplasies, or metazoan pleisiomor- phies that were all lost in the demosponge lineage leading to A. queenslandica. Although it is possible that the loss of The lack of a C-terminal WRPY motif in AmqRunx was ver- ified by expressed sequence data. Based on alignment with genomic DNA, EST sequence 2941805_1 was found to encode the last 115 bp of the AmqRunx coding sequence, the stop codon, and an additional 626 bp of 3' UTR spanning two exons. In order to confirm that this EST was transcribed from AmqRunx, oligonucleotide primers – forward primer in the Runt domain and reverse primer in Schematic of the 20 kb genomic sequence contig bearing AmqRunx Figure 4 Schematic of the 20 kb genomic sequence contig bearing AmqRunx. Predicted exons are shown as black boxes. The syntenic relationship between Runx and Supt3h is conserved between demosponge (A. queenslandica) and mouse (Mus muscu- lus), and is also found (at least) in the genomes of a cnidarian (N. vectensis), a basal chordate (B. floridae), and a teleost (Takifugu rubripes; [29]). 1 kb Runx Supt3h DNApolG Conserved synteny Conserved synteny Schematic Figure 4 g q g g q g Schematic of the 20 kb genomic sequence contig bearing AmqRunx. Predicted exons are shown as black boxes. The syntenic relationship between Runx and Supt3h is conserved between demosponge (A. queenslandica) and mouse (Mus muscu- lus), and is also found (at least) in the genomes of a cnidarian (N. vectensis), a basal chordate (B. floridae), and a teleost (Takifugu rubripes; [29]). Page 7 of 9 (page number not for citation purposes) BMC Research Notes 2009, 2:59 http://www.biomedcentral.com/1756-0500/2/59 Scenarios for Runx-WRPY evolution mapped onto alternative metazoan phylogenies Figure 5 Scenarios for Runx-WRPY evolution mapped onto alternative metazoan phylogenies. (A) Conventional phylogeny wherein the demosponge Amphimedon queenslandica (Amq) and the homoscleromorph sponge Oscarella carmela (Osc) are both classified as demosponges within the phylum Porifera. Additional material Additional File 1 Bioinformatics and Cloning Details. This file provides a detailed description of the methods used to obtain the Runx gene sequences and phylogenetic trees presented in this paper. Click here for file [http://www.biomedcentral.com/content/supplementary/1756- 0500-2-59-S1.doc] Additional File 1 Bioinformatics and Cloning Details. This file provides a detailed description of the methods used to obtain the Runx gene sequences and phylogenetic trees presented in this paper. Click here for file [http://www.biomedcentral.com/content/supplementary/1756- 0500-2-59-S1.doc] Authors' contributions [http://www.biomedcentral.com/content/supplementary/1756- 0500-2-59-S2.doc] AJR performed BLAST searches, sequence assemblies, alignments, and computational construction of gene models. CL independently verified the A. queenslandica contig assembly and Runx gene model, performed the phylogenetic analyses, and obtained the PCR amplicon of AmqRunx cDNA. BMD performed some sequence assem- blies, provided intellectual guidance and assisted in the writing of the manuscript. JAC performed some of the BLAST searches and sequence alignments, and drafted the http://www.biomedcentral.com/1756-0500/2/59 This scenario suggests that the WRPY motif was lost in the demosponge sub-lineage leading to Amq. (B) Alternative phylogeny wherein sponges are paraphyletic. In this tree homoscleromorphs are a sister group of eumetazoans within Epitheliozoa [16], which would imply that Runx gained the WRPY motif in the ancestors of the latter group following their divergence from demosponges, either prior to or after divergence from calcisponges. Runx+WRPY Runx Amq Eumetazoans Epitheliozoa Calcisponges Demosponges Osc Eumetazoans Calcisponges Porifera Amq Osc A B Runx+WRPY ? Runx loses WRPY True epithelia Runx+WRPY Demosponges Osc Eumetazoans Calcisponges Porifera Amq A Runx loses WRPY Runx Amq Eumetazoans Epitheliozoa Calcisponges Osc B Runx+WRPY ? True epithelia B A Epitheliozoa Runx+WRPY Scenarios Figure 5 pp p y g g Scenarios for Runx-WRPY evolution mapped onto alternative metazoan phylogenies. (A) Conventional phylogeny wherein the demosponge Amphimedon queenslandica (Amq) and the homoscleromorph sponge Oscarella carmela (Osc) are both classified as demosponges within the phylum Porifera. This scenario suggests that the WRPY motif was lost in the demosponge sub-lineage leading to Amq. (B) Alternative phylogeny wherein sponges are paraphyletic. In this tree homoscleromorphs are a sister group of eumetazoans within Epitheliozoa [16], which would imply that Runx gained the WRPY motif in the ancestors of the latter group following their divergence from demosponges, either prior to or after divergence from calcisponges. manuscript and figures. All authors read and approved the final manuscript. multiple characters occurred within the demosponge lin- eage, it is unlikely that body plan simplification is in itself sufficient to relax the selection pressure for maintaining the Runx-WRPY linkage, as evidenced by its maintenance in placozoans. The more parsimonious scenario is that the C-terminal WRPY motif of Runx proteins, and presuma- bly the consequent recruitment of Groucho to a subset of Runx target cis-regulatory modules, originated in eumeta- zoan ancestors following their divergence from the sponge lineage leading to A. queenslandica (Fig. 5B). An interesting possibility is that the Runx associated WRPY motif originated in Epitheliozoa {eumetazoans and homoscleromorphs} [16], which would suggest that Runx-WRPY mediated cis-regulatory recruitment of Grou- cho is functionally linked to the evolution and develop- ment of an epithelium. Testing this possibility awaits the sequencing of a calcisponge Runx gene. Additional File 2 Sequences of Runx genes listed in Table 1. This file provides gene, CDS, mRNA, and/or predicted peptide sequences of each of the Runx genes that are described for the first time (or corrected, in the case of SpRunt-1) in this report. For the two sea urchin genes, URLs are given to the scaffold coordinates on the SpBase genome browser, as well as to the original genome annotations. For gene sequences obtained from JGI genome projects, links are provided to the scaffold coordinates on the JGI genome browser. Additional File 3 AmqGroucho sequence. This file provides an A. queenslandica genomic trace sequence that encodes peptides homologous to Groucho, identified by tBLASTn using the TLE-domain (pfam03920: TLE_N), and confirmed by reciprocal BLASTx. Click here for file [http://www.biomedcentral.com/content/supplementary/1756- 0500-2-59-S3.doc] Page 8 of 9 (page number not for citation purposes) Page 8 of 9 (page number not for citation purposes) Competing interests p g The authors declare that they have no competing interests. Click here for file References 1. Coffman JA: Runx transcription factors and the developmen- tal balance between cell proliferation and differentiation. Cell Biol Int 2003, 27(4):315-324. 22. King N, Westbrook MJ, Young SL, Kuo A, Abedin M, Chapman J, Fair- clough S, Hellsten U, Isogai Y, Letunic I, et al.: The genome of the choanoflagellate Monosiga brevicollis and the origin of meta- zoans. Nature 2008, 451(7180):783-788. ( ) 2. Coffman JA: Is Runx a linchpin for developmental signaling in metazoans? J Cell Biochem 2009, 107(2):194-202. ( ) 2. Coffman JA: Is Runx a linchpin for developmental signaling in metazoans? J Cell Biochem 2009, 107(2):194-202. ( ) 23. Larroux C, Luke GN, Koopman P, Rokhsar DS, Shimeld SM, Degnan BM: Genesis and expansion of metazoan transcription factor gene classes. Mol Biol Evol 2008, 25(5):980-996. J ( ) 3. Kagoshima H, Shigesada K, Kohara Y: RUNX regulates stem cell proliferation and differentiation: insights from studies of C. elegans. J Cell Biochem 2007, 100(5):1119-1130. 24. Levanon D, Glusman G, Bangsow T, Ben-Asher E, Male DA, Avidan N, Bangsow C, Hattori M, Taylor TD, Taudien S, et al.: Architecture and anatomy of the genomic locus encoding the human leukemia-associated transcription factor RUNX1/AML1. Gene 2001, 262(1–2):23-33. g J ( ) 4. Nimmo R, Woollard A: Worming out the biology of Runx. Dev Biol 2008, 313(2):492-500. ( ) 5. Westendorf JJ, Hiebert SW: Mammalian runt-domain proteins and their roles in hematopoiesis, osteogenesis, and leuke- mia. J Cell Biochem 1999:51-58. ( ) 25. Sun L, Vitolo MI, Qiao M, Anglin IE, Passaniti A: Regulation of TGFbeta1-mediated growth inhibition and apoptosis by RUNX2 isoforms in endothelial cells. Oncogene 2004, 23(27):4722-4734. J 6. Rennert J, Coffman JA, Mushegian AR, Robertson AJ: The evolution of Runx genes I. A comparative study of sequences from phy- logenetically diverse model organisms. BMC Evol Biol 2003, 3:4. 7. Sullivan JC, Sher D, Eisenstein M, Shigesada K, Reitzel AM, Marlow H, Levanon D, Groner Y, Finnerty JR, Gat U: The evolutionary origin of the Runx/CBFbeta transcription factors–studies of the most basal metazoans. BMC Evol Biol 2008, 8:228. ( ) 26. Tsuji K, Noda M: Identification and expression of a novel 3'- exon of mouse Runx1/Pebp2alphaB/Cbfa2/AML1 gene. Bio- chem Biophys Res Commun 2000, 274(1):171-176. 27. Larroux C: Genome content and developmental expression of transcription factor genes in the demosponge Amphimedon queenslandica: insights into the first multicellular animal. In PhD Thesis Brisbane, Australia: The University of Queensland; 2007. 8. Additional File 4 AmqRunx cDNA sequence. This file provides the cDNA sequence of AmqRunx containing the N-terminus, Runt domain, predicted C-termi- nus, and some 3' UTR, obtained by RT-PCR from adult and embryonic RNA. 17. Levanon D, Glusman G, Bettoun D, Ben-Asher E, Negreanu V, Bern- stein Y, Harris-Cerruti C, Brenner O, Eilam R, Lotem J, et al.: Phylo- genesis and regulated expression of the RUNT domain transcription factors RUNX1 and RUNX3. Blood Cells Mol Dis 2003, 30(2):161-163. 18. Putnam NH, Butts T, Ferrier DE, Furlong RF, Hellsten U, Kawashima T, Robinson-Rechavi M, Shoguchi E, Terry A, Yu JK, et al.: The amphioxus genome and the evolution of the chordate kary- otype. Nature 2008, 453(7198):1064-1071. 19. 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References Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 9 of 9 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 8 of 9 BMC Research Notes 2009, 2:59 http://www.biomedcentral.com/1756-0500/2/59 Additional File 4 AmqRunx cDNA sequence. This file provides the cDNA sequence of AmqRunx containing the N-terminus, Runt domain, predicted C-termi- nus, and some 3' UTR, obtained by RT-PCR from adult and embryonic RNA. Click here for file [http://www.biomedcentral.com/content/supplementary/1756- 0500-2-59-S4.doc] 16. Sperling EA, Pisani D, Peterson KJ: Poriferan paraphyly and its implications for Precambrian paleobiology. In The Rise and Fall of the Eidiacaran Biota Volume 286. Edited by: Vickers-Rich P, Komar- ower P. 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Saudi Arabia, pharmacists and COVID-19 pandemic
Journal of pharmaceutical policy and practice
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© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. * Correspondence: alhossan@ksu.edu.sa Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Saudi Arabia, pharmacists and COVID-19 pandemic Ajaz Ahmad, Khalid M. Alkharfy, Ziyad Alrabiah and Abdulaziz Alhossan* Ahmad et al. Journal of Pharmaceutical Policy and Practice (2020) 13:41 https://doi.org/10.1186/s40545-020-00243-1 Ahmad et al. Journal of Pharmaceutical Policy and Practice (2020) 13:41 https://doi.org/10.1186/s40545-020-00243-1 Open Access Abstract The latest outbreak of Covid-19 pandemic has placed a significant effect on health care system around the world. This article discusses the role of pharmacists in Saudi Arabia during the current Covid-19 pandemic. Pharmacists are an important part of everyday healthcare in Saudi Arabia. Pharmacists helped to protect the public from Covid-19 pandemic disease by participating in various initiatives including health education and promotion, medication dispensing, medication reconciliation, medication and patient counselling, training for self-management in current outbreak and emergency preparedness. Full utilization of skills of pharmacists boosted the safety response of Saudi Arabia to Covid-19 pandemic. Keywords: Saudi Arabia, Pharmacist, Covid-19, Pandemic, Pharmaceutical services Keywords: Saudi Arabia, Pharmacist, Covid-19, Pandemic, Pharmaceutical services Introduction actively involved in community services like encouraging the public to wear surgical masks, maintaining social dis- tance, usage of hand sanitizers and avoiding social gatherings. The COVID-19 was declared a Public Health Emergency of International Concern on 30 January 2020, and on 8 March 2020, the WHO declare the Covid-19 as a pan- demic [1]. At this time, there are no specific vaccines or treatments for Covid-19. However, there are many on- going clinical trials evaluating potential treatments. WHO continues to provide updated information as soon as clinical findings become available. The pharmacists were also participating in online webinars, following WHO guidelines on routine bases and discussing new strategies in combating the current pandemic. Pharmacist were performing pa- tient counseling and drug reconciliation utilizing dif- ferent interactive platforms during Covid-19 [4]. Strengthening pharmaceutical workforce can help to overcome COVID-19 and to achieve objectives of the Universal Health Coverage [5, 6]. A total of 10,464,141 confirmed cases with 509,361 deaths linked to this pathogen as of June 30, 2020 have been reported [2]. Pharmacists played an important role in timely refilling of medications which reduced un- necessary hospital visits where individuals were at high risk of being exposed to Covid-19 [3]. Individuals or those undertaking home quarantine or isolation were provided home delivery service of medicines through community pharmacies [3]. Pharmacists recognized the incidence, dissemination and avoidance of spreading of Covid-19 pandemic. Apart from providing the pharma- ceutical services, the pharmacy professionals were Following are the measures to effectively utilize phar- macists’ expertise during the Covid 19 pandemic. Those involve clarifying pharmacist positions, coordinating and collaborating with pharmacists and maintaining capabil- ities for the workplace. Pandemic response from China, the USA, Canada, Japan and the UK are seen as indica- tors of how new regulations might be changed to en- hance the practice of pharmacists through pandemic response [1]. The Kingdom stopped exporting all medical devices and products, including diagnostic or protective agents, to ensure their availability in Covid-19 pandemic. Many hospitals in different provinces were assigned in Saudi Arabia to treat Covid-19 incidents. The primary respon- sibility for handling a Covid-19 in Saudi Arabia lies with the Ministry of Health (MoH) and hospital organiza- tions, each agreeing on the most appropriate approach to prepare and react. The Kingdom of Saudi Arabia began to take many pre- cautionary measures to combat the Covid-19. The im- mediate action was to set up a committee of various governmental organizations to determine and implement the actions needed against Covid-19 [8]. Since there is no vaccine available for the outbreak yet, avoidance is the only approach to protect the transmission of the virus; Consequently, the MoH and other departments carried out major initiatives to inform the public on vari- ous forms of stopping the transmission of the virus followed by the Ministry of Health (MoH) punch line campaign “We all are responsible”. These control mea- sures played a significant role in restricting the transmis- sion of SARS-CoV-2 with less mortalities. The mortality rate is very low in Saudi Arabia compared to other countries due to best health care facilities available at all levels in the Kingdom of Saudi Arabia [9]. During the time of public emergencies, the inclusion of pharmacists has helped to alleviate the burden on the other healthcare workers in health systems [10]. Phar- macists are the most accessible healthcare workers and thus understand the hardship of any outbreak like Covid-19 pandemic. They played a vital role in inform- ing patients, encouraging the avoidance of diseases and referring any suspicious cases to suitable health care fa- cilities in a timely manner [11]. Saudi Arabia has a well- equipped and advanced health system in the world [12], and improving use of medicines and medicines policy is an important part of healthcare [13] g Community pharmacists are the most available health- care providers to the general population, so they have a great deal to say in responding to Covid-19 pandemic. This has led to significant changes in the health care sys- tems of many countries [19, 20]. Community pharma- cists in Saudi Arabia played a crucial role in proper medication usage that improved patient outcomes, and prevented misuse of medications. Page 2 of 3 Page 2 of 3 Ahmad et al. Journal of Pharmaceutical Policy and Practice (2020) 13:41 The pharmacists in Saudi Arabia are providing a huge contribution by keeping patients safe at homes and pro- vided them medications by mail as well as by offering continuous counseling remotely during Covid-19 [15]. During current Covid-19 crises, pharmacists in Saudi Arabia led to a broad delivery of medication manage- ment activity in clinical and non-clinical settings. In current Covid-19 crises, hospital pharmacists in Saudi Arabia formulated emergency medications, tracked and addressed product shortages, built remote pharmacy sys- tems to avoid human-to-human infections, provided event-driven pharmaceutical treatment, informed the public on infection control and disease detection, helped in medication adherence, and counselled the patients for medication review and follow-up. Patients were being di- rected to use mobile applications for sending messages for required medications from pharmacies [16]. Several hospitals in Saudi Arabia have launched “drive-through pharmacy,” services allowing medicine to be picked up at the hospital so that there is no need for visitors to enter the premises. Once a doctor has prescribed the medications to a patient via a telephone or online con- sultation, the pharmaceutical team were notified. The hospital pharmacist then reviews the prescription order and ascertains the doses. Once the drug is ready, the pharmacist calls the patient to determine the best way of delivering the medicine. The means used for this phar- macy service included postal delivery and the drive- through pharmacy. Since the launch of the drive- through pharmacies, around thousands of patients were being serviced daily [17, 18]. Prevention and preparation for the Covid-19 pandemic are essential not only for the general public, but also for healthcare personnel in the clinical setting. Since the healthcare providers are at risk to get the disease, the hospital pharmacists encouraged the co-workers to wear personal protective equipment including gowns, goggles, face shields, facemasks and gloves Saudi Arabia, COVID-19 pandemic and pharmacist In Saudi Arabia, there are more than 190,800 reported cases of Covid-19 with 1649 deaths as of June 30, 2020 [2]. The first case of Covid-19 took place in Saudi Arabia on 2 March 2020. The outbreak led to temporary clos- ure of air traffic, business establishments, government offices, educational institutions and public transport. People were no longer allowed to visit the two holy mos- ques in Makkah (for Umrah) and Almadinah [7]. Their involvement has enhanced patient outcomes, quality of life, disease and drug knowledge and reduced utilization of health care services [21]. In Saudi Arabia, the community pharma- cist role was not restricted to the pharmacy only but ad- equate reporting of suspected Covid-19 individuals to the concerned authorities. The community pharmacists in Saudi Arabia helped in the management of chronic illness, making sure that the medications are available Pharmacists are an important part of everyday health- care in Saudi Arabia and have the ability to serve a num- ber of positions during this latest Covid-19 pandemic [14]. Pharmacists are acknowledged for their importance in preventive care and in Covid-19 response; they stayed at the forefront for public safety by functioning as pri- mary contact points. Ahmad et al. Journal of Pharmaceutical Policy and Practice (2020) 13:41 Page 3 of 3 and refilled, promoted continuous medication adherence and prescribed OTC medications which reduced the un- necessary hospital visits, where an individual is at high risk of being exposed to Covid-19. In Saudi Arabia the community pharmacies prepared information materials like posters, leaflets, app alerts and text messages in order to simplify the MOH guidelines related to the dis- ease. Community pharmacies in Saudi Arabia also of- fered door to door step delivery of the medications, online counselling especially for the high-risk individuals and for those undertaken home quarantine or isolation. The pharmacists in Saudi Arabia played a key role in this endeavour. The eventual effect of the Covid-19 pan- demic is likely to lead to a significant reorganization of global health care. It is necessary to keep up to date with current information about Covid-19, in order to tackle the containment of this pandemic [22, 23]. an Example. Saudi Pharmaceutical Journal. 2020. https://doi.org/10.1016/j. jsps.2020.05.005. an Example. Saudi Pharmaceutical Journal. 2020. https://doi.org/10.1016/j. jsps.2020.05.005. 10. Aruru M, Truong HA, Clark S. Pharmacy Emergency Preparedness and Response (PEPR): a proposed framework for expanding pharmacy professionals’ roles and contributions to emergency preparedness and response during the COVID-19 pandemic and beyond. Res Social Adm Pharm. 2020 Apr 10:S1551-7411(20)30323-5. 11. Dalton K, Byrne S. Role of the pharmacist in reducing healthcare costs: current insights. Integr Pharm Res Pract. 2017;6:37–46. 12. Mirza AH, Hasan SS. El-Dahiyat, Baber ZUD. Medicines policy issues for Saudi Arabia: priorities and model. Journal of Pharmaceutical Health Services Research. 2019;10(4):443–5. 12. Mirza AH, Hasan SS. El-Dahiyat, Baber ZUD. Acknowledgements h h d h The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for this work through research group no. RG-1441- 455. We are thankful for Prof. Zaheer-Ud-Din Babar for providing constructive remarks on this article. 18. Arab News, 2020: https://www.arabnews.com/node/1652126/saudi-arabia. 18. Arab News, 2020: https://www.arabnews.com/node/1652126/saudi-arabia. 19. International Pharmaceutical Federation (Health Advisory). Coronavirus 2019-nCoV outbreak. Information and interim guidelines for pharmacists and the pharmacy workforce 2020 The Netherlands. https://www.fip.org/ files/content/priority-areas/coronavirus/Coronavirus-guidance-update- ENGLISH.pdf. Medicines policy issues for Saudi Arabia: priorities and model. Journal of Pharmaceutical Health Services Research. 2019;10(4):443–5. 13. Babar Z, Francis S. Identifying priority medicines policy issues for New Zealand: a general inductive study. BMJ Open. 2014;4:e004415. https://doi. org/10.1136/bmjopen-2013-004415D. 13. Babar Z, Francis S. Identifying priority medicines policy issues for New Zealand: a general inductive study. BMJ Open. 2014;4:e004415. https://doi. org/10.1136/bmjopen-2013-004415D. 14. Rasheed K, Hasan SS, Babar ZU. Community pharmacist’s knowledge, attitude, roles and practices towards patient-centered care in Saudi Arabia: a systematic review of the literature. Journal of Pharmaceutical Health Services Research. 2018;10(1):101–15. 14. Rasheed K, Hasan SS, Babar ZU. Community pharmacist’s knowledge, attitude, roles and practices towards patient-centered care in Saudi Arabia: a systematic review of the literature. Journal of Pharmaceutical Health Services Research. 2018;10(1):101–15. 15. CGC, 2020. www.cgc.gov.sa/ Saudi Arabia’s ruthless fight against coronavirus. 15. CGC, 2020. www.cgc.gov.sa/ Saudi Arabia’s ruthless fight against coronavirus. 16. Saudi Gazette, 2020: https://www.saudigazette.com.sa/article/591011. 16. Saudi Gazette, 2020: https://www.saudigazette.com.sa/article/591011. 17. Khaleej Today, 2020: https://alkhaleejtoday.co/saudi-arabia/69882/Saudi- medical-centers-implement-delivery-services-drive-throughs-to-combat- COVID-19.html. Consent for publication ll h d h Consent for publication All authors approved the manuscript. 22. Christian MD, Sprung CL, King MA. Triage: care of the critically ill and injured during pandemics and disasters: CHEST consensus statement. Chest. 2014;2020(April):e61S–74S. 22. Christian MD, Sprung CL, King MA. Triage: care of the critically ill and injured during pandemics and disasters: CHEST consensus statement. Chest. 2014;2020(April):e61S–74S. Competing interests Competing interests The authors declare no conflict of interest. 23. Care P. Strategies for Scarce Resource Situations. Minnesota Department of Health; St. Paul. 2019; https://www.health.state.mn.us/communities/ep/ surge/crisis/standards.pdf. 23. Care P. Strategies for Scarce Resource Situations. Minnesota Department of Health; St. Paul. 2019; https://www.health.state.mn.us/communities/ep/ surge/crisis/standards.pdf. Received: 28 May 2020 Accepted: 2 July 2020 Received: 28 May 2020 Accepted: 2 July 2020 Authors’ contributions ll h d All authors participated equally in the conceptualization, writing and revision of the manuscript. All authors have read and approved the final draft of the manuscript. 20. Alaqeel S, Abanmy NO. Counselling practices in community pharmacies in Riyadh, Saudi Arabia: a cross-sectional study. BMC Health Serv Res. 2015;15: 557. 20. Alaqeel S, Abanmy NO. Counselling practices in community pharmacies in Riyadh, Saudi Arabia: a cross-sectional study. BMC Health Serv Res. 2015;15: 557. 21. Erick Wesley Hedima, Michael Samuel Adeyemi and Nasiru Yakubu Ikunaiye, Community pharmacists: on the frontline of health service against COVID- 19 in LMICs. Research in Social and Administrative Pharmacy, 2020, https:// doi.org/10.1016/j.sapharm.2020.04.017. Ethics approval and consent to participate Not applicable Ethics approval and consent to participate Not applicable References Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1. WHO. World Health Organisation. Pandemic Covid-19 preparedness and response: a WHO guidance document. Geneva: World Health Organisation; 2020. https://www.who.int/health-topics/coronavirus. 2020. https://www.who.int/health-topics/coronavirus. 2. Coronavirus Updates, 2020: www.worldometer.com/corona. 3. Bukhari N, Rasheed H, Nayyer B, Babar ZUD. Pharmacists at the frontline beating the COVID-19 pandemic. J of Pharm Policy and Pract. 2020;13:8. 4. Kretchy IA, Asiedu-Danso M, Kretchy JP. Medication management and adherence during the COVID-19 pandemic: perspectives and experiences from low-and middle-income countries. Res Social Adm Pharm. 2020;S1551- 7411(20):30332–6. 5. Chan AHY, Rutter V, Ashiru-Oredope D, Tuck C, Babar ZU. Together we unite: the role of the Commonwealth in achieving universal health coverage through pharmaceutical care amidst the COVID-19 pandemic. J of Pharm Policy and Pract. 2020;13:13. https://doi.org/10.1186/s40545-020- 00214-6. 6. Bukhari N, Maanzoor M, Rasheed H, Nayyer B, Malik M, Babar Z-U-D. A step towards gender equity to strengthen the pharmaceutical workforce during COVID-19. J of Pharm Policy and Pract. 2020;13:15. 7. MOH, 2020. COVID-19 Monitoring Committee Holds Its Meeting. https:// www.moh.gov.sa/en/Ministry/MediaCenter/News/Pages/News. 8. SPA, 2020. Kingdom’s Government Sets Preventive, Precautionary Measures to Prevent COVID-19 Infection Transmission. https://www.spa.gov.sa. 9. Alshammari TM, Altebainawi AF, Alenzi KA. Importance of early precautionary actions in avoiding the spread of COVID-19: Saudi Arabia as
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Exploring the role of T helper subgroups and their cytokines in the development of pregnancy-induced hypertension
Frontiers in immunology
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Introduction According to the modern tenets of reproductive immunology, pregnancy can be regarded as a triumphant outcome of a natural allograft. The fetus successfully evades rejection owing to the presence of the immune barricade of the placenta, immunosuppressive cells, and immunomodulators within the maternal environment. Thus, a successful pregnancy depends on the balance of immunity between the fetus and the mother. In other words, maternal-fetal tolerance is the key to a successful pregnancy. Furthermore, the interaction between the maternal and fetal systems, as well as the impact of fetal cells circulating within the maternal bloodstream and inducing an inflammatory response, warrants consideration (1). Loss of maternal immune tolerance, causing immune rejection, may lead to pregnancy pathology such as pregnancy-induced hypertension (PIH) (2), abortion, and other conditions (3). PIH is a pregnancy-specific disorder characterized by the onset of hypertension (systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg) after 20 weeks of gestation in the absence of proteinuria or other organ dysfunction. The severity of PIH is classified into three stages: mild, moderate, and severe. PIH is a serious condition that can lead to significant maternal and fetal morbidity and mortality. The risks associated with PIH include placental abruption, fetal growth restriction (FGR), preterm birth, and maternal organ damage, such as liver and kidney dysfunction (4). If left untreated, PIH can progress to preeclampsia (PE) or eclampsia, which are even more severe conditions that can be life-threatening for both mother and fetus (5). Adopting an immunological perspective can be instrumental in uncovering and comprehending the etiology and pathogenesis of PIH, thereby providing valuable insights for the prevention, diagnosis, treatment, and care of PIH. © 2023 Zhou, Wu and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. © 2023 Zhou, Wu and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution The most recognized model of PIH is poor placentation due to abnormal spiral artery formation, while immune modulation of trophoblast invasion is thought to play an important role in the pathogenesis of PIH (6). TYPE Opinion PUBLISHED 05 June 2023 DOI 10.3389/fimmu.2023.1126784 TYPE Opinion PUBLISHED 05 June 2023 DOI 10.3389/fimmu.2023.1126784 OPEN ACCESS OPEN ACCESS EDITED BY Yin Tailang, Wuhan University, China REVIEWED BY Susanta Pahari, Texas Biomedical Research Institute, United States Nishel Mohan Shah, Imperial College London, United Kingdom *CORRESPONDENCE Dongmei Zhang 13818808552@163.com Youcheng Wu 1341927901@qq.com Qianqian Zhou, Youcheng Wu* and Dongmei Zhang* Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China KEYWORDS pregnancy-induced hypertension (PIH), Th1 cell, Th2 cell, cytokines, immune Zhou Q, Wu Y and Zhang D (2023) Exploring the role of T helper subgroups and their cytokines in the development of pregnancy-induced hypertension. Front. Immunol. 14:1126784. doi: 10.3389/fimmu.2023.1126784 Exploring the role of T helper subgroups and their cytokines in the development of pregnancy- induced hypertension OPEN ACCESS EDITED BY Yin Tailang, Wuhan University, China REVIEWED BY Susanta Pahari, Texas Biomedical Research Institute, United States Nishel Mohan Shah, Imperial College London, United Kingdom *CORRESPONDENCE Dongmei Zhang 13818808552@163.com Youcheng Wu 1341927901@qq.com RECEIVED 18 December 2022 ACCEPTED 16 May 2023 PUBLISHED 05 June 2023 CITATION Zhou Q, Wu Y and Zhang D (2023) Exploring the role of T helper subgroups and their cytokines in the development of pregnancy-induced hypertension. Front. Immunol. 14:1126784. doi: 10 3389/fimmu 2023 1126784 Introduction The maternal immune response to fetal alloantigens is dynamic and ideally shifts between immune suppression and response during placentation, with pregnancy gestation, and then birth (7). In a normal pregnancy, Frontiers in Immunology frontiersin.org 01 Zhou et al. 10.3389/fimmu.2023.1126784 the maternal immune system undergoes changes to accommodate the fetus and prevent it from being recognized as foreign (8). This includes the production of certain immune cells and molecules that suppress the immune response and promote tolerance to the fetus. The rejection reaction, on the other hand, occurs when the immune system recognizes the fetus as foreign and launches an attack against it. However, in pregnant women with PIH, there is a decrease in the protective response and an increase in the rejection reaction (9). This means that the mother’s immune system is more likely to perceive the fetus as a threat and attack it, potentially leading to complications. Chemokine gene silencing in decidual stromal cells restricts the entry of T cells into the maternal-fetal interface. Effector T cells cannot accumulate in the decidua, the special stromal tissue that surrounds the fetus and placenta (10). Moreover, the regulation and recruitment of inducible regulatory T cells by trophoblast cells occur during early pregnancy (11). A large number of studies have shown that the T helper (Th) cells subgroup and their secreted cytokines play a core regulatory role in pregnancy immunity and are closely related to the occurrence of PIH (7, 9, 12, 13).Therefore, even in the presence of a small number of T cells, the effects of T cells themselves and their secreted cytokines are noteworthy. can promote the invasion of extracellular villus trophoblast cells and inhibit their apoptosis (57). Treg cells promote CD4+CD25-T differentiation to CD4+CD25 + Treg by secreting inhibitory cytokines such as TGF-b, showing the advantage of Treg cells at the mother-fetal interface and indirectly playing the role of immunomodulator (58). Therefore, normal physiological pregnancy mainly presents a Th2-type immune advantage and the Treg cell amplification phenomenon. Th1/Th2 and Th17/Treg balance is an essential condition for maintaining normal pregnancy. On the other hand, Th1/Th2 and Th17/Treg balance play a role in maintaining maternal-fetal immune tolerance (14). Research has reported that the number and function of Th cells and the ratio of Th1/Th2 in patients with PE were significantly decreased (59). Introduction Therefore, paying attention to the changes of Th1/Th2 in PIH that may exist before the occurrence of PE is of great significance for understanding the development mechanism of PIH during pregnancy. Overview of cytokines secreted from T helper subsets T helper subsets primarily achieve their physiological functions through the release of cytokines. These cytokines have broad biological effects, including regulating the activation and proliferation of immune cells, regulating inflammatory responses, and affecting biological processes such as cell proliferation, differentiation, and apoptosis. In this article, we mainly focus on the effects of cytokines produced by T helper subsets on the placental trophoblast and other immune cells, and the association of these effects with the development of PIH (Figure 1). In this perspective, we will summarize the role of T helper subsets (TH1/2/ 17 and Treg) in PIH and explore the role of relevant cytokines in the pathogenesis of PIH. Cytokines play a complex role in the pathophysiology of PIH and PE (60). The direct effects of cytokines on myocardiocytes to suppress contractility can also have negative consequences for maternal and fetal cardiovascular function (61). The endothelial injury caused by PE can lead to peripheral edema and other complications (62). Overall, the effects of cytokines on the maternal-fetal interface and cardiovascular system in PE are complex and require further research to fully understand. In this article, we will discuss selected cytokines produced by T helper subsets. We will focus on their effects on the immune environment, inflammatory processes, and trophoblast invasion. Frontiers in Immunology Cytokines secreted from Th1 The Th cell-mediated adaptive immune response represents a critical component in the intricate mechanism of maternal-fetal immune tolerance. Upon encountering a diverse array of cytokine stimuli, the Th0 cells, which originate from initial CD4+ T cells, undergo differentiation into distinct subsets including Th1, Th2, Th17, and Treg cells, each of which assumes discrete biological functions (13). Th1 cells mainly secrete IFN-g, TNF-a, and other cytokines, which have cytotoxic effects, and can inhibit the invasion of trophoblasts and induce their apoptosis. (53) Th1 cells can also inhibit embryo implantation by enhancing the vitality of decidual macrophages, which is not conducive to the maintenance of pregnancy (54). In contrast to Th1 cells, Th2 cells possess the capacity to produce IL-4 cytokines, which act to promote trophoblast cell proliferation and invasion, enhance uterine receptivity, and confer immune-nutritive and protective benefits to the fetus (55, 56). IFN-g Interferon-gamma (IFN-g), a cytokine produced by various immune cells, has been implicated in the pathogenesis of PIH. Studies have shown that PIH patients have elevated levels of IFN-g, indicating that the dysregulation of IFN-g may be involved in the pathogenesis of PIH (65). Endothelial dysfunction is a hallmark of PIH, and IFN-g has been shown to induce endothelial cell dysfunction (17). Specifically, IFN-g can increase endothelial cell apoptosis, impair endothelial cell proliferation (18), and promote the production of reactive oxygen species (ROS) (19), all of which contribute to the development of PIH. In normal pregnancies, trophoblast cells invade the maternal decidua and remodel the maternal spiral arteries to promote fetal growth (66). However, in PIH, trophoblast invasion is impaired, leading to inadequate placental perfusion and subsequent hypoxia (67). IFN-g has been shown to inhibit trophoblast invasion by inducing apoptosis of extravillous trophoblast cells and suppressing the expression of invasion-related genes (20). Overexpression of Th1 at the maternal- fetal interface can activate NK cells to up-regulate the expression of HLA-G in the placenta, which is prone to immune rejection, by stimulating the secretion of IFN-g (21). PIH is associated with an inflammatory response (68), and IFN-g has been shown to stimulate the secretion of pro-inflammatory cytokines and chemokines (22), promoting the development of an inflammatory environment that contributes to the pathogenesis of PIH. Therefore, the dysregulation of IFN-g plays a critical role in the pathogenesis of PIH by regulating immune responses, inducing endothelial dysfunction, impairing trophoblast invasion, and promoting an inflammatory response. At the same time, TNF-a could activate neutrophils to release elastic proteinase and promote neutrophils to adhere to vascular endothelial cells (26), leading to vascular endothelial damage. It can also directly activate vascular endothelial cells, induce the expression of endothelial cell surface adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), damage vascular endothelium, and further enhance the activity of neutrophils through the endothelial system (27). TNF-a has the ability to modulate anticoagulant factors (28), thereby promoting a procoagulant state in vascular endothelial cells. Activation of these pathways can potentially contribute to the pathogenesis of PIH. Research has revealed that increased placental synthesis and secretion of TNF-a in patients with PIH can lead to augmented apoptosis of placental trophoblast cells, which in turn impairs their capacity to invade the decidua and spiral arteries, resulting in shallow placental implantation (29). IL-2 IL-2 Notably, IL-2 is a critical cytokine produced by Th1 cells. Studies have demonstrated that Th1 cells are generated from the trophoblast cell and decidual lymphocytes during pregnancy, and their expression is augmented in the placental microenvironment (63). Specifically, studies have shown that the loss or reduction of IL-2 in pregnancy can suppress total natural killer (NK) cell activation, including non-cytolytic NK cells that may play a protective role in the fetal environment (64). Cytotoxic NK cells have been implicated in the development of PE as they are thought to contribute to endothelial dysfunction and inflammation. On the other hand, noncytotoxic NK cells have been shown to play a protective role in maintaining a healthy pregnancy by regulating trophoblast invasion and promoting placental development. Loss or reduction of IL-2 can lead to an increase in cytotoxic NK cells and a decrease in noncytotoxic NK cells, which can contribute to the Th17 cells mainly mediate inflammatory diseases and autoimmune diseases, and the cytokine IL-17 secreted by Th17 Frontiers in Immunology 02 frontiersin.org 10.3389/fimmu.2023.1126784 Zhou et al. could affect trophoblast infiltration of the maternal spiral artery (SA), resulting in blocked angioplasty of SA, stenosis of the vascular cavity, increased resistance, and sensitivity to vasoactive substances. Conrad et al. conducted a study to investigate the association between circulatory inflammatory cytokines and the pathogenesis of PE (24). The study found that the median concentration of plasma TNF-a was twofold higher in women with PE compared to normal third-trimester pregnancy (P < 0.001) and gestational hypertension (P < 0.04). FGR and PE are frequently linked to abnormal maternal inflammation, deficient SA remodeling, and altered uteroplacental perfusion. Cotechini et al. (25) revealed a novel mechanistic association between abnormal maternal inflammation and the development of FGR with features of PE. By administering low-dose lipopolysaccharide (LPS) to pregnant rats during gestational days 13.5-16.5, they demonstrated that abnormal inflammation resulted in FGR mediated by TNF-a. The results indicated that maternal inflammation can cause severe pregnancy complications through a mechanism involving increased maternal levels of TNF-a (25). development of PIH and PE. In addition, IL-2 plays an important role in regulating immune responses and maintaining immune balance, and its abnormal expression may lead to immune dysregulation, promoting the occurrence and development of PIH (14). Hama et al. IFN-g This pathological process can cause a restructuring of the uterine spiral artery architecture, leading to placental ischemia, hypoxia, and metabolic disturbances (30). In recent years, it has been found that TNF-a can also regulate plasma leptin levels in PIH (31). High levels of TNF-a and leptin may act on trophoblast cells and vascular endothelial cells together to impair their functions and lead to the occurrence of PIH (32). Leptin can induce oxidative stress and inflammation in endothelial cells, leading to endothelial dysfunction and injury (33). Leptin can also impair trophoblast invasion by inhibiting the expression of adhesion molecules and enzymes required for trophoblast invasion, such as integrins and matrix metalloproteinases (71). Additionally, leptin can induce the production of pro-inflammatory cytokines and chemokines, which further impair trophoblast invasion (72). IL-2 found that IL-2 played a coordinating role in the destruction of trophoblast cells due to a decrease in the nonclassical human leukocyte antigen-1 (HLA-G) in vitro experiments (15). Furthermore, it has been observed that lymphokine-activated killer (LAK) cells derived from decidua are capable of inducing the secretion of vascular endothelial growth factor (VEGF) by nourishing cells (16). Interestingly, heightened IL-2 expression in the decidual milieu has been shown to dampen VEGF release. Frontiers in Immunology TNF-a Among several Th1 cytokines, TNF-a is the most closely related to the occurrence of PIH (29, 69, 70). Under physiological conditions, there is typically a low expression of TNF-a mRNA in the endometrial glandular epithelium, basement membrane, and ovarian stroma of females. However, during gestation, both the developing fetus and decidual tissue are capable of producing TNF- a, which plays a pivotal role in mediating maternal-fetal immune regulation (23). However, TNF-a increased significantly when PIH occurred. Conrad (24) and Cotechini (25) believed that TNF-a The upregulation of TNF-a and other cytokines causes the involvement of trophoblast cells and decreased infiltration ability (34), shallow placental implantation resulting in placental ischemia and hypoxia, enhanced local cellular immune response (35), activation of white blood cells in the villus space leading to vascular endothelial injury, and eventually the occurrence of PIH (73). Evidently, immunological factors play a crucial role in the Frontiers in Immunology 03 frontiersin.org 10.3389/fimmu.2023.1126784 Zhou et al. contribute to the pro-inflammatory state seen in PIH (43). It can be concluded that IL-4 is involved in the regulation of vascular function, immune response, and inflammation, which are important implications in the pathogenesis of PIH. pathogenesis of shallow placental implantation, vascular endothelial injury, and other related factors in PIH. In the future, continued investigation of molecular immunology is anticipated to elucidate the underlying mechanisms of TNF-a in the context of PIH. The observed increase in Th1 cytokines in PIH patients is likely a consequence of the pathology, rather than a cause. Systemic inflammation resulting from tissue injury can lead to an upregulation of inflammatory cytokines, including TNF-a, IL-2, IFN-g, and IL-4. These cytokines can then contribute to the pathogenesis of PIH through various mechanisms, such as impairing trophoblast invasion and causing endothelial dysfunction. In order to deeply understand the relationship between Th cell subsets and their differentiated cytokines and PIH, Saito et al. (44) investigated Th1 and Th2 cytokines secreted by peripheral blood mononuclear cells (PBMC) of patients with hypertensive diseases during pregnancy by enzyme-linked immunosorbent assay (ELISA). The results showed that the level of Th1 cytokines secreted by PBMC in PIH patients was significantly higher than that in the normal control group, and the ratios of TNF-a/IL-4, IL- 2/IL-4, and IFN-g/IL-4 were also significantly higher. Moreover, the concentrations of the three Th1 cytokines were positively correlated with patients’ MAP. IL-6 IL-6 can stimulate B cells to produce antibodies to stimulate the proliferation and differentiation of cytotoxic T lymphocytes (CTL) (75). Both the placenta and decidua in early pregnancy contain IL-6 mRNA, suggesting that IL-6 may work in conjunction with other factors to facilitate the fusion of maternal and nourishing cells (36, 37). IL-6 also participates in the formation of the placental blood vessels. IL-6 can promote the proliferation and migration of endothelial cells, and stimulate the release of angiogenic factors, such as vascular endothelial growth factor (VEGF), which further promote angiogenesis (38). Excessive IL-6 in late pregnancy is involved in the pathological process of PIH (39). TNF-a Systemic inflammation resulting from tissue injury can lead to an upregulation of inflammatory cytokines, including TNF-a, IL-2, IFN-g, and IL-4. Abnormal secretion of these cytokines can then contribute to the pathogenesis of PIH through various mechanisms, such as impairing trophoblast invasion and causing endothelial dysfunction. IL-4 IL-4 is also an important cytokine involved in immune regulation and inflammation and has been found to be decreased in the serum and placenta of women with PE compared to those with normal pregnancy, suggesting that it may be involved in the pathogenesis of the disease (68, 76). IL-4 is known to play a role in the maintenance of vascular integrity and endothelial function, which are key factors in the development of PIH (40). Reduced IL-4 levels could therefore result in impaired eNOS activity and decreased NO-mediated vasodilation, contributing to hypertension and other cardiovascular complications in pregnancy (41). Furthermore, IL-4 may also modulate the immune response and contribute to the development of PIH through its effects on T-helper cell differentiation and cytokine production (42). Specifically, decreased IL-4 levels have been associated with an imbalance in the Th1/Th2 ratio, which may Cytokines secreted from Th2 Cytokines secreted by Th2, such as IL-4, IL-6, and IL-10, can inhibit the Th1 immune response and the activation of NK cells to protect the fetus (74). These cytokines mainly participate in B cell proliferation and maturation, which can increase the antibody- mediated immune response. A further study showed that compared with normal pregnant women, the ratio of Th1 and Th2 cells increased and the content of Th2 cells decreased in patients with PIH during late pregnancy. The results of this study are consistent with Saito’s report (77). The reciprocal regulation between Th1 and Th2 cells plays a pivotal role in the maintenance of immune homeostasis, particularly in the context of transplant immunology (78). Therefore, aberrant maternal immune responses may serve as a trigger for the onset of PIH (9). Recent investigations have demonstrated that the Th1/ Th2 cell ratio in patients with PIH exhibits a tendency towards heightened Th1 activity (79). Cytokines from Th17 Th17 cells secrete a number of pro-inflammatory cytokines, including IL-17, IL-21, and IL-22, which have been found to be elevated in the circulation of women with PIH (80). These cytokines may contribute to the development of hypertension and endothelial dysfunction, which are key features of PIH. IL-10 IL-10 is a kind of cytokine that has a variety of biological activities and its most important role is in immunosuppression. Many researchers have found that IL-10 can inhibit the expression of TNF-a, INF-g, major histocompatibility complex (MHC-II) molecules, and B7 adhesion molecules on phagocytes, and block the killing effect of NK (51). The relative lack of IL-10 will increase the content of immune factors, resulting in the breakdown of the Th1/Th2 balance, resulting in the enhancement of the Th1 immune response, leading to the occurrence of PIH (52). Taken together, the cytokines produced by Th17 cells are important contributors to the pathogenesis of PIH, promoting inflammation, oxidative stress, endothelial dysfunction, and vascular damage. Targeting these cytokines may provide a potential therapeutic strategy for the prevention and treatment of PIH. IL-17 IL-17, in particular, has been implicated in the pathogenesis of PIH (81). It induces the production of other pro-inflammatory cytokines and chemokines, such as IL-6 and TNF-a (45), which contribute to the development of hypertension and endothelial dysfunction. IL-17 also stimulates the production of reactive oxygen species (ROS) (46), which can lead to oxidative stress and endothelial damage. Additionally, IL-17 promotes the infiltration of neutrophils and macrophages into the placenta (47), which can further contribute to inflammation and tissue damage. Frontiers in Immunology frontiersin.org 04 Zhou et al. 10.3389/fimmu.2023.1126784 FIGURE 1 A summary of how imbalanced T helper cell functions can lead to both systemic changes as well as local changes contributing to PIH pathology. The dysregulation of Th cells containing Th1, Th2, Th17, and Tregs subsets alters the cytokine environment, which can promote a local and systemic inflammatory response that is associated with the occurrence of PIH. Furthermore, locally during placentation, an imbalance of T helper cells and related cytokines can hinder the infiltration of trophoblasts and induce the dysfunction of vascular endothelial cells, and further prompt the generation of PE. FIGURE 1 A summary of how imbalanced T helper cell functions can lead to both systemic changes as well as local changes contributing to PIH pathology. The dysregulation of Th cells containing Th1, Th2, Th17, and Tregs subsets alters the cytokine environment, which can promote a local and systemic inflammatory response that is associated with the occurrence of PIH. Furthermore, locally during placentation, an imbalance of T helper cells and related cytokines can hinder the infiltration of trophoblasts and induce the dysfunction of vascular endothelial cells, and further prompt the generation of PE. Cytokines from Tregs Tregs are a subset of T cells that regulate immune responses and maintain tolerance to self-antigens. They produce cytokines such as IL-10 and TGF-b, which have anti-inflammatory and immunosuppressive effects. Several studies have reported lower levels of Tregs in women with PIH compared to normotensive pregnant women (82). This suggests that a deficiency in Tregs may contribute to the development of PIH. In addition, decreased production of IL-10 and TGF-b has been observed in women Frontiers in Immunology IL-21 and IL-22 with PIH (83), further supporting the role of Tregs in the development of PIH. Moreover, it has been shown that administration of TGF-b can ameliorate PIH in a rat animal models study (50). This suggests that Tregs and their cytokines may have therapeutic potential for the treatment of PIH. IL-21 and IL-22 have also been shown to be elevated in women with PIH. IL-21 promotes the differentiation and activation of Th17 cells, and can enhance the production of IL-17 and other pro- inflammatory cytokines (48). IL-22, on the other hand, has been implicated in the regulation of angiogenesis and VEGF signaling (49), which are important processes in the development of placental vascularization and function. Discussion and conclusion In brief, the etiology and pathogenesis of PIH in immunological investigations are posited to arise from the dysregulation of maternal- fetal immune homeostasis or immune tolerance, characterized by diminished Th2-mediated immunosuppression and/or heightened Th1-mediated cellular immune activation Table 1. frontiersin.org 05 Zhou et al. 10.3389/fimmu.2023.1126784 TABLE 1 The association of Th cells and cytokines with PIH. TABLE 1 The association of Th cells and cytokines with PIH. Cell Type cytokines The association with PIH References Th1 cell IL-2 Immune dysregulation (14) Destruction of trophoblast cell (15) Regulates the secretion of VEGF (16) INF-g Induces endothelial cell dysfunction and apoptosis (17, 18) Promotes production of ROS (19) Inhibits trophoblast invasion (20) Up-regulates the expression of HLA-G (21) Promotes the development of an inflammatory environment (22) TNF-a Mediating maternal-fetal immune regulation (23) Affects trophoblast infiltration of maternal SA (24, 25) Activates neutrophils to release elastic proteinase and promote neutrophils (26) Vascular endothelial damage (27) Modulates anticoagulant factors (28) Augments apoptosis of placental trophoblast cells (29, 30) Regulates plasma leptin levels (31–33) Causes the involvement of trophoblast cells and decreased infiltration ability (34) Enhances local cellular immune response (35) Th2 cell IL-6 Facilitates the fusion of maternal and nourishing cells. (36, 37) Participates in the formation of the placental blood vessels (38, 39) IL-4 Maintenance of vascular integrity and endothelial function (40, 41) Modulates the immune response (42) Imbalances in the Th1/Th2 ratio (43, 44) Th17 IL-17 Induces the production of other pro-inflammatory cytokines and chemokines (45) Stimulates the production of ROS (46) Promotes the infiltration of neutrophils and macrophages (47) IL-21 Differentiation and activation of Th17 cells (48) IL-22 Regulates angiogenesis and VEGF (49) Tregs TGF-b Ameliorates PIH (50) IL-10 Immunosuppression (51, 52) PIH represents a prevalent condition in obstetrics and has been a subject of intense research. Numerous etiological and pathogenic mechanisms have been proposed, including the immune response theory, oxidative stress theory, capillary endothelial injury theory, and others. However, thus far, none of these theories have fully accounted for the multifaceted pathophysiology of PIH. Here, we briefly summarized the close relationship between Th1 cells, Th2 cells, and the released cytokines and PIH from the perspective of immunology, and discussed the possible occurrence and development mechanism. Overall, although PIH is caused by many factors, the immune factor plays a pivotal role. Therefore, it may be more effective to prevent, diagnose, treat, and care for PIH by focusing on immunological indicators. Discussion and conclusion The pathogenesis of PIH is still unclear, which makes clinical diagnosis and treatment difficult. The current diagnosis of PIH and PE mainly focuses on hypertension, proteinuria, serum biochemical abnormalities, and fetal growth. It can be seen from this review that the pathogenesis of PIH may be related to immune factors to a certain extent. In recent years, researchers have suggested that the serum levels of inflammatory factors associated with the pathogenesis of PIH and PE may become part of the diagnostic criteria. For example, a study by Li et al. found that serum levels of Frontiers in Immunology frontiersin.org 06 Zhou et al. Zhou et al. 10.3389/fimmu.2023.1126784 Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. References 1. Frascoli M, Coniglio L, Witt R, Jeanty C, Fleck-Derderian S, Myers DE, et al. Alloreactive fetal T cells promote uterine contractility in preterm labor via IFN-g and TNF-a. Sci Transl Med (2018) 10(438):eaan2263. doi: 10.1126/scitranslmed.aan2263 trophoblast cells. Am J Reprod Immunol (2012) 67(1):17–27. doi: 10.1111/j.1600- 0897.2011.01056.x trophoblast cells. Am J Reprod Immunol (2012) 67(1):17–27. doi: 10.1111/j.1600- 0897.2011.01056.x 12. 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Am J Reprod Immunol (1999) 41 (2):153–8. doi: 10.1111/j.1600-0897.1999.tb00088.x 5. Roberts JM, August PA, Bakris G, Barton JR, Bernstein IM, Druzin M, et al. Hypertension in pregnancy. Report of the American college of obstetricians and gynecologists’ task force on hypertension in pregnancy. Obstet Gynecol (2013) 122 (5):1122–31. doi: 10.1097/01.AOG.0000437382.03963.88 16. Matsubara K, Nagamatsu T, Fujii T, Kozuma S, Taketani Y. Author contributions IL-6 and TNF-a were significantly higher in women with early- onset PIH compared to normotensive pregnant women (29). They proposed that serum levels of IL-6 and TNF-a could be used as potential biomarkers for early diagnosis of PIH. QZ, YW, and DZ wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version. Significantly, immune modulation is anticipated to emerge as a novel therapeutic target for PIH and PE in clinical management and holds considerable promise in ameliorating maternal mortality. This approach aims to modulate the immune response by regulating key immune pathways and cytokines, such as interleukin-6 (IL-6) and interleukin-10 (IL-10), and may improve outcomes for women with PIH and PE. Immune modulation strategies for the treatment of PIH and PE include the use of immunomodulatory agents and the development of novel targeted therapies. For example, Tinsley et al. (84) used a PIH rat model of deoxycorticosterone acetate (DOCA)/salt-low renin, which exhibits features of hypertension, proteinuria, endothelial dysfunction, and intrauterine growth restriction (IUGR). Furthermore, suppression of the immune system with either azathioprine (Aza) or mycophenolate mofetil (MMF) during the second half of pregnancy significantly reduced hypertension, proteinuria, and endothelial dysfunction, as well as increased the proinflammatory Th1 cytokine profile in rats treated with DOCA/salt, which alleviated the development of PIH. Medicationsthattargettheimmunesystem,suchasanti-inflammatory drugs or targeted immunotherapy agents, may be beneficial for PIH patients with an overactive immune system. However, medication interventionsshouldbetailoredtotheindividualpatientbasedontheir immunological profile and other medical conditions. Funding This work was supported by the Foundation of Tongren Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (No. 2020TRYJ(LB)07). 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Sykes L, Macintyre DA, Yap XJ, Teoh TG, Bennett PR. The Th1:th2 dichotomy of pregnancy and preterm labour. Mediators Inflammation (2012) 2012:967629. doi: 10.1155/2012/967629 73. Carpentier PA, Dingman AL, Palmer TD. Placental TNF-a signaling in illness- induced complications of pregnancy. Am J Pathol (2011) 178(6):2802–10. doi: 10.1016/ j.ajpath.2011.02.042 73. Carpentier PA, Dingman AL, Palmer TD. Placental TNF-a signaling in illness- induced complications of pregnancy. Am J Pathol (2011) 178(6):2802–10. doi: 10.1016/ j.ajpath.2011.02.042 80. Mora-Palazuelos C, Bermúdez M, Aguilar-Medina M, Ramos-Payan R, Ayala- Ham A, Romero-Quintana JG. Cytokine-polymorphisms associated with preeclampsia: a review. Med (Baltimore) (2022) 101(39):e30870. doi: 10.1097/MD.0000000000030870 74. Bretscher PA. The regulatory functions of CD4+ and CD8+ T-cell subsets in immune class regulation. Res Immunol (1991) 142(1):45–50. doi: 10.1016/0923-2494 (91)90011-7 81. Orejudo M, Rodrigues-Diez RR, Rodrigues-Diez R, Garcia-Redondo A, Santos- Sánchez L, Rández-Garbayo J, et al. Interleukin 17A participates in renal inflammation associated to experimental and human hypertension. Front Pharmacol (2019) 10:1015. doi: 10.3389/fphar.2019.01015 75. Dos Santos Fagundes I, Brendler EP, Nunes Erthal I, Eder Ribeiro RJ, Caron- Lienert RS, Machado DC, et al. Total Th1/Th2 cytokines profile from peripheral blood lymphocytes in normal pregnancy and preeclampsia syndrome. Hypertens Pregnancy (2022) 41(1):15–22. doi: 10.1080/10641955.2021.2008424 82. ZhangZJ,DingLT,ZouJ,LyuGZ.[ChangesofhelperTlymphocytes17andregulatory T lymphocytes in peripheral blood of patients with extensive burn at early stage in august 2nd kunshan factory aluminum dust explosion accident and the significance]. Zhonghua Shao Shang Za Zhi (2018) 34(6):360–4. doi: 10.3760/cma.j.issn.1009-2587.2018.06.009 76. Zhu J. T Helper 2 (Th2) cell differentiation, type 2 innate lymphoid cell (ILC2) development and regulation of interleukin-4 (IL-4) and IL-13 production. Cytokine (2015) 75(1):14–24. doi: 10.1016/j.cyto.2015.05.010 83. Del Gobbo V, Giganti MG, Zenobi R, Villani V, Premrov MG. The immunosuppressive cytokines influence the fetal survival in patients with pregnancy-induced hypertension. Am J Reprod Immunol (2000) 44(4):214–2. doi: 10.1111/j.8755-8920.2000.440405.x 84. Tinsley JH, Chiasson VL, South S, Mahajan A, Mitchell BM. Immunosuppression improves blood pressure and endothelial function in a rat model of pregnancy-induced hypertension. Am J Hypertens (2009) 22(10):1107–14. 71. Sánchez-Aranguren LC, Prada CE, Riaño-Medina CE, Lopez M. Endothelial dysfunction and preeclampsia: role of oxidative stress. Front Physiol (2014) 5:372. doi: 10.3389/fphys.2014.00372 72. Muralimanoharan S, Maloyan A, Myatt L. Evidence of sexual dimorphism in the placental function with severe preeclampsia. Placenta (2013) 34(12):1183–9. doi: 10.1016/j.placenta.2013.09.015 73. Carpentier PA, Dingman AL, Palmer TD. Placental TNF-a signaling in illness- induced complications of pregnancy. Am J Pathol (2011) 178(6):2802–10. doi: 10.1016/ j.ajpath.2011.02.042 74. Bretscher PA. The regulatory functions of CD4+ and CD8+ T-cell subsets in immune class regulation. Res Immunol (1991) 142(1):45–50. doi: 10.1016/0923-2494 (91)90011-7 75. Dos Santos Fagundes I, Brendler EP, Nunes Erthal I, Eder Ribeiro RJ, Caron- Lienert RS, Machado DC, et al. Total Th1/Th2 cytokines profile from peripheral blood lymphocytes in normal pregnancy and preeclampsia syndrome. Hypertens Pregnancy (2022) 41(1):15–22. doi: 10.1080/10641955.2021.2008424 76. Zhu J. T Helper 2 (Th2) cell differentiation, type 2 innate lymphoid cell (ILC2) development and regulation of interleukin-4 (IL-4) and IL-13 production. Cytokine (2015) 75(1):14–24. doi: 10.1016/j.cyto.2015.05.010 77. Saito S, Sakai M, Sasaki Y, Tanebe K, Tsuda H, Michimata T. Quantitative analysis of peripheral blood Th0, Th1, Th2 and the Th1:Th2 cell ratio during normal human pregnancy and preeclampsia. Clin Exp Immunol (1999) 117(3):550–5. doi: 10.1046/j.1365-2249.1999.00997.x 77. Saito S, Sakai M, Sasaki Y, Tanebe K, Tsuda H, Michimata T. Quantitative analysis of peripheral blood Th0, Th1, Th2 and the Th1:Th2 cell ratio during normal human pregnancy and preeclampsia. Clin Exp Immunol (1999) 117(3):550–5. doi: 10.1046/j.1365-2249.1999.00997.x 76. Zhu J. T Helper 2 (Th2) cell differentiation, type 2 innate lymphoid cell (ILC2) development and regulation of interleukin-4 (IL-4) and IL-13 production. Cytokine (2015) 75(1):14–24. doi: 10.1016/j.cyto.2015.05.010 References doi: 10.1038/ajh.2009.125 09 09 Frontiers in Immunology frontiersin.org
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https://zenodo.org/records/1877444/files/article.pdf
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Records of Graduates of Flint Medical College.
JAMA
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public-domain
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Population of Los Angeles. Los Angeles, Cal., Aug. 27, 1906. Ed i l N b g , g To the Editor:\p=m-\Onpage 613 of the Educational Number (August 25) of The Journal, under the California heading, the city of Los Angeles is given a population of 116,420. h Respectfully submitted, T Respectfully submitted, T Respectfully submitted, TORALD SOLLMAN, Edgar D. Brown. y g g p p Since clinical facilities are dependent somewhat on the amount of population, a correction is in order if a proper im- pression is to be given. The population of Los Angeles at the present time is over 240,000 instead of 116,420. I refer you to the enclosed article from to-day's Los Angeles Times showing the most recent figures. The school census last spring showed a population of 230,000. Further, the city water department has 50,977 service pipes in use and five persons to a service pipe is a conservative average. The monthly health reports are based on a population of 200,000, in order to allow a safe margin, but this estimate will shortly be increased. h d, TORALD SOLLMAN, Edgar D. Brown. The Teaching of Medical Jurisprudence in New York. The Teaching of Medical Jurisprudence in New York. Jersey City, Aug. 28, 1906. g To the Editor:\p=m-\InThe Journal, Aug. 25, 1906, page 586, you make the editorial statement that "The New York schools, Columbia, Bellevue and Cornell, make no mention of the sub- ject whatever. Medical jurisprudence is not among the courses required by the New York Board of Regents." I call Downloaded From: http://jama.jamanetwork.com/ by a RYERSON UNIVERSITY LIBRARY User on 06/15/201 Cocain in Tucker Asthma Cure. Shiloh, O., Aug. 28, 1906. Shiloh, O., Aug. 28, 1906. g To the Editor:\p=m-\Referringto the article of Dr. Vickery on "Cocain in Tucker Asthma Cure" in The Journal A. M. A., Aug. 4, 1906. I wish to thank the author for his report and to commend his suggestion as to what ought to be done to sup- press such a "cure." I had a case here, with all the symptoms of acute cocain poisoning, as the result of using the above preparation. The patient was a child 5 years of age, and I can assure you that a "remedy" that will produce as marked symptoms of poisoning in as short a time as does the Tucker Asthma Cure, needs to be removed from the market, or at least labeled as to its contents, and not retailed to an un- suspecting public as a harmless asthma cure. N P M G margin, y Los Angeles then is not in the 100,000 but rather in the 250.000 class, and has clinical facilities commensurate with a city of that size. George H. Kress. commensurate with George H. Kress. y [Editor's Note:—We are very glad to have this informa¬ tion. As stated in the Educational Number, the figures used were the estimates of the government's Census Bureau for 1903.] attention to the fact that Hon. Millard Bartlett of the ap- pellate division of the Supreme Court delivers fifteen lectures each year at the Long Island College Hospital to the third year class. Walter T. Dannreuther, M.D. Preparation 1, Preparation 1, 6 Preparation 2, Préparât on 2, b Preparation 3, Preparation 3, b Preparation 4, a Preparation 4, b Efficiency of Fresh Dilution. 70 70 100 86 si; ttt 95 0 After Standing One Day. 25 Active. . 0 0 15 0 After two Days. 0 Active . 0 Discolor¬ ation in One Day. None . . . None Dark pink. Dark pink. None . . . None . . . Light pink Light pink In Two Days. None. None. CONCLUSIONS. Preparation 1, Preparation 1, 6 Preparation 2, Préparât on 2, b Preparation 3, Preparation 3, b Preparation 4, a Preparation 4, b Efficiency of Fresh Dilution. 70 70 100 86 si; ttt 95 0 After Standing One Day. 25 Active. . 0 0 15 0 After two Days. 0 Active . 0 Discolor¬ ation in One Day. None . . . None Dark pink. Dark pink. None . . . None . . . Light pink Light pink In Two Days. None. None. CONCLUSIONS. CONCLUSIONS. In view of the variability in the commercial samples, as re¬ vealed in our examination, definite conclusions concerning the comparative activity of the different makes of suprarenal solu¬ tions are not warranted. Theoretically, all samples of "1 to 1,000 solution of suprarenal alkaloid" should be of identical strength, just as are all 1 to 1,000 solutions of strychnin. It is evident that this is not the case in practice, and that the physician, in injecting this powerful substance, is at present unable to use a perfectly exact dosage. Our samples of prepa¬ rations 1 and 2 exhibited less variation than preparations 3 and 4, but we can not be certain that a larger series of samples might not have shifted the relative position. This is also true of the average efficiency, which decreases in the first series of samples from preparation 1 (100) to preparation 4 (95), to preparation 3 (86), to preparation 1 (70) ; in the second series, from preparation 2 (86), to preparation 1 (70), to preparation 3 ( (63), to preparation 4 (0). We repeat that we would not lay any emphasis on the comparative efficiency of the different makes on account of the variability. Pollantin in hay fever has given me no relief. E. S. McKee. ef. E. S. McKee. Records of Graduates of Flint Medical College. New Orleans, Aug. 27, 1906. New Orleans, Aug. 27, 1906. S g To the Editor:\p=m-\At the May meeting of the Louisiana State Board seven of the eight 1906 graduates of Flint Medical Col- lege presented themselves for examination, and only one failed to pass. This gives a 14 2/7 per cent, of failures and should place us in Table J of your statistics instead of Table K. In justice to our school and its present administration, may I ask you to publish this statement? B h D A. D. Bush, Dean. [Editor's Note.\p=m-\TablesJ and K, to which Dr. Bush refers, appear in the annual Educational Number of The Journal, August 25, and are a part of the detailed summaries of the results of the state board examinations in all states for the year 1905. This May examination, at which he says a better record was made, is, of course, a portion of the record for 1906, and this will be included in a summary of the year 1906 to appear early in 1907.] y The results as to the stability of the solutions are more constant. Preparation 1 is the most stable, then comes prepa¬ ration 3, then preparation 4, then preparation 2. This subject of stability, however, has probably little practical importance, since the dilutions of all the samples are practically worthless after twenty-four hours. y We are unable to state whether the samples vary in strength when they leave the makers' hands, or whether the differences are due to subsequent deterioration. The former would be quite inexcusable. The latter would seem to demand a dating and returning system similar to that in vogue for antitoxic sera. Hay Fever and Ivy Poisoning. Cincinnati, Aug. 28, 1906. f Treatment of Hay Fever by Securing Normal Nostrils. Hartford, Conn., Aug. 20, To the Editor:\p=m-\InThe Journal, August 18, Dr. Dunbar Roy writes that at the session of the Association held at Atlantic City in 1904 I claimed to cure hay fever "by simply forcing patients to become absolute nose breathers." Dr. Roy is in error. I did and do claim to cure hay fever by giving my patients nostrils as nearly normal as possible, and then insist- ing that they breathe through the nose as Nature intended they should. The perusal of my monograph, "Mouth Breath- ing," should make clear to the reader what I consider neces- sary in a general way, not only for the relief but cure of the N. P. McGay. Cincinnati, Aug. 28, 1906. f g To the Editor:\p=m-\Ayear ago, while suffering from hay fever, I was poisoned with ivy. Though in the midst of the season the hay fever disappeared immediately on the advent of the ivy poisoning and gave me no further trouble that year. Was this a coincidence or a consequence? If the latter, why? Have any more of your numerous readers made the same observation?
https://openalex.org/W2109814284
https://europepmc.org/articles/pmc4065763?pdf=render
English
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Pharmacology of Hallucinations: Several Mechanisms for One Single Symptom?
BioMed research international
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cc-by
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1 Department of Addictionology, CHU Lille, 59037 Lille, France Department of Addictionology, CHU Lille, 59037 Lille, France 2 Department of Pharmacology, Univ Lille Nord de France, EA 1046, 59000 Lille, France 3 Functional Neurosciences & Disorders Laboratory, Universit´e Lille Nord de France, EA 4559, 59000 Lille, France 4Department of Pediatric Psychiatry, CHU Lille, 59037 Lille, France 5 Department of Psychiatry, CHU Lille, 59037 Lille, France 2 Department of Pharmacology, Univ Lille Nord de France, EA 1046, 59000 Lille, France 3 Functional Neurosciences & Disorders Laboratory, Universit´e Lille Nord de France, EA 4559, 59000 Lille, France 4Department of Pediatric Psychiatry, CHU Lille, 59037 Lille, France 5 Department of Psychiatry, CHU Lille, 59037 Lille, France Correspondence should be addressed to Benjamin Rolland; benjrolland@gmail.com Received 18 February 2014; Accepted 11 May 2014; Published 4 June 2014 Received 18 February 2014; Accepted 11 May 2014; Published 4 June 2014 Academic Editor: Changyang Gong Academic Editor: Changyang Gong Copyright © 2014 Benjamin Rolland et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hallucinations are complex misperceptions, that principally occur in schizophrenia or after intoxication induced by three main classes of drugs: psychostimulants, psychedelics, and dissociative anesthetics. There are at least three different pharmacological ways to induce hallucinations: (1) activation of dopamine D2 receptors (D2Rs) with psychostimulants, (2) activation of serotonin 5HT2A receptors (HT2ARs) with psychedelics, and (3) blockage of glutamate NMDA receptors (NMDARs) with dissociative anesthetics. In schizophrenia, the relative importance of NMDAR and D2R in the occurrence of hallucinations is still debated. Slight clinical differences are observed for each etiology. Thus, we investigated whether the concept of hallucination is homogenous, both clinically and neurobiologically. A narrative review of the literature is proposed to synthesize how the main contributors in the field have approached and tried to solve these outstanding questions. While some authors prefer one explanatory mechanism, others have proposed more integrated theories based on the different pharmacological psychosis models. In this review, such theories are discussed and faced with the clinical data. In addition, the nosological aspects of hallucinations and psychosis are addressed. We suggest that if there may be common neurobiological pathways between the different pharmacological systems that are responsible for the hallucinations, there may also be unique properties of each system, which explains the clinical differences observed. Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 307106, 9 pages http://dx.doi.org/10.1155/2014/307106 Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 307106, 9 pages http://dx.doi.org/10.1155/2014/307106 Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 307106, 9 pages http://dx.doi.org/10.1155/2014/307106 2. The Three Main Pharmacological Models of Hallucinations Three different types of psychoactive drugs can induce hallucinations in humans. In each case, a specific phar- macological process is involved (Table 1). Psychostimulants stimulate D2Rs, dissociative anesthetics block NMDARs, and psychedelics stimulate H5T2ARs. In schizophrenia, both D2Rs and NMDARs are involved. In each type of situation, slight clinical features are observed (Table 1). 2.2. The Glutamate Model: Hallucinations, Dissociative Anes- thetics, and Schizophrenia. Shortly after the synthesis of a new class of anesthetic drugs at the end of the 1950s, it was observed that these drugs could induce schizophrenia-like symptoms, with a combination of hallucinations, negative symptoms, and dissociative symptoms. These drugs were consequently known as “dissociative anesthetics” [18]. The main molecules of this class are phencyclidine (PCP) and ketamine. 2.1. The Dopamine Model: Hallucinations, Antipsychotics, and Schizophrenia . Schizophrenic hallucinations are mainly auditory verbal [2, 3]. However, notable exceptions include early-onset forms in which visual and multisensory halluci- nations are more frequent [4]. Schizophrenic hallucinations combined with other psychotic symptoms are commonly classified within the “positive symptoms” of schizophrenia [5]. It is these positive symptoms on which antipsychotic drugs have the most blatant therapeutic effects [6], and successive studies from the 1960’s revealed that this effect could be due to antagonistic action on D2Rs, which is shared by all antipsychotic molecules [7]. Consequently, the hypoth- esis that positive symptoms may be related to an excessive transmission of dopamine has become the main pharmaco- logical model of positive symptoms in schizophrenia [5]. This hypothesis has been reinforced by contemporaneous imaging techniques, which have confirmed that positive symptoms were associated with an increase of dopaminergic activity in the striatum [8]. For a while, the other dopamine receptors, notably the D1 receptors, were suggested as other receptors possibly implicated in positive symptoms of schizophrenia [9], but it appeared that they were probably more involved in negative symptoms of schizophrenia, which consist of blunted effects and social withdrawal [10, 11]. D2Rs thus emerged as the primary dopaminergic modulators underly- ing positive symptoms [7]. It has been demonstrated that PCP exhibits antagonistic effects on NMDARs [19]. As a result, a new pharmacological model of hallucinations and other schizophrenic symptoms was introduced [20, 21]. Subsequently, a progressive amount of evidence indicated that many susceptibility genes for schizophrenia were related to the functioning of NMDARs [20, 21] and that glutamate may have a more central place in the pathophysiology of schizophrenia than dopamine [22]. 1. Introduction completely different. Psychostimulants-induced hallucinat- ions result from increased dopamine transmission and hype- ractivation of dopamine D2 receptor (D2R). Furthermore, “dissociative anesthetics” drugs induce complex schizo- phrenia-like clinical pictures, including hallucinations, that result from the blockade of glutamate NMDA receptors (NMDAR). Lastly, psychedelics act by stimulating the serotoninergic 5HT2A receptor (5HT2AR). In schizophrenia, although antipsychotic blocking studies suggest that halluci- nations result from D2R hyperstimulation, there are also numerous arguments for NMDAR dysfunction, which may be a potential and specific hallucinatory mechanism. A hallucination is a type of misperception that can be defined as “the perception of an object without an object to perceive” [1]. While hallucinations may occasionally occur in diverse psychiatric and neurological pathologies, they are partic- ularly characteristic of schizophrenia-related disorders, in which antipsychotic drugs are commonly used to treat them. However, hallucinations may also be triggered by at least three different kinds of drugs: psychostimulants (i.e., cocaine or amphetamine), the so-called “dissociative anesthetics” (i.e., phencyclidine (PCP) or ketamine), and psychedelics, (i.e., lysergic diethylamid (LSD) and psilocybin). Initially, the existence of these different pharmacological systems underlying hallucinations appears incompatible with Depending on which situation is considered, the pharmacological hypotheses underlying the symptoms are BioMed Research International 2 dopaminergic-enhanced transmission, and may pave the way for the emergence of psychosis [7]. a unified conception hallucination. It is necessary to artic- ulate these three mechanisms into an integrated model, or, alternatively, there may be different forms of hallucinations, that are mediated by different pharmacological supports and neurobiological circuits. It has thus been proposed that dopamine is the pharmaco- logical keystone of all psychotic states, which is the expression of a process of dopamine supersensitivity, because of an increase in the high-affinity states of the D2Rs, or D2High receptors [16]. According to this theory, the clinical activity of the hallucinogenic drugs may result from the property of these drugs to target D2High receptors, and this action may be the fundamental pharmacological mechanism underlying psychosis [17]. This “all-dopamine” conception of psychosis will be discussed below. 2. The Three Main Pharmacological Models of Hallucinations The role of NMDAR in schizophrenia was also highlighted by the effectiveness of several NMDAR regulators on both pos- itive and negative symptoms of schizophrenia [23]. All these arguments have progressively led to an increased interest in the role of NMDARs concerning the whole pathophysiology of schizophrenia [24]. Currently, NMDAR hypofunction is considered by several leading researchers of the field to be a major neurobiological hypothesis for schizophrenia [25]. Apart from the use of PCP or ketamine, other nonschizophrenic NMDAR-related psychoses have been reported [26]. NDMAR-related psychosis is thus not confined to the spectrum of schizophrenia. Moreover, the sole blockade of NMDAR, without any relation with the dopaminergic system may be sufficient to induce psychosis [21]. In these types of states, mixed positive and negative symptoms are observable, which is in contrast to what happens with the use of dopaminergic drugs. As these states also include hallucinations, it could be concluded that purely NMDAR-related hallucinations are conceivable, without any relation with the dopaminergic system. It has secondarily been hypothesized that all forms of D2R overstimulation in striatum could trigger psychosis, even beyond the scope of schizophrenia. The clinical pic- ture of psychosis induced by psychostimulant drugs [12], which result from sustained dopamine action [13], appears relevant to this hypothesis [14]. Psychotic symptoms can also result from the use of dopaminergic receptors agonists in Parkinson’s disease [15]. Thence, it has been suggested that striatal dopaminergic hyperfunction may better fit with psychosis than with schizophrenia, as nonpsychotic forms of schizophrenia are not linked with such striatum- based anomalies [7]. Psychotic symptoms in schizophrenia may be related to a neurobiological pathological process, incentive salience, which is the cognitive consequence of 2.3. The Serotonin Model: Hallucinations, Psychedelics, and Schizophrenia. Psychedelics constitute a heterogeneous class of molecules, among which LSD and psilocybin are the two most well-known and best studied molecules [27]. Psychedelics induce phenomenologically complex pictures, which can mix visual hallucinations, synesthesia, and pecu- liar altered states of consciousness with mystical feelings [28]. 2.3. The Serotonin Model: Hallucinations, Psychedelics, and Schizophrenia. Psychedelics constitute a heterogeneous class of molecules, among which LSD and psilocybin are the two most well-known and best studied molecules [27]. Psychedelics induce phenomenologically complex pictures, which can mix visual hallucinations, synesthesia, and pecu- liar altered states of consciousness with mystical feelings [28]. 3 BioMed Research International 3 Table 1: Characteristics of main pictures of hallucinations. 3. Confrontations between Models and Attempts for Integration Although there has been much debate regarding the psychedelics’ exact pharmacological mechanism [29], the most commonly admitted mode of activity of this class of drugs is the stimulation of serotonin 5HT2AR on cortical neurons [30, 31]. Cortical 5HT2AR hyperactivation may affect the functioning of the cortico-striato-thalamo-cortical loops and triggers a disruption in the thalamic gating of sensory and cognitive information [28]. It has been proposed that this process triggers a breakdown of cognitive integrity and results in the subsequent occurrence of aberrant feelings and perceptions [28]. Because three different cerebral receptors contribute to the triggering of different hallucinatory processes, it is necessary to assess whether these three receptors are part of the same global neural circuitry, which would preserve the conceptual unity of hallucinations from a pharmacological perspective, of whether they belong to pathways that induce hallucina- tions separately, which would mean that there are several pharmacological forms of hallucinations, with each having specific clinical expressions. This has led scientists to question each model in front of the two other ones. We will first focus on discussions of two-system interactions (NMDAR-D2R, 5HT2AR-D2R, and 5HT2AR-NMDAR) and then move to theories that attempt to integrate all of the three receptors. As soon as the serotoninergic-based mode of action of LSD was discovered, a serotoninergic hypothesis of schizophrenia was proposed, prior to being supplanted by the dopaminergic hypothesis [32]. Today, several authors have proposed a reappraisal of the role of the 5HT2AR in both schizophrenia and psychosis [28, 31]. However, psychedelics-induced forms of psychosis sensibly differ from schizophrenia-like psychosis, in particular regarding the clinical aspect of hallucinations. Visual hallucinations are typical with psychedelics, whereas auditory hallucina- tions are much more rare [33]. Furthermore, “pseudohal- lucinations” (i.e., misperceptions with intact reality test- ing and insightfulness) are very frequent [33], although insight into hallucinations in schizophrenia is quite poor [34]. 3.1. Glutamate/Dopamine Interactions. Glutamate and dopamine hypotheses of schizophrenia may be considered as rival models, particularly in regards to the pathophysiology of positive symptoms in schizophrenia. Several experts believe that one of the models is more superior than the others. According to the supporters of the “all-dopamine” hypothesis, the role of dopamine is central, and the schizophrenia-like effects of ketamine and PCP may be at least partially explained by the affinity of the drugs to the D2High receptors [7]. 2. The Three Main Pharmacological Models of Hallucinations Psychostimulants (cocaine, amphetamine) Dissociative anesthetics (PCP, ketamine) Psychedelics (LSD, psilocybin) Schizophrenia (paranoid) Supposed pharmacological mode of action D2R activation NMDAR blockage 5HT2AR activation D2R activation/NMDAR blockage Main type of hallucinations Auditory Visual Visual Auditory Most frequent associated delusions Paranoid paranoid Mystical Paranoid Most frequent associated behaviour Agitation Social withdrawal Mystical feelings Variable Insightfulness No No Yes No Table 1: Characteristics of main pictures of hallucinations. 3. Confrontations between Models and Attempts for Integration involving both 5HT2AR and D2R has been observed on the membranes of mouse striatal neurons, which may result in a functional crosstalk between the two neurotransmission sys- tems [47]. In this theory, however, the 5HT2ARs implicated in psychosis are located in the striatum and not in the prefrontal cortex, which suggests that they interact with D2Rs. Yet, other arguments support that psychedelic-induced hallucinations are not related to the modulation of D2Rs. Haloperidol was found unable to block the psychotomimetic effects of psilocybin, whereas the 5HT2AR antagonist ketanserin was able to do so, notably regarding VH [48, 49]. Consequently, it remains very unclear whether 5HT2ARs and D2Rs may interact in schizophrenia hallucinations if D2Rs are not involved in psychedelic-induced hallucinations. D2R-NMDAR interactions in the striatum still remain to be confirmed. However, recent studies question the potential influences of D2Rs on ketamine-induced abnormalities in the striatum [41]. Whether the dopaminergic and the glu- tamatergic systems function jointly or separately in striatum remains a key issue toward understanding the ability of both NMDARs and D2Rs modulators in inducing or reducing hallucinations. 3.3. Glutamate/Serotonin Interactions. Both NMDAR antag- onists and 5HT2AR agonists induce hallucinations and have been used in drug-induced experimental models of schizophrenia. In addition, some specific cognitive functions, such as the inhibition of return, are impaired in schizophre- nia and are disrupted with both NDMAR antagonists and HT2AR agonists [50]. However, other cognitive impairments involved in schizophrenia, including deficits in mismatch negativity, are only reported when administering NDMAR antagonists [51]. Moreover, the prepulse inhibition of the acoustic startle reflex, which is reduced in schizophrenic patients, appears to be increased only by NMDAR antagonists and is unmodified following administration of 5HT2AR agonists in humans [52]. Clinically, NDMAR antagonists also induce negative symptoms, and this class of drugs may present a more sustained face of validity with schizophrenia than psychedelics. 3.2. Dopamine/Serotonin Interactions. The role of 5HT2ARs has been recently reintroduced in schizophrenia, since many second-generation antipsychotics are both D2R and 5HT2AR antagonists [35]. This suggests that the blockade of 5HT2AR may underlie the antipsychotic effects of these drugs, in accordance with the 5HT2AR-mediated hallu- cinogenic properties of psychedelics [31]. However, second- generation antipsychotics are characterized by the triggering of lesser side effects that result from the blockade of D2Rs in the non-limbic areas, such as extrapyramidal symptoms or galactorrhea [35]. 3. Confrontations between Models and Attempts for Integration This observation has led to the hypothesis that 5HT2AR blockade reverses the effects of D2R blockade only in these areas [42], whereas the D2R antagonistic effects of second-generation antipsychotics are preserved in the limbic system, which preserves the therapeutic activity of these drugs on positive symptoms of schizophrenia [35]. Consequently, it is not obvious that the blockade of 5HT2ARs is responsible for the effects of these drugs on positive symp- toms. Furthermore, first generation antipsychotics, which have a lack or little activity on 5HT2ARs, exhibit the same level of efficacy on the positive symptoms compared with more recent drugs [6, 43]. Nevertheless, several attempts for integrating NMDAR and 5HT2AR into a common neurobiological framework for psychosis have been proposed. According to some of these theories, abnormalities in one of the two neurotransmission systems could trigger dysfunctions in the other. For example, noncompetitive antagonists NMDAR appear to potentiate the activation of serotoninergic receptors [53], while pos- itive modulators of NMDARs could inhibit serotoninergic activation [54]. Thus, psychosis may be at least partially the expression of mechanisms in series, in which NMDAR dys- function leads to the enhanced activation of 5HT2ARs [53]. Other models hypothesize that psychosis results from a final common pathway that is equally disrupted by both NMDAR hypoactivation or 5HT2AR hyperactivation. For example, it is thought that an impairment in the cognitive function of inhibition of return is responsible for the occurrence of psychosis and results from dysfunctions in several different psychopharmacological pathways, that is, NDMAR blockade or 5HT2AR stimulation [50].f However, several studies have investigated the D2R- related theory of positive symptoms in schizophrenia and the activity of psychedelics. As previously described, some researchers have justified this issue with the idea that psychedelics function because of their stimulating effect on D2Rs [17]. Several studies have supported this notion. For example, LSD exhibits biphasic activity in mice; the first phase involves only 5HT2ARs, and the second phase, which is related to the psychotic symptoms observed in humans, involves only D2R [44, 45]. Nevertheless, according to other studies, psychedelic-induced stimulation of 5HT2ARs in the prefrontal cortex is responsible for a downstream activation of dopaminergic neurons that are located in the ventral tegmental area [46]. Moreover, the formation of heteromers More recently, different types of interconnections between glutamatergic and serotoninergic neurotransmis- sion systems have been proposed to explain psychosis. 3. Confrontations between Models and Attempts for Integration In this hypothesis, activation of D2Rs is indispensable to induce any form of psychosis [16] and consequently any form of hallucinations. Despite these numerous clinical differences, there are some arguments that suggest a role of 5HT2AR in schizophrenia. The level of expression of 5TH2AR is upregulated in young and untreated patients with schizophrenia, and because visual hallucinations frequently occur in the early phases of schizophrenia, it has been proposed that psychedelic-induced pictures may be related to early forms of schizophrenia [31]. Furthermore, many second-generation antipsychotics have an antagonistic action on the 5HT2AR, which highlights the role of 5HT2ARs in schizophrenia [35]. However, the antipsychotic action of this antagonist effect remains questionable and will be discussed in the next chapter.i However, other authors note that psychostimulants (i.e., pure dopaminergic drugs) are far less hallucinogenic com- pared with dissociative anaesthetics [36] and that many symptoms induced by NMDAR antagonists have been reported not to be linked with an increase of dopamine trans- mission in the striatum [21]. Moreover, in animal models, only few antipsychotic drugs can reverse the effects of acute and chronic administration of PCP on prepulse inhibition [37], which is a cognitive parameter used as a model of positive symptoms [38]. NMDAR blockade is thus considered as an independent mechanism for the induction of psychosis [24]. Consequently, it appears that some specific forms of hal- lucinations may result from 5HT2AR activation, in addition to other specific clinical abnormalities. At first glance, these particular clinical pictures do not appear to be linked with the participation of NMDARs or D2Rs. Interconnections and reciprocal regulations between the two systems are also possible. The prefrontal cortex may trigger NMDAR-mediated decrease of the dopaminergic tone 4 4 BioMed Research International in striatum [39]. Moreover, dopaminergic and glutamatergic systems may have opposite effects in the striatum, which may explain that both D2R activation and NMDAR blockade induces hallucinations in a similar manner [36]. Animal stud- ies appear to validate this hypothesis, as NMDAR modulation limits some behavioural effects induced by amphetamine [40]. However, this may also indicate a two-way interaction, as recent studies have reported that activation of D2Rs induces a multimodal downregulation of NMDAR in the striatum [40], resulting in reciprocal regulations between the dopaminergic and glutamatergic systems. As discussed below, the role of 5HT2ARs has been suspected for its involvement within this complex neural circuitry. 3. Confrontations between Models and Attempts for Integration Recent work has shown that the action of psychedelic drugs on 5HT2ARs requires the indispensable formation of a complex between 5HT2AR and the metabotrop-ic glutamatergic mGlu2 receptor (mGlu2R) [55]. Furthermore, mGlu2R and mGlu3R agonists experimentally reverse the BioMed Research International 5 D2R NMDAR Limbic striatum mPFC VTA ? Thalamus 5HT2AR 5HT2AR Figure 1: 5HT2AR/D2R/NMDAR interactions. Simplified version of the Geyer and Vollenweider model of psychosis [25], which supposes a disruption in the cortico-striato-thalamo-cortical loops. This model tries to connect 5HT2R, D2R, and NMDAR in a unified neurobiological system which could be impaired in psy- chosis. Abbreviations. mPFC: medial prefrontal cortex; VTA: ventral tegmental area; NMDAR: N-methyl-D-aspartate receptor; D2R: dopamine-2 receptor; 5HT2R: 5-hydroxytryptamine-2A receptor. D2R NMDAR Limbic striatum mPFC VTA ? Thalamus 5HT2AR 5HT2AR effects of NMDAR antagonist drugs [56] and appear to have antipsychotic effects in human, notably on positive symptoms of schizophrenia [57]. This suggests that a common pharmacological process involving mGlu2R and mGlu3R hypoactivation may be the missing link between the clinical activities of both NMDAR antagonists and 5HT2AR agonists [58]. The role of dopamine and the mode of action of current antipsychotic drugs are still unclear in relation to this theory, and attempts at a more unified theory would require a multipharmacological model. 3.4. NMDAR/DR2/5HT2AR Interactions. Because the three pharmacological systems described above that obviously have close interactions between each other, it could be assumed that they belong actually to a complex and integrated neuro- biological circuit, whose impairment could occur at various levels in case of psychosis. Experimental studies have shown that the three different systems appear interdependent in inducing psychosis-like behavioral abnormalities in rodents [53, 59]. Recently, several leaders of the field have proposed an entirely integrated model that includes several different neurotransmission systems, most notably the three that are discussed here [28].h Figure 1: 5HT2AR/D2R/NMDAR interactions. Simplified version of the Geyer and Vollenweider model of psychosis [25], which supposes a disruption in the cortico-striato-thalamo-cortical loops. This model tries to connect 5HT2R, D2R, and NMDAR in a unified neurobiological system which could be impaired in psy- chosis. Abbreviations. mPFC: medial prefrontal cortex; VTA: ventral tegmental area; NMDAR: N-methyl-D-aspartate receptor; D2R: dopamine-2 receptor; 5HT2R: 5-hydroxytryptamine-2A receptor. 3. Confrontations between Models and Attempts for Integration This model is constructed around the hypothesis that psychotic symptoms could result from filtering disruptions within the cortico-striato-thalamo-cortical loops, which underlie the level of awareness and attention that is dedicated at any time in the brain to an external stimulus [28] (Figure 1). By disorganizing the filtering processes, different kinds of drugs could trigger psychotic symptoms, and any of D2Rs, 5HT2ARs, and NMDARs may constitute unspecific vulnera- bility points in this circuitry. D2Rs and NMDARs act directly in the limbic striatum, whereas prefrontal 5HT2AR regulate the striatal activity via the modulation of cortical pyramidal neurons. separated, are met in identical types of pathological states. Moreover, it has been contested that misperceptions and false beliefs rely on radically separated cognitive processes [60]. Dopaminergic theories suggest that both types of symptoms result from the increased dopaminergic transmission in the limbic striatum, even if there could be also subtle differences in their respective neural circuitry [2]. In this perspective, delusions and hallucinations are not separate clinical entities but nondissociable components of psychosis.hi Such a scheme allows the synthesis of many of the disparate data that have been previously enumerated between the different neurotransmission systems. Thus, an integrated explanation for the occurrence of psychosis is proposed, with respect to the implication of the different aforementioned pharmacological systems. Nevertheless, this theory struggles to properly explain the subtle clinical dissimilarities that are observed depending on the underlying disorder or the ingested drug. The first concern with that standpoint is that there are many definitions of what is psychosis (Figure 2) [61]. While thought disorders are sometimes considered to belong to psychotic symptoms, the most restrictive definition of psychosis is “delusions or prominent hallucinations in the absence of insight into their pathological nature” [61]. 4. Discussion Whether hallucinations occur in schizophrenia or after drug intoxication, they are very often clinically associated with a collection of other symptoms, including delusions, thought disorders, and loss of insight. All these symptoms are usually pooled into the general concept of psychosis. A particular issue about hallucinations is whether such a symptom can be nosologically distinguished from psychosis, or whether it is intrinsically linked in its occurrence with other psychotic symptoms such as delusions. Hallucinations are phenomenologically different from delusions, as halluci- nations are misperceptions, while delusions are false beliefs. Nevertheless, both frequently appear mixed together or, if Moreover, whereas thought disorders are not always considered to be included in psychosis, situations in which hallucinations appear isolated from cognitive disorders are very rare. The state of consciousness is often clinically altered in some way. Even for hallucinations that occur in the general population, the state of consciousness appears to always be associated with infraclinical cognitive impairments in the executive functions and language abilities associated with the symptoms [62]. Thus, it appears difficult to affirm that hallu- cinations exist outside the scope of psychosis. Furthermore, a distinction has been proposed in the literature, between “hallucinations,” which would refer to “psychotic” states (i.e., 6 BioMed Research International 6 Loss of insightfulness Thought disorders Hallucinations Delusions Figure 2: Different scopes of psychosis. The straightest definition of psychosis includes hallucinations or delusions with a loss of insight and thought disorders (1). A second definition is delusions or hallucinations with a sole loss of insightfulness (2). At last, several authors consider isolated hallucinations to belong to psychosis. Other investigations found that the same brain areas were similarly disrupted by NMDAR antagonist drugs [68, 69]. However, psilocybin-induced visual hallucinations and synesthesia have been repeatedly associated with occipi- toparietal cortex activity [49, 66, 70], which has not been the case either for VH induced either by NDMAR antagonists, or for VH of schizophrenia [71] or first psychosis episode [72]. Loss of insightfulness Thought disorders Hallucinations Delusions pi p y p Nonperceptive symptoms induced by psychedelics are also very specific. These mystical feelings consist of a merging with the external world. This phenonenon, called “oceanic boundleness” [33], is not commonly reported with other classes of hallucinogenic drugs. We assume that the origin of such feelings is derived from a cognitive reconstruction following preliminary visual disruptions. 4. Discussion Indeed, serotoner- gic synesthesia is defined as projections of nonvisual percepts onto the visual field. If one hypothesizes that both acoustic and kinesthetic information can be projected onto the visual field during psychedelic intoxication, then the subject could pathologically overlap sensations of corporal identity with visual perception and thus experience a feeling of merging with the outside world. Of course, such a presumption would require additional experimental support. Figure 2: Different scopes of psychosis. The straightest definition of psychosis includes hallucinations or delusions with a loss of insight and thought disorders (1). A second definition is delusions or hallucinations with a sole loss of insightfulness (2). At last, several authors consider isolated hallucinations to belong to psychosis. associated with anxiety, disorganization and loss of control, and insight upon the symptoms [61]), and “pseudohallucina- tions” or “nonpsychotic hallucinations” [63], which refer to misperceptions with no anxiety and insightfulness that the misperceptions are not real [33]. This distinction deserves full attention because “pseudohallucinations” are frequent with psychedelics [33]. Additionally, they have not been reported with psychostimulants or dissociative anesthetics and are rare in acute schizophrenia either. It appears, however, that there could be a threshold effect with psychedelics, above which pseudohallucinations become vivid hallucinations, with loss of insight and increased anxiety [64]. Future studies should precise whether “pseudohallucinations” occur exclusively with 5HT2AR-related drugs or whether there is a dose-effect mechanism that underlies all types of hallucinogenic drugs. If the former scenario was true, it would imply that only psychedelics could induce misperceptions without psychosis, which would restrict such clinical patterns to the sole activity of the HT2ARs. Even if all of the hallucinogenic drugs act by modulating the stimuli filtering and integrator system [28], it is also possible that each pharmacological system also specifically acts on other cerebral processes, which could confer quite a specific phenomenological pattern to the disruption of one system compared to the other. Thus, activation of 5HT2ARs could disrupt the information filtering system and induce at the same time a specific process of multisensory attraction by the visual system. On the other hand, NMDAR antago- nists could disrupt the information filtering system, thereby enhancing the risk that hallucinations could appear, but at the same time cause interference in several cognitive processes and induce a loss of insight and social withdrawal. 4. Discussion Lastly, dopaminergic stimulation may involve a specific dimension of excitement and motor agitation, as it is the main effect of psychostimulants drugs, which is not observed in other drug intoxications. In conclusion, all three hallucinatory mechanisms—D2R activation, 5HT2AR activation, and NMDAR blockage— are proposed to trigger partial overlapping neurobiologi- cal processes whose hallucinations, among other psychotic symptoms, are the clinically expressed. In addition, the mod- ulation of each of these three receptors induces characteristic cognitive impairments that give each class of hallucinatory drug a specific clinical tonality. Increased insight does not appear to be the only feature of HT2AR-induced symptoms. The visual component of symptomatology appears to occupy a much larger place than in other hallucinatory pictures, particularly those observed in schizophrenic-related disorders. Furthermore, visual hallucinations occurring in Parkinson’s disease have also been related to 5HT2ARs [65], and recent studies note that the serotoninergic system plays a central role in the visual processing [66]. The occurrence of synesthesia, which is almost uniquely observed with psychedelics and with other hallucinogenic drugs, is relevant to this hypothesis. It appears that, compared with other hallucinogenic drugs, only psychedelics impair the integrity of visual functioning. Consequently, it could be presumed that psychedelics do not trigger strictly the same types of neurobiological processes that are triggered by NMDAR antagonists or even dopamin- ergic drugs. However, there could be some overlapping, since a recent neuroimaging study has found that psychedelic activity may be related to a disruption in the network relating the prefrontal cortex with the posterior cingulate cortex [67]. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. References [1] J. D. Blom, A Dictionary of Hallucinations, Springer, New York, NY, USA, 2009. 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Efficacy of capecitabine and oxaliplatin versus S-1 as adjuvant chemotherapy in gastric cancer after D2 lymph node dissection according to lymph node ratio and N stage
BMC cancer
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Shin et al. BMC Cancer (2019) 19:1232 https://doi.org/10.1186/s12885-019-6433-3 Shin et al. BMC Cancer (2019) 19:1232 https://doi.org/10.1186/s12885-019-6433-3 Open Access Efficacy of capecitabine and oxaliplatin versus S-1 as adjuvant chemotherapy in gastric cancer after D2 lymph node dissection according to lymph node ratio and N stage Kabsoo Shin1, Se Jun Park1, Jinsoo Lee1, Cho Hyun Park2,3, Kyo Young Song2,3, Han Hong Lee2,3, Ho Seok Seo2,3, Yoon Ju Jung2,3, Jae Myung Park3,4, Sung Hak Lee3,5, Sang Young Roh6 and In-Ho Kim1,3,6* Abstract Background: We sought to assess the prognostic significance of lymph node ratio (LNR) and N stage in patients undergoing D2 gastrectomy and adjuvant chemotherapy, S-1, and XELOX and to compare the efficacy of them according to LNRs and N stages to evaluate the clinical impact of using LNRs compared with using N staging. Methods: Patients undergoing D2 gastrectomy with adequate lymph node dissection and adjuvant chemotherapy for stage II/III gastric cancer between Mar 2011 and Dec 2016 were analysed. Of the 477 patients enrolled, 331 received S-1 and 146 received XELOX. LNR groups were segregated as 0, 0–0.1, 0.1–0.25, and > 0.25 (LNR0, 1, 2, and 3, respectively). Propensity score matching (PSM) was used to minimise potential selection bias and compare DFS and OS stratified by LNRs and N stages in the two treatment groups. Results: After PSM, the sample size of each group was 110 patients, and variables were well balanced. All patients had more than 15 examined lymph nodes (median 51, range 16~124). In multivariate analysis, LNR (> 0.25) and N stage (N3) showed independent prognostic value in OS and DFS, but LNR (> 0.25) showed better prognostic value. In subgroup analysis, the LNR3 group showed better 5-year DFS (20% vs 54%; HR 0.29; p = 0.004) and 5-year OS (26% vs 67%; HR 0.28; p = 0.020) in the XELOX group. The N3 group showed better 5-year DFS (38% vs 66%; HR 0.40; p = 0.004) and 5-year OS (47% vs 71%; HR 0.45; p = 0.019) in the XELOX group. Stage IIIC showed better 5-year DFS (22% vs 57%; HR 0.32; p = 0.004) and 5-year OS (27% vs 68%; HR 0.32; p = 0.009) in the XELOX group. The LNR3 group within N3 patients showed better 5-year DFS (21% vs 55%; HR 0.31; p = 0.004) and 5-year OS (27% vs 68%; HR 0.34; p = 0.018) in the XELOX group. (Continued on next page) * Correspondence: ihkmd@catholic.ac.kr 1Division of Medical Oncology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, South Korea 3Department of Gastric Cancer Centre, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea Full list of author information is available at the end of the article * Correspondence: ihkmd@catholic.ac.kr 1Division of Medical Oncology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, South Korea 3Department of Gastric Cancer Centre, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea Full list of author information is available at the end of the article Background mainly in South Korea, patients with Stage II, III gas- tric cancer were treated with D2 gastrectomy, and showed an HR for 3-year disease-free survival (DFS) of 0.56 (95% CI, 0.44–0.72; p < 0.0001) and for OS of 0.72 (95% CI, 0.52–1.00; p = 0.049) in the comparison of 1) adjuvant capecitabine and oxaliplatin for 6 months after D2 gastrectomy versus 2) surgery alone after a median follow-up of 34 months [2, 14]. Despite this evidence, there has been no prospective study that directly compare S-1 and XELOX. Previous studies suggested that XELOX would be more bene- ficial for more aggressive disease with higher N stage [15, 16]. Gastric cancer is the fifth most common cancer and the third leading cause of cancer death worldwide, ac- counting for over 1,000,000 newly diagnosed cancer patients and over 783,000 cancer-related deaths annu- ally [1]. Radical gastrectomy with extended lymphade- nectomy (D2 gastrectomy) is the standard of care for gastric cancer in many countries in East Asia [2, 3]. Although the safety and utility of extended lymph node dissection have been debated for a long time in Europe and the US, D2 gastrectomy is recommended based on several trials (especially the Dutch D1D2 study), which showed a reduction in cancer-related deaths with D2 gastrectomy [4–6]. In addition to the TNM staging system, the ratio of positive and total examined lymph nodes (lymph node ratio, LNR) has been proposed as a simple and con- venient tool for identifying subgroups of gastric can- cer patients with similar prognosis. It can also be used to adjust stage migration from current tumour, node, metastasis (TNM) staging of gastric cancer. Cut-off values of 0.1 and 0.25 have been adopted in several studies and have been found to be in good agreement to the N1, N2, and N3 stages of the 6th and 7th UICC/TNM staging system [17–21]. How- ever, the significance of LNR has not been evaluated for patients with adjuvant chemotherapy after D2 gastrectomy. Furthermore, whether LNR is more accurate prognostic and predictive than N stage is not clear in these patients. However, the recurrence rate of D2 gastrectomy is high. Approximately 40% of patients relapse within 2 years of surgery, necessitating adjuvant treatment [7–9]. Adjuvant treatments for gastric cancer differ by geographical region. In the UK and other European countries, perioperative chemotherapy is recom- mended as a standard treatment [10]. Trial registration: Not applicable (retrospective study). Trial registration: Not applicable (retrospective study). Keywords: Tegafur, Capecitabine, Oxaliplatin, Gastric cancer, Lymph node ratios, N stage, Propensity score matching Keywords: Tegafur, Capecitabine, Oxaliplatin, Gastric cancer, Lymph node ratios, N stage, Propensity score matching Background In the USA, the recommended adjuvant therapy is postoperative che- moradiation or chemotherapy, depending on the type of lymph node dissection [11]. The evidence of post- operative chemoradiation is based on the UK Medical Research Council Adjuvant Gastric Infusional Chemo- therapy (MAGIC) trial [12] and the US Intergroup- 0116 trial [13]. Both studies assessed the survival ben- efits of adjuvant therapy after limited dissection of the regional lymph nodes. Therefore, we sought to 1) assess the prognostic significance of LNR and N stage in patients undergoing D2 gastrectomy and adjuvant chemotherapy, S-1, and XELOX and 2) assess the efficacy of adjuvant S-1 and XELOX according to LNRs and N stages to evaluate the clinical impact of using LNRs compared with using N staging. The evidence of postoperative chemotherapy is based on two randomised controlled trials that inves- tigated the efficacy of adjuvant chemotherapy after D2 gastrectomy compared to D2 gastrectomy alone in patients with resectable gastric cancer [2, 14]. In the ACTS-GC trial in Japan, patients with Stage II, III gastric cancer were treated with D2 gastrectomy, and showed a hazard ratio (HR) for 5-year overall survival (OS) of 0.669 [95% confidence intervals (CI), 0.540– 0.828] in the comparison of 1) surgery and adjuvant chemotherapy treatment with oral fluoropyrimidine S- 1 for 1 year versus 2) surgery alone and a 5-year follow-up. In the CLASSIC trial, which took place (Continued from previous page) (Continued from previous page) Conclusions: LNR showed better prognostic value than N staging. LNR3, N3 and stage IIIC groups showed the superior efficacy of XELOX to that of S-1. And the LNR3 group within N3 patients showed more survival benefit from XELOX. LNR > 0.25, N3 stage and stage IIIC were the discriminant factors for selecting XELOX over S-1. Trial registration: Not applicable (retrospective study). Trial registration: Not applicable (retrospective study). © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shin et al. BMC Cancer (2019) 19:1232 Page 2 of 14 Follow-up evaluation Tumour assessments were performed with abdominal computed tomography (CT) or magnetic resonance imaging (MRI) every two or three cycles of treatment with tumour marker; CEA, CA 19–9. After finishing adjuvant chemotherapy, tumour assessments were performed every 6 months for the first 3 years and yearly thereafter. When signs or symptoms indicated a possible recurrence or development of new gastric cancer, additional imaging or biopsies were performed to confirm the presence of malignancy. Patients in the XELOX group received oral capecita- bine (1000 mg/m2 twice daily (on days 1–14 of each cycle) plus intravenous oxaliplatin (130 mg/m2 on day 1 of each cycle) every 3 weeks. The duration of XELOX was eight cycles (6 months). Patients in the S-1 group re- ceived daily doses of 80 mg, 100 mg or 120 mg of S-1. Those with a body-surface area of less than 1.25 m2 re- ceived 80 mg daily; those with a body-surface area of 1.25 m2 or more but less than 1.5 m2 received 100 mg daily; and those with a body-surface area of 1.5 m2 or more received 120 mg daily. In each six-week cycle, S-1 was administered for 4 weeks, followed by a two-week resting period. The duration of S-1 was eight cycles (12 months). Disease-free survival (DFS) was defined as the inter- val between the time from the curative resection of gastric cancer until the date of disease recurrence at locoregional and/or distant sites, or the date of death from any cause. Overall survival (OS) was measured as the time from the curative resection of the gastric cancer until death from any cause or until the last follow-up date. Methods We retrospectively investigated the data of 798 patients who underwent curative resection for gastric cancer and diagnosed as stage II or III between Mar 2011 and Dec 2016 at the Catholic University of Seoul St. Mary’s hospital. Shin et al. BMC Cancer (2019) 19:1232 Page 3 of 14 Fig. 1 Study flow diagram according to the eligible criteria. After 321 of 798 patients were excluded, data from 477 patients were analysed retrospectively. The propensity score matching was performed between XELOX group and S-1 group ig. 1 Study flow diagram according to the eligible criteria. After 321 of 798 patients were excluded, data from 477 patients etrospectively. The propensity score matching was performed between XELOX group and S-1 group on retrospective analyses of existing administrative and clinical data. Among these patients, eligible patients (1) were aged 18 years or older, (2) had histologically con- firmed gastric adenocarcinoma after radical gastrec- tomy with D2 lymph node dissection and R0 resection (3) had stage II or III disease (based on the 7th edition of the American Joint Committee on Can- cer criteria) and (4) had no prior treatment for cancer other than the initial gastric resection for the primary lesion. After 321 of 798 patients were excluded, 477 met the eligibility criteria and received XELOX or S-1. (Fig. 1). Statistical analyses To directly compare the efficacies of S-1 and XELOX chemotherapies, DFS and OS were determined and 5- year DFS and 5-year OS were compared. To minimise the influence of potential confounders on selection The Institutional Review Board of the Catholic Univer- sity of Seoul Saint Mary’s Hospital approved the study (KC18RESI0596, KC19RASI0751). Requirement for in- formed consent was waived because the study was based Shin et al. Statistical analyses BMC Cancer (2019) 19:1232 Page 4 of 14 Table 1 Baseline characteristics of the patients before and a Before propensity score matching (n = 477) S-1 (n = 331) XELOX (n = 146) p value* Absolut differen Age (years) < 65 181 (54.7) 104 (71.2) 0.001 34.7 ≥65 150 (45.3) 42 (28.8) 34.7 Sex Male 225 (68.0) 101 (69.2) 0.795 2.6 Female 106 (32.0) 45 (30.8) 2.6 ECOG 0 241 (72.8) 124 (84.9) 0.004 30.0 ≥1 90 (27.2) 22 (15.1) 30.0 ASA score 1 to 2 308 (93.1) 138 (94.5) 0.549 3.4 ≥3 23 (6.9) 8 (5.5) 3.4 Location EGJ 11 (3.3) 7 (4.8) 0.437 7.4 Other 320 (96.7) 139 (95.2) 7.4 Stage (AJCC 7th edition) IIA 109 (32.9) 5 (3.4) < 0.001 82.8 IIB 73 (22.1) 19 (13.0) 23.9 IIIA 52 (15.7) 39 (26.7) 27.2 IIIB 53 (16.0) 48 (32.9) 40.0 IIIC 44 (13.3) 35 (24.0) 27.7 T stage T1 26 (7.9) 3 (2.1) 0.001 27.0 T2 51 (15.4) 10 (6.8) 27.5 T3 129 (39.0) 56 (38.4) 1.3 T4a,b 125 (37.8) 77 (52.7) 30.4 N stage N0 87 (26.3) 9 (6.2) < 0.001 56.7 N1 67 (20.2) 28 (19.2) 2.7 N2 103 (31.1) 38 (26.0) 11.3 N3 74 (22.4) 71 (48.6) 57.1 Number of dissected lymph nodes mean ± sd 47.0 ± 18.8 52.4 ± 17.1 < 0.001 30.0 median (IQR) 43 (35–55) 52 (39–65) LNR group LNR 0 88 (26.6) 9 (6.2) < 0.001 40.2 LNR 1 127 (38.4) 49 (33.6) 40.2 LNR 2 78 (23.6) 47 (32.2) LNR 3 38 (11.5) 41 (28.1) < 0.001 57.4 Tumor size (cm) 10.0 < 6 250 (75.5) 83 (56.8) 19.3 Table 1 Baseline characteristics of the patients before and after propensity score matching Before propensity score matching (n = 477) After propensity score matching §(n = 220) S-1 (n = 331) XELOX (n = 146) p value* Absolute‡ Standardized difference in % S-1 (n = 110) XELOX (n = 110) p value† Absolute‡ Standa difference in % Age (years) < 65 181 (54.7) 104 (71.2) 0.001 34.7 70 (63.6) 68 (61.8) 0.889 3.7 ≥65 150 (45.3) 42 (28.8) 34.7 40 (36.4) 42 (38.2) 3.7 Sex Male 225 (68.0) 101 (69.2) 0.795 2.6 76 (69.1) 76 (69.1) > 0.999 < 0.001 Female 106 (32.0) 45 (30.8) 2.6 34 (30.9) 34 (30.9) < 0.001 ECOG 0 241 (72.8) 124 (84.9) 0.004 30.0 86 (78.2) 89 (80.9) 0.738 6.7 ≥1 90 (27.2) 22 (15.1) 30.0 24 (21.8) 21 (19.1) 6.7 ASA score 1 to 2 308 (93.1) 138 (94.5) 0.549 3.4 100 (90.9) 103 (93.6) 0.615 9.2 ≥3 23 (6.9) 8 (5.5) 3.4 10 (9.1) 7 (6.4) 9.2 Location EGJ 11 (3.3) 7 (4.8) 0.437 7.4 107 (97.3) 108 (98.2) > 0.999 6.1 Other 320 (96.7) 139 (95.2) 7.4 3 (2.7) 2 (1.8) 6.1 Stage (AJCC 7th edition) IIA 109 (32.9) 5 (3.4) < 0.001 82.8 3 (2.7) 4 (3.6) 0.982 5.2 IIB 73 (22.1) 19 (13.0) 23.9 21 (19.1) 19 (17.3) 4.7 IIIA 52 (15.7) 39 (26.7) 27.2 28 (25.5) 26 (23.6) 4.2 IIIB 53 (16.0) 48 (32.9) 40.0 33 (30.0) 34 (30.9) 2.0 IIIC 44 (13.3) 35 (24.0) 27.7 25 (22.7) 27 (24.5) 4.3 T stage T1 26 (7.9) 3 (2.1) 0.001 27.0 4 (3.6) 3 (2.7) > 0.999 5.2 T2 51 (15.4) 10 (6.8) 27.5 8 (7.3) 8 (7.3) 0.0 T3 129 (39.0) 56 (38.4) 1.3 40 (36.4) 40 (36.4) 0.0 T4a,b 125 (37.8) 77 (52.7) 30.4 58 (52.7) 59 (53.6) 1.8 N stage N0 87 (26.3) 9 (6.2) < 0.001 56.7 9 (8.2) 9 (8.2) 0.986 0.0 N1 67 (20.2) 28 (19.2) 2.7 16 (14.5) 17 (15.5) 2.5 N2 103 (31.1) 38 (26.0) 11.3 39 (35.5) 36 (32.7) 5.8 N3 74 (22.4) 71 (48.6) 57.1 46 (41.8) 48 (43.6) 3.7 Number of dissected lymph nodes mean ± sd 47.0 ± 18.8 52.4 ± 17.1 < 0.001 30.0 51.4 ± 21.4 51.5 ± 16.5 0.493 0.7 median (IQR) 43 (35–55) 52 (39–65) 45 (37–64) 52 (39–62) LNR group LNR 0 88 (26.6) 9 (6.2) < 0.001 40.2 68 (61.8) 66 (60.0) 0.89 3.7 LNR 1 127 (38.4) 49 (33.6) 40.2 42 (38.2) 44 (40.0) 3.7 LNR 2 78 (23.6) 47 (32.2) LNR 3 38 (11.5) 41 (28.1) < 0.001 57.4 9 (8.2) 9 (8.2) 0.994 0.0 Tumor size (cm) 10.0 36 (32.7) 35 (31.8) 1.9 < 6 250 (75.5) 83 (56.8) 19.3 39 (35.5) 41 (37.3) 3.8 ≥6 81 (24.5) 63 (43.2) 42.6 26 (23.6) 25 (22.7) 2.2 Table 1 Baseline characteristics of the patients before and after propensity score matching Before propensity score matching (n = 477) After propensity score matching §(n = 220) S-1 (n = 331) XELOX (n = 146) p value* Absolute‡ Standardized difference in % S-1 (n = 110) XELOX (n = 110) p value† Absolute‡ Standardized difference in % Shin et al. Statistical analyses BMC Cancer (2019) 19:1232 Page 5 of 14 Table 1 Baseline characteristics of the patients before and after propensity score matching (Continued) Before propensity score matching (n = 477) After propensity score matching §(n = 220) S-1 (n = 331) XELOX (n = 146) p value* Absolute‡ Standardized difference in % S-1 (n = 110) XELOX (n = 110) p value† Absolute‡ Standardized difference in % Differentiation Well to moderately 114 (34.4) 34 (23.3) 0.015 24.8 25 (22.7) 28 (25.5) 0.753 6.4 Poorly 217 (65.6) 112 (76.7) 24.8 85 (77.3) 82 (74.5) 6.4 Lauren classification Intestinal 118 (35.6) 39 (26.7) 0.111 19.4 30 (27.3) 34 (30.9) 0.732 8.0 Diffuse 96 (29.0) 43 (29.5) 1.0 37 (33.6) 32 (29.1) 9.8 Mixed 117 (35.3) 64 (43.8) 17.4 43 (39.1) 44 (40.0) 1.9 Lymphovascular invasion no 90 (27.2) 13 (8.9) < 0.001 50.2 8 (7.3) 13 (11.8) 0.359 8.9 yes 241 (72.8) 133 (91.1) 50.2 102 (92.7) 97 (88.2) 8.9 Perineural invasion no 161 (48.6) 49 (33.6) 0.002 30.9 40 (36.4) 39 (35.5) > 0.999 1.9 yes 170 (51.4) 97 (66.4) 30.9 70 (63.6) 71 (64.5) 1.9 Completion of planned chemotherapy no 69 (20.8) 42 (28.8) 0.059 18.4 25 (22.7) 26 (23.6) > 0.999 2.1 yes 262 (79.2) 104 (71.2) 18.4 85 (77.3) 84 (76.4) 2.1 CEA (ng/ml) < 5 315 (95.2) 140 (95.9) 0.728 3.5 106 (96.4) 106 (96.4) > 0.999 < 0.001 ≥5 16 (4.8) 6 (4.1) 3.5 4 (3.6) 4 (3.6) < 0.001 CA 19–9 (U/ml) < 37.0 308 (93.1) 132 (90.4) 0.320 9.6 100 (90.9) 102 (92.7) 0.806 9.7 ≥37.0 23 (6.9) 14 (9.6) 9.6 10 (9.1) 8 (7.3) 9.7 Data are presented as the n (%) for categorical variable, unless otherwise indicated *P value from Wilcoxon rank sum test for continuous variables or Chi-square test, for categorical variables in before Propensity score matching data †P value from Wilcoxon signed rank sum test for continuous variables or Chi-square test, for categorical variables in matched data ‡no covariates would be considered imbalanced if the threshold was set at either 0.10 (Normand et al. Statistical analyses 2001) or 0.25 (Rubin 2001) §matched using digit-based greedy (“greedy”) ine characteristics of the patients before and after propensity score matching (Continued) matching data †P value from Wilcoxon signed rank sum test for continuous variables or Chi-square test, for categorical variables in matched data ‡no covariates would be considered imbalanced if the threshold was set at either 0.10 (Normand et al. 2001) or 0.25 (Rubin 2001) §matched using digit-based greedy (“greedy”) bias, propensity score matching (PSM) was performed. The propensity scores were elicited from matched pa- tients at 1:1 ratio using greedy matching algorithms without replacement. Age, sex, ECOG (Eastern Co- operative Oncology Group) performance status, ASA (American Society of Anesthesiologists) score, location of the tumour, stage (based on the 7th AJCC guide- lines), T stage, N stage, number of dissected lymph nodes, tumour size, LNR group, differentiation, Lau- ren classification, lymphovascular invasion, perineural invasion, completion of planned chemotherapy, pre- operative CEA and CA 19–9 were used to calculate propensity scores for each patient using logistic re- gression. Standardized differences were estimated for all covariates before and after matching to assess pre- match imbalance and post-match balance. A Wilcoxon rank sum test for continuous variables or Chi-square test for categorical variables was used to compare the demographics between treatment arms in before PSM data. A Wilcoxon signed rank sum test for continuous variables or Chi-square test for categorical variables was used in matched data. The Kaplan-Meier method was used to estimate cumulative survival. The treatment groups were com- pared with a two-sided log-rank test. Estimates of treatment effect were calculated with 95% Cis using Cox proportional hazards models. Univariate and multivariate analysis models of pa- tient and tumour characteristics in association with DFS and OS were based on Cox-proportional hazards regression analyses. P values of less than 0.05 were considered to indicate statistical significance. All Shin et al. BMC Cancer (2019) 19:1232 Page 6 of 14 Table 2 Univariate, multivariate cox proportional hazards regression in the PSM cohort. Statistical analyses (n = 220) Overall survival Disease-free survival univariate multivariate univariate multivariate HR (95%CI) p value HR (95%CI) p value HR (95%CI) p value HR (95%CI) p value Treatment S-1 1 1 XELOX 0.71 0.40–1.26 0.240 0.65 0.39–1.09 0.101 Age (years) < 65 1 1 1 ≥65 1.93 1.11–3.35 0.02 1.33 0.72–2.46 0.363 1.58 0.96–2.60 0.07 Sex Female 1 1 Male 1.14 0.64–2.04 0.66 1.25 0.85–2.11 0.393 ECOG 0 1 1 1 1 ≥1 2.32 1.31–4.12 0 1.54 0.80–3.00 0.198 2.17 1.28–3.66 0 1.72 0.99–2.98 0.051 ASA 1 to 2 1 1 ≥3 0.85 0.58–1.26 0.420 1.1 0.85–1.43 0.462 Location Other 1 1 EGJ 0.54 0.13–2.23 0.398 2.82 0.88–9.01 0.081 T stage T1,T2 1 1 T3,T4 3.19 0.78–13.12 0.108 2.65 0.83–8.47 0.1 N stage N0,1,2 1 1 1 1 1 N3 1.69 1.37–2.09 < 0.001 1.4 1.09–1.80 0.009 1.54 1.29–1.84 < 0.001 1.26 1.00–1.58 0.05 LNR group LNR0,1,2 1 1 1 1 LNR3 1.7 1.41–2.04 < 0.001 1.36 1.09–1.70 0.006 1.67 1.41–1.97 < 0.001 1.44 1.16–1.78 0 Tumor size < 6 1 1 1 1 ≥6 1.95 1.13–3.39 0.02 1.07 0.97–1.18 0.209 1.91 1.16–3.13 0.01 1.049 0.96–1.15 0.288 Differntiation Well to moderately 1 1 Poorly 0.81 0.44–1.51 0.512 0.95 0.53–1.69 0.855 Lauren classification Intestinal 1 1 Diffuse/Mixed 0.94 0.70–1.26 0.681 0.99 0.75–1.30 0.923 Lymphovascular invasion no 1 1 yes 3.13 0.76–12.88 0.114 2.53 0.79–8.07 0.117 Perineural invasion no 1 1 1 1 yes 2.72 1.36–5.43 0.01 2.39 1.18–4.82 0.015 2.05 1.15–3.66 0.02 1.47 0.81–2.66 0.205 Shin et al. BMC Cancer (2019) 19:1232 Page 7 of 14 Table 2 Univariate, multivariate cox proportional hazards regression in the PSM cohort. Statistical analyses (n = 220) (Continued) Overall survival Disease-free survival univariate multivariate univariate multivariate HR (95%CI) p value HR (95%CI) p value HR (95%CI) p value HR (95%CI) p value Chemotherapy completion no 1 1 1 1 yes 0.43 0.24–0.77 0 0.5 0.28–0.91 0.023 0.36 0.21–0.59 < 0.001 0.36 0.21–0.61 < 0.001 CEA (before surgery) normal 1 1 elevated 1.31 0.32–5.38 0.711 1.02 0.25–4.19 0.975 CEA (after surgery) normal 1 1 elevated 1.14 0.28–4.68 0.86 0.91 0.22–3.74 0.902 CA 19–9 (before surgery) normal 1 1 1 elevated 1.87 0.84–4.16 0.123 2.66 1.36–5.24 0.01 1.81 0.88–3.74 0.107 CA 19–9 (after surgery) normal 1 1 elevated 1.32 0.32–5.41 0.705 2.09 0.65–6.67 0.213 Univariate analysis and multivariate survival analysis were performed using Cox proportional hazard model, and P values < 0.05 were considered to indicate statistical significance Abbreviations: CI confidence interval, HR hazard ratio. Significant values are in boldface type multivariate cox proportional hazards regression in the PSM cohort. (n = 220) (Continued) number of dissected lymph nodes, LNR group, tumour size, differentiation, lymphovascular invasion, perineural invasion. statistical analyses were conducted using SAS software ver. 9.4 (SAS Institute Inc., Cary, NC, USA) and R version 3.5.3 (http://www.r-project.org). The XELOX group had a younger age than the S-1 group (S-1 vs XELOX, median age 58 vs 55 years, p < 0.001). The XELOX group had a smaller number of patients aged more than 65 years than the S-1 group (S-1 vs XELOX, 45.3% vs 28.8%, p = 0.001). The XELOX group had a smaller number of patients with ECOG PS ≥1 than the S-1 group (S-1 vs XELOX, 27.2% vs 15.1%, p = 0.004). Compared with the S-1 group, the XELOX group had patients with more advanced T and N stages of gastric cancer (p = 0.001, < 0.001 respectively), had patients with an increased number of dissected lymph nodes (S-1 vs XELOX, median (IQR) 43(35–55) vs 52(39–65), Clinical characteristics Of the 477 patients eligible for this study, 331 re- ceived S-1 and 146 received XELOX. The median age was 57 years (range 22 ~ 79), and the male: female ra- tio was 326 (68.3%): 151 (31.7%). The median follow- up duration was 52.3 months. The baseline character- istics of the patients in the two groups are sum- marised in Table 1. Before PSM, the two groups differed significantly in age, ECOG performance sta- tus, cancer stage (AJCC 7th edition), T stage, N stage, Table 3 DFS, OS of XELOX and S-1 in the PSM cohort total event 3 year 5 year HR(95% CI)a p value Ovarall survival 3-year OS % (95% CI) 5-year OS % (95% CI) TS-1 110 31 78 (70–86) 72 (64–81) 1 0.240 XELOX 110 20 86 (80–93) 77 (68–88) 0.71 (0.40–1.26) Disease-free survival 3-year DFS % (95% CI) 5-year DFS % (95% CI) TS-1 110 38 71 (63–80) 66 (57–75) 1 0.101 XELOX 110 25 79 (72–88) 74 (66–84) 0.65 (0.39–1.09) aHR of XELOX adjuvant chemotherapy for recurrence of gastric cancer compared with S-1 as the reference was calculated using Cox’s proportional hazards model Abbreviations: CI confidence interval, HR hazard ratio. Significant values are in boldface type aHR of XELOX adjuvant chemotherapy for recurrence of gastric cancer compared with S-1 as the reference was calculated using Cox’s proportional hazards model Abbreviations: CI confidence interval, HR hazard ratio. Significant values are in boldface type Shin et al. BMC Cancer (2019) 19:1232 Page 8 of 14 p = 0.023) were shown as independent prognostic fac- tors of survival. p < 0.001), and had a greater number of patients in the higher LNR groups (median LNR 0.06 vs 0.13, p < 0.001). An increased number of patients with tumour size (≥6 cm) was observed in the XELOX group compared to the S-1 group (S-1 vs XELOX, 24.5% vs 43.2%, p < 0.001). The percentage of patients assigned a ‘poorly differenti- ated’ histologic grade was also higher in the XELOX group than in the S-1 group (S-1 vs XELOX, 65.6% vs 76.7% p = 0.015). In addition, ECOG performance status (0 vs ≥1), N stage (N0,1,2 vs N3), LNR group (LNR0,1,2 vs LNR3), tumour size (≥6 cm), perineural invasion, completion of planned chemotherapy, and elevated preoperative CA 19–9 were shown as prognostic factors associated with recurrence. Clinical characteristics After adjusting for covariates in multivariate analysis, N3 stage (HR 1.26; 95% CI, 1.00–1.58; p = 0.049), LNR3 group (HR 1.44; 95% CI, 1.16–1.78; p = 0.001), and completion of planned chemotherapy (HR 0.36; 95% CI, 0.21–0.61; p < 0.001) were shown as independent prognostic fac- tors of recurrence. Lymphovascular invasion and perineural invasion were more significantly more frequently observed in the XELOX group than in the S-1 group (S-1 vs XELOX, 72.8% vs 91.1, 51.4% vs 66.4%, respectively). The rate of chemotherapy completion in the S-1 group showed tendency to be higher than that in the XELOX group (S-1 vs XELOX, 79.2% vs 71.2%, p = 0.059). After PSM, each group was one-to-one matched so that there were 110 patients per group. Each variable was well balanced, without significant difference in terms of absolute standardised difference (Table 1). Subgroup analysis of the PSM cohort. S-1 vs XELOX After PSM, OS and DFS were higher in the XELOX group than in the S-1 group, with HR of 0.71 (95% CI 0.40–1.26; p = 0.240) and 0.65 (95% CI 0.39–1.09; p = 0.101). The 5-year DFS rate in the S-1 group versus the XELOX group was 66% versus 74%. The 5-year OS rate in the S-1 vs XELOX groups was 72% versus 77%. Both DFS and OS rates were not signifi- cantly different between the two groups. (Table 3, Fig. 2). Univariate and multivariate analyses of DFS and OS in the PSM cohort. (Table 2) Upon univariate analysis of all patients after PSM, age (< 65 vs ≥65), ECOG performance status (0 vs ≥1), N stage (N0,1,2 vs N3), LNR group (LNR0,1,2 vs LNR3), tumour size (≥6 cm), lymphovascular inva- sion, perineural invasion, and completion of planned chemotherapy were shown as prognostic factors asso- ciated with survival. After adjusting for covariates in multivariate analysis, N stage (HR 1.40; 95% CI, 1.09–1.80; p = 0.009), LNR group (HR 1.36; 95% CI, 1.09–1.70; p = 0.006), perineural invasion (HR 2.39; 95% CI, 1.18–4.82; p = 0.015) and completion of planned chemotherapy(HR 0.50; 95% CI, 0.28–0.91; Subgroup analysis of the PSM data set revealed that the XELOX group, compared with the S-1 group, showed significantly better 5-year DFS (S-1 vs XELOX, 22% vs 57%, HR 0.32, 95% CI 0.15–0.70; p = 0.004) and better 5-year OS (27% vs 68%, HR 0.32, 95% CI 0.14–0.76; p = 0.009) in stage IIIC patients. All stage III patients showed better DFS and OS in the XELOX group than in the S-1 group, but statistically not significant. (DFS 60% vs 69%, OS 67% vs 73%). (Table 4, Fig. 3, Additional file 1; survival curves of XELOX and S-1 in Stage IIIA, B, C). Fig. 2 OS and DFS of S-1 and XELOX in the PSM cohort Shin et al. Univariate and multivariate analyses of DFS and OS in the PSM cohort. (Table 2) BMC Cancer (2019) 19:1232 Page 9 of 14 Table 4 Subgroup analysis of the PSM cohort (n = 220) number of patients Overall survival Disease-free survival 5-year OS % (95% CI) HR(95% CI) p value* 5-year DFS % (95% CI) HR(95% CI) p value* S-1 XELOX S-1 XELOX Sex Male 152 73 (63–84) 78 (68–90) 0.63 (0.32–1.27) 0.196 66 (56–78) 77 (67–88) 0.60 (0.32–1.14) 0.117 Female 68 71 (57–89) 76 (59–97) 0.93 (0.35–2.48) 0.890 64 (49–83) 68 (49–94) 0.75 (0.32–1.77) 0.507 Age (years) < 65 138 78 (68–88) 87 (79–96) 0.66 (0.29–1.50) 0.316 71 (61–83) 77 (67–90) 0.73 (0.36–1.44) 0.361 ≥65 82 62 (48–80) 64 (48–85) 0.69 (0.32–1.51) 0.358 56 (42–74) 69 (55–87) 0.55 (0.261–1.18) 0.125 Stage (AJCC 7th) IIA 7 100 (100–100) 100 (100–100) NA NA 100 (100–100) 100 (100–100) NA NA IIB 40 89 (64–97) 92 (57–99) 0.67 (0.06–7.48) 0.747 85 (61–95) 92 (54–99) 0.38(0.04–3.68) 0.405 IIIA 54 89 (70–96) 75 (33–93) 1.56 (0.31–7.96) 0.593 85 (66–94) 77 (53–90) 1.58(0.43–5.76) 0.487 IIIB 67 78 (65–94) 72 (55–94) 1.35 (0.50–3.69) 0.554 66 (51–85) 74 (60–92) 0.84 (0.34–2.04) 0.697 IIIC 52 27 (10–46) 68 (51–90) 0.32 (0.14–0.76) 0.009 22 (8–41) 57 (39–84) 0.32 (0.15–0.70) 0.004 All II 47 90 (78–100) 94 (83–100) 0.58 (0.05–6.40) 0.655 87(74–100) 93 (82–100) 0.35 (0.04–3.34) 0.360 All III 173 67 (58–78) 73 (62–86) 0.73 (0.40–1.31) 0.285 60 (50–71) 69 (59–81) 0.67 (0.40–1.13) 0.133 N stage N0 18 100 (100–100) 100 (100–100) NA NA 100 (100–100) 100 (100–100) NA NA N1 33 93 (59–99) 81 (52–94) 3.40 (0.35–32.86) 0.290 87 (56–96) 80 (50–93) 1.81(0.30–10.96) 0.519 N2 75 86 (71–94) 77 (42–92) 1.40 (0.36–5.41) 0.623 82 (65–91) 78 (57–90) 1.18(0.42–3.34) 0.757 N3 94 47 (34–65) 71 (58–86) 0.45 (0.23–0.87) 0.019 38 (26–55) 66 (52–82) 0.40 (0.21–0.75) 0.004 T stage T1 7 100 (100–100) 100 (100–100) NA NA 75 (13–96) 100 (100–100) 0.42(0.00–41.43) 0.712 T2 16 86 (33–98) 86 (33–98) 0.87 (0.05–13.85) 0.919 88 (39–98) 86 (33–98) 0.93(0.06–14.83) 0.957 T3 80 80 (68–93) 81 (66–99) 0.75 (0.25–2.23) 0.604 74 (62–90) 80 (67–95) 0.78 (0.30–2.05) 0.617 T4a,b 117 64 (52–78) 72 (59–88) 0.69 (0.35–1.37) 0.290 56 (42–68) 67 (50–79) 0.61 (0.33–1.14) 0.121 LNR group LNR 0 18 100 (100–100) 100 (100–100) NA NA 100 (100–100) 100 (100–100) NA NA LNR 1 71 94 (78–98) 86 (61–96) 3.01 (0.55–16.59) 0.205 88 (72–95) 85 (65–94) 1.43(0.41–4.99) 0.579 LNR 2 80 74 (57–85) 72 (50–86) 0.84 (0.34–2.10) 0.705 66 (49–79) 74 (56–85) 0.73(0.32–1.68) 0.464 LNR 3 51 26 (12–55) 67 (50–89) 0.28 (0.11–0.71) 0.020 20 (9–47) 54 (35–82) 0.29 (0.13–0.65) 0.004 *The hazard ratio of the XELOX group using the S-1 group as the reference and the 95% CIs were calculated using Cox’s proportional hazards model †NA = not evaluable Abbreviations: CI confidence interval, HR hazard ratio. Univariate and multivariate analyses of DFS and OS in the PSM cohort. (Table 2) Significant values are in boldface type Table 4 Subgroup analysis of the PSM cohort (n = 220) two regimens. The LNR3 group showed significantly better 5-year DFS in the XELOX group (20% vs 54%, HR 0.29, 95% CI 0.13–0.65; p = 0.004). The 5-year OS was also statistically different (26% vs 67%, HR 0.28, 95% CI 0.11–0.71; p = .0.020) (Table 4, Fig. 5, Add- itional file 3; survival curves of XELOX and S-1 in LNR1, 2, 3). When stratified by N stage in the PSM cohort, the XELOX group showed no difference in OS and DFS compared to the S-1 group in the N0, N1, and N2 groups. The N3 group showed significantly better 5-year DFS (38% vs 66%, HR 0.40, 95% CI 0.21–0.75; p = 0.004) and better 5-year OS (47% vs 71%, HR 0.45, 95% CI 0.23–0.87; p = 0.019) in the XELOX group (Table 4, Fig. 4, Additional file 2; survival curves of XELOX and S-1 in N1, 2, 3). Discussion When stratified by LNR group, LNR0, 1, 2 showed no significant difference in OS and DFS between the In this study, we analysed clinical impact of LNRs and N stages as prognostic factors and as clinical Shin et al. BMC Cancer (2019) 19:1232 Page 10 of 14 Fig. 3 OS and DFS of XELOX and S-1 in Stage IIIC. XELOX regimen showed significantly better efficacy compared to S-1 in Stage IIIC patients in terms of OS and DFS Fig. 3 OS and DFS of XELOX and S-1 in Stage IIIC. XELOX regimen showed significantly better efficacy compared to S-1 in Stage IIIC patients in terms of OS and DFS trial compared XPRT with XP, and showed that XPRT was better in patients who had an N ratio of > 25% [23]. determinants for selecting XELOX or S-1 in the PSM cohort of gastric cancer patients after D2 gastrectomy with adequate lymph node dissection. In this study, cut-off values of 0.1 and 0.25 have been adopted for categorizing four tiers of LNRs from Nitti’s study. The cut-off value for discriminating LNR3 from others was 0.25, which is similar to the 0.26 value calculated by a maximal chi-square method to identify optimal cutting point to discriminate all the PSM cohort patients into poor- and good- prognosis subgroups in terms of DFS [24]. And all the PSM cohort in this study underwent D2 gastrec- tomy, with more than 15 lymph nodes were examined (median 51, range 16~124), which is relatively higher than that examined in previous studies that showed prognostic value of LNR [21]. Although LNR is Perineural invasion was independent prognostic factors for survival consistent with previous studies that showed prognostic factors of gastric cancer [22]. N3, LNR3 and completion of planned chemotherapy showed the prognostic significance for both survival and recurrence. Nitti et al. proposed a four-tier categorisation for N ratio (0, 1%~ 9, 10%~ 25, and > 25%) in gastric cancer, and reported that N ratio was an independent pre- dictor of survival in their series [19]. Marchet et al. deduced the same conclusion with their Italian study [20]. Further, categorisation by N ratio has previously been utilised in clinical trials. Especially, the ARTIST Fig. 4 OS and DFS of XELOX and S-1 in N3. XELOX regimen showed significantly better efficacy compared to S-1 in N3 patients in terms of OS and DFS Fig. Discussion 4 OS and DFS of XELOX and S-1 in N3. XELOX regimen showed significantly better efficacy compared to S-1 in N3 p and DFS Shin et al. BMC Cancer (2019) 19:1232 Page 11 of 14 Fig. 5 OS and DFS of XELOX and S-1 in LNR3. XELOX regimen showed significantly better efficacy compared to S-1 in LNR3 patients in terms of OS and DFS Fig. 5 OS and DFS of XELOX and S-1 in LNR3. XELOX regimen showed significantly better efficacy compared to S-1 in LNR3 patients in terms of OS and DFS vs 55% and 5-year OS 27% vs 68%, Fig. 6) This indicated that LNR3 can distinguish patients who can be more beneficial with XELOX regimen from N3 patients. Thus, for selecting XELOX or S-1, LNRs might have more clinical impact than N3 stage. However, its usefulness in patients with limited lymph node evaluation (examined LN ≤15) needs to be investigated further. considered to have more prognostic value when the number of examined lymph nodes is less than 15, several studies showed that LNR has prognostic value regardless of retrieved lymph node and the LNR3 group in this study showed more prognostic value compared to N3 stage in both recurrence and survival in multivariate analysis [25–27]. Additionally, when stratified by stage (AJCC 7th edition) in the subgroup analysis of the PSM cohort, the XELOX group showed better DFS in stage IIIC patients. This re- sult is consistent with that of a previous multi-centred, retrospective PSM study that compared XELOX and S-1. In the study, Kim et. all showed that XELOX was statisti- cally more beneficial than S-1 in terms of 3-year DFS in stage IIIB, IIIC, and all stage III sub-types [15]. However, our study did not show the difference in DFS between the two regimens in stage IIIB and all stage III. The reason is that the sample size was too small to show statistical power. In the study, the 3-year DFS for S-1 vs XELOX in stage IIIB was 65.8% (95% CI, 61.2–70.4) vs 68.6% (95% CI, 55.9–81.3) (p = 0.019), and stage IIIB patients were 126 for S-1 and 48 for XELOX. Such a slim yet statistically significant difference might be explained by the relatively small sample size of this study, which included 33 patients for S-1 and 34 patients for XELOX in stage IIIB. Discussion (B) LNR3 within N3. XELOX regimen showed significantly better efficacy within N3, but not in LNR1,2 within N3 Fig. 6 OS and DFS of XELOX and S-1 within N3. (A) LNR1,2 within N3. (B) LNR3 within N3. XELOX regimen showed significantly better efficacy compared to S-1 in LNR3 within N3, but not in LNR1,2 within N3 and stage IIIC were the discriminant factors for selecting XELOX over S-1. as possible in propensity matching, unmeasured variables might have still existed, resulting in unadjusted bias. Moreover, this study only included patients with adju- vant chemotherapy. Thus, prognosis of the patients in this study should be interpreted with caution. Further- more, a relatively small number of stage IIA (7 patients, 3.2% of the PSM cohort) was included in the PSM co- hort even though their baseline characteristics were well-balanced after PSM. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12885-019-6433-3. Additional file 1: Figure S1. DFS and OS of XELOX and S-1 in stage IIIA, B, C. (A) Stage IIIA, (B) Stage IIIB, (C) Stage IIIC. Additional file 2: Figure S2. DFS and OS of XELOX and S-1 in N1, 2, 3. (A) N1 (B) N2 (C) N3. Additional file 3: Figure S3. DFS and OS of XELOX and S-1 in LNR1, 2, 3. (A) LNR1 (B) LNR2 (C) LNR3. pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12885-019-6433-3. y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12885-019-6433-3. Additional file 1: Figure S1. DFS and OS of XELOX and S-1 in stage IIIA, B, C. (A) Stage IIIA, (B) Stage IIIB, (C) Stage IIIC. Additional file 2: Figure S2. DFS and OS of XELOX and S-1 in N1, 2, 3. (A) N1 (B) N2 (C) N3. Additional file 2: Figure S2. DFS and OS of XELOX and S-1 in N1, 2, 3. (A) N1 (B) N2 (C) N3. Additional file 3: Figure S3. DFS and OS of XELOX and S-1 in LNR1, 2, 3. (A) LNR1 (B) LNR2 (C) LNR3. Conclusion In gastric cancer patients underwent D2 gastrectomy with adequate lymph node dissection and adjuvant chemotherapy, LNR showed better prognostic value than N staging. Stage IIIC, LNR3 and N3 groups showed the superior efficacy of XELOX to that of S-1 in terms of DFS and OS. And the LNR3 group within N3 patients showed more survival benefit from XELOX. It suggests that using LNR might be useful for selecting patients for adjuvant chemotherapy regimens. LNR > 0.25, N3 stage Discussion And all stage III patients were 469 for Kim et al.‘s study and 173 patients for this study. Furthermore, our study showed that the XELOX group showed significantly better OS in stage IIIC, compared to the S-1 group. In the N3 group, XELOX showed significant benefit for DFS and OS. This is consistent with the result of the CLASSIC trial and ACT-GC trial. The former showed a greater benefit in patients with node posi- tive disease than in those whose disease was limited to N0, and the latter showed a minimal or no benefit when positive lymph node was equal to or more than three, even though they were deduced from subgroup analysis [2, 14]. In the PSM cohort, the number of LNR3 patients were 51 (23.2%) and 48 of them classified to the N3 stage. (Table 5) When N3 group was divided into two groups; LNR3 group and LNR1,2 group, the XELOX and the S-1 in LNR1,2 group didn’t show difference in OS and DFS. However, LNR3 within N3 stage still showed significant survival benefit of the XELOX regimen (5-year DFS 21% Table 5 The distribution of the lymph node ratio and N stage in the PSM cohort LNR0 LNR1 LNR2 LNR3 total N stage N0 18 18 N1 31 2 33 N2 39 33 3 75 N3 1 45 48 94 total 18 71 80 51 220 Table 5 The distribution of the lymph node ratio and N stage in the PSM cohort This study had several limitations. Because this study used retrospective, single-centre data, it had the limitation of se- lection bias. Despite several efforts to reduce selection bias, including using multivariable analyses and PSM, unadjusted bias may have still been present between the two groups. Even though this study included as many clinical variables Page 12 of 14 Shin et al. BMC Cancer (2019) 19:1232 Shin et al. BMC Cancer Fig. 6 OS and DFS of XELOX and S-1 within N3. (A) LNR1,2 within N3. (B) LNR3 within N3. XELOX regimen showed significantly better efficacy compared to S-1 in LNR3 within N3, but not in LNR1,2 within N3 Fig. 6 OS and DFS of XELOX and S-1 within N3. (A) LNR1,2 within N3. (B) LNR3 within N3. XELOX regimen showed signif compared to S-1 in LNR3 within N3, but not in LNR1,2 within N3 OX and S-1 within N3. (A) LNR1,2 within N3. Abbreviations AJCC: American Joint Committee on Cancer; CA 19–9: Carbohydrate antigen 19–9; CEA: Carcinoembryonic antigen; CI: Confidence interval; CT: Computed tomography; DFS: Disease-free survival; ECOG: Eastern Cooperative Oncology Group; EGJ: Esophagogastric junction; HR: Hazard ratio; IQR: Interquartile range; LN: Lymph node; LNR: Lymph node ratio; MRI: Magnetic resonance imaging; OS: Overall survival; PSM: Propensity score matching; XELOX: Capecitabine and oxaliplatin Page 13 of 14 Page 13 of 14 Page 13 of 14 Shin et al. BMC Cancer (2019) 19:1232 Shin et al. BMC Cancer (2019) 19:1232 Availability of data and materials g 11. NCCN. NCCN Clinical Practice Guidelines in Oncology. Gastric Cancer. Version 2.2019[cited, 2019]. NCCN. http://www.nccn.org. Published 2019. 11. NCCN. NCCN Clinical Practice Guidelines in Oncology. Gastric Cancer. Version 2.2019[cited, 2019]. NCCN. http://www.nccn.org. Published 2019. The data that support the findings of this study are available from the corresponding author but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the corresponding author upon reasonable request and with permission of Institutional Review Board of the Seoul St. Mary’s Hospital. 12. Iveson TJ, Cunningham D, Stenning SP, et al. Perioperative chemotherapy versus surgery alone for Resectable Gastroesophageal Cancer. N Engl J Med. 2006. https://doi.org/10.1056/nejmoa055531. 12. Iveson TJ, Cunningham D, Stenning SP, et al. Perioperative chemotherapy versus surgery alone for Resectable Gastroesophageal Cancer. N Engl J Med. 2006. https://doi.org/10.1056/nejmoa055531. 13. Macdonald JS, Smalley SR, Benedetti J, et al. Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction. N Engl J Med. 2001. https://doi.org/10.1056/ NEJMoa010187. 13. Macdonald JS, Smalley SR, Benedetti J, et al. Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction. N Engl J Med. 2001. https://doi.org/10.1056/ NEJMoa010187. Funding Th h The author(s) wish(es) to acknowledge the financial support of the Catholic Medical Center Research Foundation made in the program year of 2018. The sponsors of the study had no involvement in study design, data collection, analysis, or decision to submit the article for publication. g g 9. Gunderson LL. Gastric cancer - patterns of relapse after surgical resection. Semin Radiat Oncol. 2002. https://doi.org/10.1053/srao.2002.30817. 9. Gunderson LL. Gastric cancer - patterns of relapse after surgical resection. Semin Radiat Oncol. 2002. https://doi.org/10.1053/srao.2002.30817. 10. Smyth EC, Verheij M, Allum W, et al. Gastric cancer: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2016. https:// doi.org/10.1093/annonc/mdw350. Acknowledgements S l l 4. Cuschieri A, Weeden S, Fielding J, et al. Patient survival after D1 and D2 resections for gastric cancer: Long-term results of the MRC randomized surgical trial. Br J Cancer. 1999. https://doi.org/10.1038/sj.bjc.6690243. Statistical consultation was supported by the Department of Biostatistics of the Catholic Research Coordinating Center. 5. Bonenkamp JJ, Hermans J, Sasako M, van de Velde CJH, The FOR. Extended lymph-node dissection for gastric cancer. Dutch Gastric Cancer Group. N Engl J Med. 1999. Authors’ contributions SKS collected and analyzed all the patient data, and was a major contributor in writing the manuscript. PSJ and LJS collected and analyzed the data. PCH, SKY, LHH, SHS and JYJ provided and analyzed the data about surgery, PJM provided and analyzed the data about endoscopic study. LSH provided and analyzed the data about pathology. RSY reviewed the analyzed data and made an adjustment. KIH analyzed and interpreted the data, and was a major contributor in interpretation of all the patient data. SKS and KIH drafted the manuscript, which has been reviewed and approved in its final form by all other authors. 6. Hartgritik HH, Van De Velde CJH, Putter H, et al. Extended lymph node dissection for gastric cancer: who may benefit? Final results of the randomized Dutch gastric Cancer group trial. J Clin Oncol. 2004. https://doi. org/10.1200/JCO.2004.08.026. 6. Hartgritik HH, Van De Velde CJH, Putter H, et al. Extended lymph node dissection for gastric cancer: who may benefit? Final results of the randomized Dutch gastric Cancer group trial. J Clin Oncol. 2004. https://doi. org/10.1200/JCO.2004.08.026. 7. D’Angelica M, Gonen M, Brennan MF, Turnbull AD, Bains M, Karpeh MS. Patterns of initial recurrence in completely resected gastric adenocarcinoma. Ann Surg. 2004. https://doi.org/10.1097/01.sla.0000143245. 28656.15. drafted the manuscript, which has been reviewed and approved in its final form by all other authors. 8. Wu CW, Lo SS, Shen KH, et al. Incidence and factors associated with recurrence patterns after intended curative surgery for gastric cancer. World J Surg. 2003. https://doi.org/10.1007/s00268-002-6279-7. Consent for publication Not applicable. Consent for publication Not applicable. Not applicable. 16. Cho JH, Lim JY, Cho JY. Comparison of capecitabine and oxaliplatin with S-1 as adjuvant chemotherapy in stage III gastric cancer after D2 gastrectomy. PLoS One. 2017;12(10):1–10. https://doi.org/10.1371/journal.pone.0186362. Competing interests The authors declare that they have no competing interests. 17. Bando E, Yonemura Y, Taniguchi K, Fushida S, Fujimura T, Miwa K. Outcome of ratio of lymph node metastasis in gastric carcinoma. Ann Surg Oncol. 2002. https://doi.org/10.1245/ASO.2002.10.011. Author details 1 1Division of Medical Oncology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, South Korea. 2Department of Surgery, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea. 3Department of Gastric Cancer Centre, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea. 4Division of Gastroenterology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea. 5Department of Clinical Pathology, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea. 6Cancer Research Institute, College of Medicine, The Catholic University of Korea, Seoul, South Korea. 18. Inoue K, Nakane Y, Iiyama H, et al. The superiority of ratio-based lymph node staging in gastric carcinoma. Ann Surg Oncol. 2002. https://doi.org/10. 1245/aso.2002.9.1.27. 19. Nitti D, Marchet A, Olivieri M, et al. Ratio between metastatic and examined lymph nodes is an independent prognostic factor after D2 resection for gastric cancer: analysis of a large European Monoinstitutional experience. Ann Surg Oncol. 2003. https://doi.org/10.1245/ASO.2003.03.520. 20. Marchet A, Mocellin S, Ambrosi A, et al. The ratio between metastatic and examined lymph nodes (N ratio) is an independent prognostic factor in gastric cancer regardless of the type of lymphadenectomy: results from an Italian multicentric study in 1853 patients. Ann Surg. 2007;245(4):543–52. https://doi.org/10.1097/01.sla.0000250423.43436.e1. Received: 31 July 2019 Accepted: 4 December 2019 Received: 31 July 2019 Accepted: 4 December 2019 21. Yamashita K, Hosoda K, Ema A, Watanabe M. Lymph node ratio as a novel and simple prognostic factor in advanced gastric cancer. Eur J Surg Oncol. 2016;42(9):1253–60. https://doi.org/10.1016/j.ejso.2016.03.001. 22. Bilici A, Seker M, Ustaalioglu BBO, et al. Prognostic significance of perineural invasion in patients with gastric cancer who underwent curative resection. Ann Surg Oncol. 2010;17(8):2037–44. https://doi.org/10.1245/s10434-010- 1027-y. 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018. https://doi.org/10. 3322/caac.21492. Ethics approval and consent to participate 14. Noh SH, Park SR, Yang HK, et al. Adjuvant capecitabine plus oxaliplatin for gastric cancer after D2 gastrectomy (CLASSIC): 5-year follow-up of an open- label, randomised phase 3 trial. Lancet Oncol. 2014;15(12):1389–96. https:// doi.org/10.1016/S1470-2045(14)70473-5. 14. Noh SH, Park SR, Yang HK, et al. Adjuvant capecitabine plus oxaliplatin for gastric cancer after D2 gastrectomy (CLASSIC): 5-year follow-up of an open- label, randomised phase 3 trial. Lancet Oncol. 2014;15(12):1389–96. https:// doi.org/10.1016/S1470-2045(14)70473-5. The Institutional Review Board of the Catholic University of Seoul Saint Mary’s Hospital approved the study (KC18RESI0596, KC19RASI0751). The Institutional Review Board of the Catholic University of Seoul Saint Mary’s Hospital approved the study (KC18RESI0596, KC19RASI0751). Requirement for informed consent was waived because the study was based on retrospective analyses of existing administrative and clinical data. Requirement for informed consent was waived because the study was based on retrospective analyses of existing administrative and clinical data. Requirement for informed consent was waived because the study was based on retrospective analyses of existing administrative and clinical data. 15. Kim I, Park S, Lee C, Kim MC. Efficacy of adjuvant S-1 versus XELOX chemotherapy for patients with gastric Cancer after D2 lymph node dissection : a retrospective Multi-Center Observational Study. Ann Surg Oncol. 2018;25(5):1176–83. https://doi.org/10.1245/s10434-018-6375-z. References 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018. https://doi.org/10. 3322/caac.21492. 23. Kim Y, Park SH, Kim KM, et al. The influence of metastatic lymph node ratio on the treatment outcomes in the adjuvant Chemoradiotherapy in stomach tumors (ARTIST) trial: a phase III trial. J Gastric Cancer. 2016;16(2):105–10. https://doi.org/10.5230/jgc.2016.16.2.105. 2. Sasako M, Sakuramoto S, Katai H, et al. Five-year outcomes of a randomized phase III trial comparing adjuvant chemotherapy with S-1 versus surgery alone in stage II or III gastric cancer. J Clin Oncol. 2011;29(33):4387–93. https://doi.org/10.1200/JCO.2011.36.5908. 2. Sasako M, Sakuramoto S, Katai H, et al. Five-year outcomes of a randomized phase III trial comparing adjuvant chemotherapy with S-1 versus surgery alone in stage II or III gastric cancer. J Clin Oncol. 2011;29(33):4387–93. https://doi.org/10.1200/JCO.2011.36.5908. 3. Kodera Y, Sano T. Japanese gastric cancer treatment guidelines 2014 (ver. 4). Gastric Cancer. 2017;20(1):1–19. https://doi.org/10.1007/s10120-016-0622-4. 25. Wang J, Dang P, Raut CP, et al. Comparison of a lymph node ratio-based staging system with the 7th AJCC system for gastric cancer: analysis of Page 14 of 14 Shin et al. BMC Cancer (2019) 19:1232 Shin et al. BMC Cancer (2019) 19:1232 18,043 patients from the SEER database. Ann Surg. 2012. https://doi.org/10. 1097/SLA.0b013e31824857e2. 26. Xu DZ, Geng QR, Long ZJ, et al. Positive lymph node ratio is an independent prognostic factor in gastric cancer after D2 resection regardless of the examined number of lymph nodes. Ann Surg Oncol. 2009. https://doi.org/10.1245/s10434-008-0240-4. 26. Xu DZ, Geng QR, Long ZJ, et al. Positive lymph node ratio is an independent prognostic factor in gastric cancer after D2 resection regardless of the examined number of lymph nodes. Ann Surg Oncol. 2009. https://doi.org/10.1245/s10434-008-0240-4. 27. Alatengbaolide, Lin D, li Y, et al. lymph node ratio is an independent prognostic factor in gastric cancer after curative resection (R0) regardless of the examined number of lymph nodes. Am J Clin Oncol Cancer Clin Trials 2013;36(4):325–30. https://doi.org/10.1097/COC.0b013e318246b4e9. 27. Alatengbaolide, Lin D, li Y, et al. lymph node ratio is an independent prognostic factor in gastric cancer after curative resection (R0) regardless of the examined number of lymph nodes. Am J Clin Oncol Cancer Clin Trials 2013;36(4):325–30. https://doi.org/10.1097/COC.0b013e318246b4e9. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Reliability of Blue-Emitting Eu2+-Doped Phosphors for Laser-Lighting Applications
Materials
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8,224
  Received: 15 June 2018; Accepted: 1 August 2018; Published: 28 August 2018 Abstract: This paper investigates the reliability of blue-emitting phosphors for Near-UV (NUV) laser excitation. By means of a series of thermal stress experiments, and of stress under high levels of optical excitation, we have been able to identify the physical process responsible for the degradation of Eu2+-activated alkaline-earth halophosphate phosphors under typical and extreme operating conditions. In particular, for temperatures equal to or greater than 450 ◦C the material exhibited a time-dependent drop in the Photo-Luminescence (PL), which was attributed to the thermally induced ionization of the Eu2+ optically active centers. Several analytical techniques, including spatially and spectrally resolved PL, Electron Paramagnetic Resonance (EPR) and X-ray Photo-emission Spectroscopy (XPS) were used to support this hypothesis and to gain insight on the degradation process. By means of further tests, evidence of this degradation process was also found on samples stressed under a relatively low power density of 3 W/mm2 at 405 nm. This indicated that the optically (and thermally) induced ionization of the optically active species is the most critical degradation process for this family of phosphorescent material. The operating limits of a second-generation Eu-doped halophosphate phosphor were also investigated by means of short-term stress under optical excitation. The experimental data showed that a threshold excitation intensity for continuous pumping exists. Above this threshold, decay of the steady-state PL performance and non-recoverable degradation of the material were found to take place. This behavior is a consequence of the extremely harsh excitation regime, mainly due to the thermal management capabilities of the substrate material employed for our experimental purposes rather than from intrinsic properties of the phosphors. Keywords: phosphors; laser-lighting; degradation; europium materials materials Reliability of Blue-Emitting Eu2+-Doped Phosphors for Laser-Lighting Applications Matteo Buffolo 1,* ID , Carlo De Santi 1 ID , Marco Albertini 2, Donatella Carbonera 2, Gian Andrea Rizzi 2 ID , Gaetano Granozzi 2 ID , Gaudenzio Meneghesso 1 ID , Enrico Zanoni and Matteo Meneghini 1 Matteo Buffolo 1,* ID , Carlo De Santi 1 ID , Marco Albertini 2, Donatella Carbonera 2, Gian Andrea Rizzi 2 ID , Gaetano Granozzi 2 ID , Gaudenzio Meneghesso 1 ID , Enrico Zanoni 1 and Matteo Meneghini 1 1 Department of Information Engineering, University of Padova, via Gradenigo 6/b, 35131 Padova, Italy; carlo.desanti@dei.unipd.it (C.D.S.); gauss@dei.unipd.it (G.M.); zanoni@dei.unipd.it (E.Z.); matteo.meneghini@dei.unipd.it (M.M.) 1 Department of Information Engineering, University of Padova, via Gradenigo 6/b, 35131 Padova, Italy carlo.desanti@dei.unipd.it (C.D.S.); gauss@dei.unipd.it (G.M.); zanoni@dei.unipd.it (E.Z.); matteo.meneghini@dei.unipd.it (M.M.) 1 Department of Information Engineering, University of Padova, via Gradenigo 6/b, 35131 Padova, Italy; carlo.desanti@dei.unipd.it (C.D.S.); gauss@dei.unipd.it (G.M.); zanoni@dei.unipd.it (E.Z.); matteo.meneghini@dei.unipd.it (M.M.) g p 2 Department of Chemical Sciences, University of Padova, via Marzolo 1, 35131 Padova, Italy albertini.marco.87@gmail.com (M.A.); donatella.carbonera@unipd.it (D.C.); i d i i@ i d it (G A R ) t i@ i d it (G G ) 2 Department of Chemical Sciences, University of Padova, via Marzolo 1, 35131 Padova, Italy; albertini.marco.87@gmail.com (M.A.); donatella.carbonera@unipd.it (D.C.); gianandrea.rizzi@unipd.it (G.A.R.); gaetano.granozzi@unipd.it (G.G.) * Correspondence: matteo.buffolo@dei.unipd.it; Tel.: +39-049-827-7625 gianandrea.rizzi@unipd.it (G.A.R.); gaetano.granozzi@unipd.it (G.G.) g p g g p * Correspondence: matteo.buffolo@dei.unipd.it; Tel.: +39-049-827-7625 * Correspondence: matteo.buffolo@dei.unipd.it; Tel.: +39-049-827-7625 1. Introduction The generation of white light by means of phosphor-converted Light-Emitting Diodes (LEDs) based on a blue-emitting Gallium Nitride chip is the common approach adopted by modern Solid-State Lighting (SSL) solutions to achieve reliable and efficient light sources for a wide range of general lighting applications. However, alternative SSL solutions based on laser diodes instead of LEDs are starting to emerge. Compared to LEDs, laser diodes offer two main advantages: an increased efficiency at high driving current densities, i.e., for high optical power single source operation, and a simplified and more effective design of the optical system. The first point is related to a reduced effect of the Materials 2018, 11, 1552; doi:10.3390/ma11091552 www.mdpi.com/journal/materials 2 of 15 Materials 2018, 11, 1552 so-called efficiency droop phenomenon in Laser Diodes (LDs). The term efficiency droop describes the set of physical mechanisms responsible for the decrease in internal quantum efficiency of the device as the injected current increases. This process, typical for LEDs, is usually ascribed to Auger recombination [1] or carrier spill-over [2]. On the other hand, in LDs operated above threshold the emission of coherent light is associated with the stimulated recombination of carriers: compared to the spontaneous emission process responsible for photon emission in LEDs, this is a much faster process, less affected by the formerly cited loss mechanisms. y y Efficiency droop ultimately limits the amount of optical power attainable from an LED for a given chip area, meaning that if high-brightness high-efficiency operation is to be achieved, the area of the semiconductor chip needs to be increased. However, not only this approach would increase the manufacturing costs, but it would also worsen the optical performance of the lighting system in terms of light collimation. In an optical system, the product of a source emitting area and the solid angle of the emitted beam, called etendue, does not decrease if the optical power is conserved. This means that the collimation of a (relatively) large area source (up to 3–4 mm2 for a modern high-power LED) featuring a large-aperture Lambertian emission pattern, would require an increase in the dimensions of the optical elements, thus increasing the complexity and the cost of the final illuminator. 1. Introduction On the other hand, laser diodes feature a very small emitting area, in the orders of tens of square microns, which allows for the design of cheaper low-divergence LD-based light sources, such as the ones employed in digital laser projectors. Laser-Activated Remote Phosphors (LARP) lighting systems rely on two main white-light generation approaches. These are either based on the partial conversion of blue light (450 nm–460 nm) through a yellow phosphor blend, or on the total phosphor conversion of NUV light (380 nm–410 nm) into blue light, which is then partially converted to longer wavelengths to attain the desired chromaticity point. In principle, assuming phosphors and LDs with comparable efficiencies, the first approach shows an intrinsic advantage due to the reduced energy loss in the phosphor conversion process (about 12.5%, considering excitation wavelengths of 400 nm and 450 nm). However, for high-power operation, the efficiency of the light source at high current densities is the dominant factor in determining the global efficiency of the LARP system. With regard to LEDs, this translates into a clear supremacy of NUV sources over conventional blue emitters [3], determined by the more prominent efficiency droop of longer wavelength devices at high injection currents. However, for LDs operating in stimulated emission regime the intrinsic inefficiency of blue emitters is less pronounced, and the choice of a specific lighting approach becomes more bound to the technological limitations of state-of-the-art devices and to the optical design of the illuminator rather than to the physical principles ruling the emission processes. This work focuses on the reliability of Eu-doped halophosphate blue-emitting phosphors for NUV (405 nm) semiconductor laser excitation. Devices featuring such emission wavelength have already proven their reliability and the capability for continuous operation at output power in excess of 7 W [4]. The high light intensity attainable by those devices requires phosphor morphologies with high thermal conductivity, to deal with the self-heating due to Stokes loss and to the non-unitary quantum efficiency, as well as an elevated chemical stability under high optical intensity excitation and high-temperature environment. Under such operating conditions several degradation processes can limit the lifetime of Eu-doped phosphors. 1. Introduction High-temperature treatment in an oxygen-rich environment can severely reduce the optical efficiency of the material by inducing the oxidation of the Eu2+ centers [5–8], of the host lattice [8] or of the cations present in correspondence of the surface of the host material [9], as well as the migration of the optically active centers towards different crystallographic sites [10]. On the other hand, the exposure to high-intensity and high-energy UV light can induce the formation of traps in the host lattice [11–14], the photo-ionization of the Eu2+ ions [13,15,16] or their migration towards the cation layer of the host lattice [17]. The operating temperature, the external environment, as well as the wavelength and the intensity of the optical excitation are all important factors that contribute in determining the dominant degradation process for a specific family of phosphorescent 3 of 15 Materials 2018, 11, 1552 materials. Therefore, aim of this work is to analyze the variation in the luminescence and in the chemical and morphological properties of Eu-doped halophosphate blue-emitting phosphors under different operating regimes. To pinpoint the root causes of degradation, and to determine the optical excitation bounds for safe Continuous Wave (CW) operation of the phosphors, several thermal and optical accelerated stress tests were performed. The experimental results showed that rapid and non-recoverable degradation of the PL properties of the material occurs once a specific excitation threshold is reached. This kind of degradation could be ascribed to the thermally and optically induced ionization of the Eu2+ centers into Eu3+ ions. The details on sample preparation and characterization, and regarding the investigation on the aforementioned degradation process are reported in the following paragraphs. 2.1. Material under Analysis The phosphorescent material under investigation is a commercial blue-emitting pigment developed to attain a peak emission wavelength of 448 nm (blue), whereas the excitation range, tuned for UV light sources, ranges from 200 nm to 400 nm, with characteristic peak excitation wavelengths at 254 nm and 365 nm (Figure 1a). The material belongs to the family of Eu2+-activated alkaline-earth halophosphates, usually employed in the past as luminescence materials for fluorescence lamps, and more recently for SSL [18]. The basic chemical composition of this material is given by Sr5 (PO 4)3Cl : Eu2+ (1) (1) where, in principle, other alkaline-earth metal ions such as Ca or Ba can be employed in conjunction with Sr to tune, within specific limits, both the emission and excitation spectra, as well as to optimize the conversion efficiency of the material and its reliability. where, in principle, other alkaline-earth metal ions such as Ca or Ba can be employed in conjunction with Sr to tune, within specific limits, both the emission and excitation spectra, as well as to optimize the conversion efficiency of the material and its reliability. Figure 1. (a) Emission and excitation spectra of the phosphor under investigation, as provided by the manufacturer. (b) Height profile map (top) and back-scattered electrons Environmental Scanning Electron Microscopy (ESEM) image (bottom) of a portion of the surface of the deposited material. Figure 1. (a) Emission and excitation spectra of the phosphor under investigation, as provided by the manufacturer. (b) Height profile map (top) and back-scattered electrons Environmental Scanning Electron Microscopy (ESEM) image (bottom) of a portion of the surface of the deposited material. As to the microstructure, the phosphorescent material is a white powder, whose typical grain size ranges from 5 µm to 40 µm, with an average diameter of about 16 µm. For experimental purposes, the powder was deposited onto a thermally conductive sapphire substrate (1/2 inch in diameter) by a low-rpm spin-coating technique. To this aim, the powder was mixed in a 50:50 ratio with benzyl alcohol, employed as a carrier fluid. A fixed amount of mixture was then spin-coated on top of the substrate. Finally, the phosphor-covered substrate was placed inside a thermal chamber at 250 ◦C for 7 min, to let the solvent evaporate. 2.1. Material under Analysis After the deposition procedure, the phosphor powder is spread in a 4 of 15 Materials 2018, 11, 1552 solid phase across the surface of the substrate: since no foreign materials are present in the sample after the evaporation of the alcohol, this procedure ensures that only the luminescent material is deposited and characterized. Despite the absence of an encapsulant may increase the risk of contamination and degradation by external agents (moisture, oxygen, dust, etc.), the possibility of analyzing only the bare material offers far more advantages. The morphological quality of the deposition was evaluated by means of a profilometer with scanning red laser interferometer (model MSA-500 from Polytec, Waldbronn, Germany). As highlighted by Figure 1b, the variation in the height of the deposition, which shows a peak-to-peak distance of 70 µm with a variance of 5.3 µm, is compatible with the dimensions of the phosphor grains: this suggests that a very good level of deposition quality could be achieved with the adopted technique. That conclusion was further demonstrated by the Environmental Scanning Electron Microscopy (ESEM) (model Quanta 200 from FEI, Hillsboro, OR, USA)) images taken on an untreated sample, here reported at the bottom of Figure 1b: the detected variations are mostly related to the different particles dimensions rather than to a non-uniform deposition. With regard to its optical performance, the material exhibits good thermal quenching behavior at low-intensity CW 375 nm excitation, showing only a moderate 4% decrease when increasing the sample temperature from 30 ◦C to 200 ◦C (Figure 2): such behavior was found to be comparable to the reported thermal quenching behavior of blue-emitting phosphors belonging to the same family [19,20]. At excitation intensity higher than 5.5 mW/mm2, a higher drop in PL efficiency is observed, possibly due to the increased self-heating of the material. Figure 2. PL emission of the blue-emitting phosphor, normalized to the respective value at 30 ◦C for each excitation intensity, as a function of the substrate temperature. The optical source employed for this measurement is a solid-state 375 nm laser (model LBX-375, Oxxius, Lannion, France). Figure 2. PL emission of the blue-emitting phosphor, normalized to the respective value at 30 ◦C for each excitation intensity, as a function of the substrate temperature. The optical source employed for this measurement is a solid-state 375 nm laser (model LBX-375, Oxxius, Lannion, France). 2.2. Experimental Details To perform optical stress and characterization of the deposited material, a custom setup for Photo-Luminescence (PL) measurement was designed (Figure 3). A thermo-controlled high-power 405 nm LD, capable of generating more than 2 W at a drive current of 1.3 A, was employed as light source. The LD was operated in constant optical power mode by maintaining constant the reading of a monitoring photodiode (PD), on which part of the emitted light was redirected by means of a beam-sampler (model BSF10-A from Thorlabs, Newton, NJ, USA). The main collimated light beam exiting from the beam-sampler was then reflected with an angled 45◦mirror, and focused onto the horizontally lying sample with a suitable focusing lens (from Thorlabs). 5 of 15 Materials 2018, 11, 1552 Figure 3. Experimental setup for optical stress and characterization under 405 nm LD excitation: top (a) and side (b) views. Figure 3. Experimental setup for optical stress and characterization under 405 nm LD excitation: top (a) and side (b) views. In order to obtain a specific optical excitation density, both the power and the spatial distribution of the excitation beam must be measured. Regarding the former, a complete optical calibration of the setup was carried out by mapping the reading of the feedback photodiode with the measurements of a factory-calibrated power-meter. The extension of the excitation spot was measured by evaluating with a Dino-Lite digital microscope (Anmo Electronics Corporation, Taipei, Taiwan) the area (at Full Width Half Maximum) of the emission spot with the LD driven above threshold. Finally, the two measured values were employed to compute the excitation density, in W/mm2, of the light beam. The surface chemical composition of the samples was investigated by XPS using a custom equipment working at a base pressure of 10−10 mbar and adopting an EA 125 Omicron electron analyzer (Scienta Omicron, Taunusstein, Germany) with a five channeltron detector. The XPS data were collected at room temperature using the Al Kα line (hv = 1486.6 eV) of a non-monochromatized dual-anode DAR400 X-ray source. High resolution spectra were acquired using 0.1 eV energy steps, and 20 eV pass energy. The multi-peak analysis of Eu 3d photo-emission lines was performed by means of Voigt function and subtracting a Tougaard background [21]. The binding energy (BE) scale was calibrated with respect to the C1s signal due to adventitious carbon contamination on sample surfaces, assuming a binding energy of 285.0 eV. 2.2. Experimental Details All samples presented a strong charging effect (the material is not conducting) of about 30 eV. Electron spin properties of the Eu centers were investigated by electron paramagnetic resonance (EPR) spectroscopy. All the measurements were performed on an X-Band Bruker Elexsys E580 spectrometer equipped with an ER4116DM dual mode resonator (both from Bruker Corporation, Billerica, MA, USA) operated in its perpendicular mode (ν = 9.815 GHz). EPR spectra were recorded at room temperature applying a 10,000 G wide magnetic field sweep centered at 5050 G; a 100 kHz modulating field of 3 G amplitude was applied to achieve proper phase sensitive detection; microwave power was set to 4.697 mW; 8192 data points per spectra were collected, resulting in a 335.5 s sweep time. 3.1. Effects of Thermal Stress Due to the Stokes shift, and to the non-unitary quantum yield, during high optical intensity operation the luminescent material can reach very high temperatures that can negatively affect both the optical performance of the material, by lowering its PL efficiency, and its long-term reliability, by inducing material degradation through temperature-activated degradation processes [5,7]. 6 of 15 Materials 2018, 11, 1552 Even though we previously demonstrated that this kind of phosphorescent material starts becoming limited by thermal quenching for temperatures higher than 200 ◦C (Figure 2), a comprehensive analysis on the effects of thermal aging is due to identify the limiting operating conditions in LARP luminaires with reduced thermal management capabilities. To this aim, a series of nominally identical phosphor samples were submitted to extended thermal cycles in temperature-controlled climate chambers. The characterization of the samples, performed at regular intervals, was carried out by means of low-intensity transmission PL measurements. The results of the long-term aging experiments at constant temperature are summarized in Figure 4. Figure 4. Trend of the PL spectra, integrated from 450 nm to 460 nm, measured during (a) moderate and (b) high-temperature treatment in air. Figure 4. Trend of the PL spectra, integrated from 450 nm to 460 nm, measured during (a) moderate and (b) high-temperature treatment in air. The degradation kinetics show no tangible worsening of the PL emission for stress temperatures lower than 170 ◦C (Figure 4a). By contrast, the material under investigation exhibited a noticeable increase in luminescence. This process is possibly caused by the annealing of the material and did not induce significant changes in the spectral shape of the emitted light, and its time constant was found to be not thermally activated. On the other hand, the degradation kinetics at high (≥450 ◦C) stress temperature, reported in Figure 4b, show remarkable PL signal degradation, even during the first 50–100 h of stress. In this case, the degradation process was found to be thermally activated, with an activation energy for the Time-To-Failure at 75% (TTF75%), the time required by the sample to lose 25% of its original PL signal strength, around 1.6 eV. As will be shown in the following paragraphs, this behavior may be explained by the thermally induced ionization of Eu2+ centers due to high-temperature baking in air environment [8], or as a consequence of optical stress under high-intensity radiation [16]. 3.2. Stress Under Optical Excitation As described previously, for high optical power density stresses a 405 nm high-power laser diode was employed as optical source. Due to the easy availability and the high efficiency of modern 405 nm solid-state sources, this excitation condition represents the sweet spot from an engineering point of view [3]. However, the reduced conversion efficiency of the phosphorescent material at this lower wavelength may pose various reliability issues, especially related to the increased self-heating of the material. To investigate the degradation processes induced by near-UV optical excitation, we started our analysis by submitting a phosphor sample to a 140 h long stress at 3.2 W/mm2, with an ambient temperature of 25 ◦C. As shown in Figure 5a, the prolonged exposure to high-intensity radiation induced a complete extinction of the PL emission in correspondence of the excitation spot. Interestingly, the non-emissive spot could only be observed by direct excitation of the phosphor surface, whereas upward illumination 7 of 15 Materials 2018, 11, 1552 induced a uniform photo-emission. Considering that no complete absorption of the excitation radiation was taking place, and thus the upper portion of the material could still be partially pumped from the back, this behavior may suggest that only the upper layers of phosphors were affected by degradation. In addition to that, a reddish PL signal, absent in untreated areas of the sample, could be detected by low-intensity (0.21 W/mm2) 405 nm laser excitation of the stressed spot (Figure 5a). Figure 5. Stress under 405 nm excitation at 3.2 W/mm2, TAMB = 25 ◦C. (a) Image of the photo-luminescence from the first excitation spot after 140 h of stress. On the left: emission from top and bottom surfaces with 405 nm LED excitation. On the right: comparison of the PL emission from the stress spot and from a portion of untreated surface (red markings highlight the approximate dimension of the red-emissive spot). (b) Trend during optical stress on a second spot of the PL spectra measured at very-low intensity (Iexct ≈0.21 W/mm2). Inset graph shows the trend of the red luminescence over stress time. Figure 5. Stress under 405 nm excitation at 3.2 W/mm2, TAMB = 25 ◦C. (a) Image of the photo-luminescence from the first excitation spot after 140 h of stress. On the left: emission from top and bottom surfaces with 405 nm LED excitation. 3.2. Stress Under Optical Excitation On the right: comparison of the PL emission from the stress spot and from a portion of untreated surface (red markings highlight the approximate dimension of the red-emissive spot). (b) Trend during optical stress on a second spot of the PL spectra measured at very-low intensity (Iexct ≈0.21 W/mm2). Inset graph shows the trend of the red luminescence over stress time. To investigate step-by-step the spectral changes in the long wavelength region and to acquire further details on the kinetics of this degradation phenomenon, the same stress experiment was repeated on a second spot of the same phosphor sample, while recording the low-intensity PL spectrum at regular stress intervals. As reported in Figure 5b, the intensity of the red luminescence peak was found to gradually increase with stress time in an almost linear fashion. Interestingly, the spurious red luminescence was found to have an emission linewidth ranging from about 609 nm to 624 nm, compatible with the characteristic peak emission wavelength of Eu3+-related optically active centers [22]. The extension of the degraded red-emitting area was then evaluated by means of spatially and spectrally resolved PL measurements carried out with 405 nm light excitation (Figure 6). By means of a scientific Electron Multiplying Charge-Coupled Device (EMCCD) camera, model Luca S DL-658M-TIL by Andor (Abington on Thames, UK), paired with a VariSpec tunable liquid crystal filter manufactured by CRI (Waltham, MA, USA), we were able to selectively map the spatial emission from the surface of material at determined wavelengths within the 400–720 nm range. The comparison between the monochromatic emission maps at 452 nm and 614 nm, respectively the peak emission wavelengths of the phosphors and of the red luminescence, showed no peculiar differences. In these hot-cold color-coded images, both measurements showed complete annihilation of the PL in correspondence of the center of the emission spot. Assuming from optical images that the red emission originates from the center of stress spot, this may indicate that under the low-intensity LED-driven excitation employed for the measurement, the process responsible for the observed spurious emission is relatively weak. This reduced emission rate may either be due to a low concentration of the chemical species that assist the spurious red emission process, or to an inherently low efficiency of the process itself, 8 of 15 Materials 2018, 11, 1552 possibly related to a non-optimized chemical configuration of the host lattice for this lower-energy optical transition. 3.3. Physico-Chemical Analysis of the Phosphors Samples To identify the physical mechanisms responsible for the optically induced degradation of the material, we performed further physico-chemical analyses on the treated phosphors samples. The drop in the PL signal could be related to an increase in a characteristic red emission around 613 nm, possibly ascribed to the photo-and/or thermally induced ionization of the Eu2+ centers into Eu3+ ions. Therefore, we further investigated this hypothesis by analyzing several samples of thermally and optically stressed phosphors by means of various material analysis techniques, including high-sensitivity optical spectroscopy, EPR and XPS. 3.2. Stress Under Optical Excitation Figure 6. Analysis of the degraded phosphor surface after stress under 405 nm, 3.2 W/mm2 excitation at TAMB = 25 ◦C. Spatially and spectrally resolved PL map of the stressed spot measured at low-intensity 405 nm laser excitation with selective filtering at 452 nm (a) and 614 nm (b); back-scattered electrons ESEM imaging of the degraded surface (c). Figure 6. Analysis of the degraded phosphor surface after stress under 405 nm, 3.2 W/mm2 excitation at TAMB = 25 ◦C. Spatially and spectrally resolved PL map of the stressed spot measured at low-intensity 405 nm laser excitation with selective filtering at 452 nm (a) and 614 nm (b); back-scattered electrons ESEM imaging of the degraded surface (c). Finally, back-scattered electrons ESEM imaging of the degraded excitation spot revealed stress-induced changes both in the morphology and in the chemical composition of the sample area subjected to high optical excitation (Figure 6). This latter consideration supports the hypothesis that the localized annihilation of the PL signal observed after moderate optical stress may be related to a variation in the chemical properties of the material. Finally, back-scattered electrons ESEM imaging of the degraded excitation spot revealed stress-induced changes both in the morphology and in the chemical composition of the sample area subjected to high optical excitation (Figure 6). This latter consideration supports the hypothesis that the localized annihilation of the PL signal observed after moderate optical stress may be related to a variation in the chemical properties of the material. 3.3.1. High-Sensitivity Optical Spectroscopy By means of a high-sensitivity spectrometer, model CAS 140CT by Instrument Systems (Munich, Germany), the emission spectrum of the luminescent material was further investigated. The results of this analysis, reported in Figure 7, show that because of the thermal and/or optical stress, characteristic spectral lines become visible or disappear, depending on the wavelength region. In region I, UV treatment and annealing at 650 ◦C in air induced the increase in the emission centered around 615 nm, which is commonly ascribed to the 5D0 →7F2 Eu3+ transition [6,22]. Similarly, in region II the same stress procedure generated a spurious emission peak around 695 nm and 705 nm, which can be related to the Eu3+ 5D0 →7F4 transitions [22,23]. On the other hand, the spectral line highlighted in region III around 800 nm, detected also on the untreated sample, was not affected by either thermal of UV stress. Considering that this emission corresponds to the 5D0 →7F6 transition of the Eu3+ compounds, we can suppose that this characteristic transition is related to the few Eu3+ centers that are generated as a consequence of the manufacturing process, and that the adopted stress conditions did not significantly contribute in increasing the concentration of the associated emissive sites. Finally, 9 of 15 Materials 2018, 11, 1552 despite the luminescence peak appearing in region IV around 870 nm could not be associated with any of the Eu2+ or Eu3+ characteristic spectral lines, its reduced intensity after high temperature or UV stress suggests a possible correlation with the former of the two species. These experimental results support the hypothesis that high levels of optical excitation, as well as high-temperature stress, can induce degradation of the material due to the ionization of the optically active Eu2+ centers into Eu3+ ions. Figure 7. Normalized low excitation intensity PL spectra in the long wavelength region recorded by means of a high-sensitivity spectrometer. The UV treatment referred to in the plot legend corresponded to the 405 nm, 3.2 W/mm2, TAMB = 25 ◦C stress described in Figure 5b. Figure 7. Normalized low excitation intensity PL spectra in the long wavelength region recorded by means of a high-sensitivity spectrometer. The UV treatment referred to in the plot legend corresponded to the 405 nm, 3.2 W/mm2, TAMB = 25 ◦C stress described in Figure 5b. Figure 7. 3.3.1. High-Sensitivity Optical Spectroscopy Normalized low excitation intensity PL spectra in the long wavelength region recorded by means of a high-sensitivity spectrometer. The UV treatment referred to in the plot legend corresponded to the 405 nm, 3.2 W/mm2, TAMB = 25 ◦C stress described in Figure 5b. 3.3.2. Results of EPR Analysis The hypothesis formulated in the previous paragraph was also supported by EPR spectroscopy on thermally treated samples. EPR spectroscopy is a material analysis technique aimed at investigating atoms, ions or molecules with unpaired valence electrons. This technique exploits the effect of an external magnetic field to separate the spin levels and of a microwave electromagnetic field to promote spin transitions. The resonant frequency depends on the surrounding environment of the paramagnetic center and give information in term of concentration, bonds, active nuclei and coordination sphere. Since in the material only the optically active Eu2+ centers, and not the Eu3+ species resulting from its degradation, feature an electronic structure with unpaired valence electrons in the 4f7 orbital, this technique proved to be suitable for our investigation. To ensure repeatability of the measurements, the material was stressed and measured in situ inside the EPR tube, a quartz-made tube-shaped holder that is inserted inside the resonant EPR cavity, suitable for probing solid and liquid-phase samples. Moreover, reference and post-stress measurements were both performed. To this aim, the degradation process was accelerated by performing 1 h long thermal stresses at the temperatures of 650 ◦C, 700 ◦C and 750 ◦C. Fluctuations of the EPR signal, due to instrumental sources, were considered by normalizing the spectra to the four sharp signals of a reference (solid) Cr3+ standard, introduced inside the tube prior to each measurement and then removed. The results of the EPR analyses are reported in Figure 8. As a consequence, to the thermal stress, the EPR signals within the 0–3000 Gauss region arising from the Eu2+ centers experienced a decrease in intensity, showing greater relative decrease for higher stress temperatures. This indicates that very high temperature short-term stress can induce a significant decrease in the concentration of the Eu2+ species within the phosphor. This thermally induced decrease was also found to be compatible with previous reports on the degradation of Eu-doped phosphors submitted to high-temperature thermal aging [7]. 10 of 15 Materials 2018, 11, 1552 Figure 8. Room-temperature EPR signal of Eu2+ centers before and after 1 h thermal stress at 650 ◦C to 750 ◦C. Figure 8. Room-temperature EPR signal of Eu2+ centers before and after 1 h thermal stress at 650 ◦C to 750 ◦C. Figure 8. Room-temperature EPR signal of Eu2+ centers before and after 1 h thermal stress at 650 ◦C to 750 ◦C. 3.3.3. Results of XPS Analysis We have previously showed how moderate optical stress only degrades the surface layers of the phosphorescent material. To further understand the effects of this localized degradation on the chemical composition of the material, we characterized both treated and untreated samples by means of XPS, a surface analysis technique capable of quantitatively evaluate the chemical composition and the electronic state of the elements within the first nanometers of the material under investigation. The results are reported in Figure 9. Figure 9. XPS data of the Eu 3d5/2 core level of untreated and thermally treated Eu-doped phosphors. The spectra have been plotted after a Tougaard background subtraction [21], a normalization between their minimum and maximum values, and shifted vertically to ease readability. Based on the adopted measuring conditions, the average information depth is of few nm. Figure 9. XPS data of the Eu 3d5/2 core level of untreated and thermally treated Eu-doped phosphors. The spectra have been plotted after a Tougaard background subtraction [21], a normalization between their minimum and maximum values, and shifted vertically to ease readability. Based on the adopted measuring conditions, the average information depth is of few nm. Figure 9. XPS data of the Eu 3d5/2 core level of untreated and thermally treated Eu-doped phosphors. The spectra have been plotted after a Tougaard background subtraction [21], a normalization between their minimum and maximum values, and shifted vertically to ease readability. Based on the adopted measuring conditions, the average information depth is of few nm. In XPS spectra, the signal intensity, i.e., the number of collected photo-emitted electrons, is proportional to the amount of a specific element inside the probing volume, whereas peaks in correspondence of specific BEs identify the electron configuration of the atoms in the material under 11 of 15 Materials 2018, 11, 1552 investigation. With XPS being a quantitative analysis technique, typically to the parts per thousand range, the experimental data suggest that thermal stress induced a variation in the amount of Eu centers near the surface of the material. In particular, looking at the Eu 3d core levels, two different valence states (+2 and +3) of Eu ions can be observed. Each set exhibits simple spin-orbit doublet peaks, which split off 30 eV from each other. 4. Operating Limits under Optical Excitation In the previous paragraph we showed that Eu-doped blue-emitting phosphor subjected to moderate levels of optical stress can degrade due to irreversible ionization of the of Eu2+ centers. From an engineering point of view, it is important to identify the limits for continuous excitation of the material, above which consistent PL efficiency decay or material degradation occurs. To this aim, an optical step-stress experiment was carried out on a second-generation Eu-doped halophosphate phosphor, sharing with the previously investigated material the (general) chemical composition and the behavior under optical excitation. A specific surface spot was submitted to 12 s long stress steps under increasing 405 nm optical excitation levels, from 0.5 W/mm2 to 3.5 W/mm2. A reference measurement at 0.5 W/mm2 was taken before and after each stress step to discriminate between thermal quenching-induced PL decay and non-recoverable phosphor degradation. A cool-down period of 300 s was employed before low-intensity characterization to let the sapphire substrate and the phosphors dissipate the heat accumulated during the stress. Finally, with the aim of attaining high temporal resolution during the acquisition of the PL signal, we employed as light detector an amplified photodiode (model PDA36A-EC from Thorlabs), carefully shielded from the 405 nm laser light reflected from the sample and connected to an oscilloscope. g p p The experimental results, reported in Figure 10, show that after an initial PL increase related to the turn-on transient of the excitation source, a sudden PL decay occurred after about 400 ms of stress at 2.25 W/mm2. Above this excitation intensity, the steady-state PL signal, i.e., the PL at the end of the 12 s stress step, drops to a fixed value corresponding roughly to the emission during 0.5 W/mm2 stress, whereas the reference PL measurements starts decreasing in amplitude, meaning that stress above 2.25 W/mm2 induced permanent degradation to the phosphor (Figure 10b). In particular, we can see how above this excitation intensity the delay between the beginning of the stress and the rapid PL decay decreases with increasing light intensity. This behavior can be explained by considering that above a certain (power-dissipation) threshold, the self-heating of the material reduces the rate of optical emission, increasing even more the quantity of incident energy converted into heat. This positive feedback rapidly increases the temperature of the stress spot, thus annihilating the emission from this area and inducing permanent degradation to the phosphor particles located nearby. 3.3.3. Results of XPS Analysis In Figure 9 we report only the lower BE Eu 3d5/2 components: the peaks located at lower BE can be assigned to Eu2+ and the higher BE one to Eu3+. In particular, thermal aging at 500 ◦C showed a comparable increase of both the Eu2+ and Eu3+ XPS peaks, suggesting a thermally driven diffusion of the two chemical species towards the surface. This phenomenon is quite common and is referred as thermal driven surface segregation. On the other hand, stress at 650 ◦C also induced an increase in the relative amount of Eu3+ with respect to the concentration of Eu2+: this observation is compatible with the oxidation of Eu2+, assisted by high-temperature and by the oxygen-rich environment near the surface of the sample [6]. A similar relative increase in Eu3+ was previously observed in literature by means of XPS on both thermally and UV-treated phosphors, as reported in [7,24]. These results confirm once again the role of oxidation of the Eu2+ centers in the degradation of the material under investigation. 4. Operating Limits under Optical Excitation When this critical stress intensity is reached, the PL becomes more dependent on the phosphorescent material surrounding the excitation spot rather than on the severely heated (and partially degraded) excitation spot itself, as testified by the constant value of the PL emission at the end of the stress for excitation intensities greater than the threshold value (Figure 10b). Materials 2018, 11, 1552 Figure 10. Optical step-stress under 405 nm excitation at TAMB = 25 ◦C. (a) Trends of the PL signal under increasing values of optical excitation. (b) Variation of the reference (low-intensity) and peak PL signals in function of the stress excitation intensity. Figure 10. Optical step-stress under 405 nm excitation at TAMB = 25 ◦C. (a) Trends of the PL signal under increasing values of optical excitation. (b) Variation of the reference (low-intensity) and peak PL signals in function of the stress excitation intensity. To prove that the excitation threshold ITH previously found represents a bound for safe CW excitation of the phosphor, we carried out two long-term stresses under optical excitation levels of 1.5 W/mm2 and 3 W/mm2, respectively below and above ITH. The PL transient was registered from the very beginning of the experiments by means of the same setup describe above. Moreover, to discriminate between thermal quenching-induced PL decay and non-recoverable phosphor degradation, a reference PL measure at a safe 0.5 W/mm2 excitation level was performed before and after the stress. The results of the experiments are reported in Figure 11. Figure 11. Stress under 405 nm excitation at 1.5 and 3 W/mm2, TAMB = 25 ◦C. (a) PL trends during stress. (b) Reference PL signal measured at 0.5 W/mm2 before and after each stress cycle. Figure 11. Stress under 405 nm excitation at 1.5 and 3 W/mm2, TAMB = 25 ◦C. (a) PL trends during stress. (b) Reference PL signal measured at 0.5 W/mm2 before and after each stress cycle. Regarding the stress under 1.5 W/mm2 optical excitation, the waveform of the PL signal acquired during stress and reported in Figure 11a shows a 10.5% difference between the peak and the steady-state value, whereas only a <0.8% decay in the reference PL measurement was registered (Figure 11b). 4. Operating Limits under Optical Excitation This suggests that the PL decay experienced by the sample is mostly related to a thermal quenching phenomenon and that 1.5 W/mm2 represents a safe pumping intensity for the given deposition conditions and the thermal management capabilities of the system. On the other hand, stress at 3 W/mm2 induced a 71.8% decay in the steady-state PL with respect to peak value (Figure 11a), as well as a non-recoverable 54.6% decrease in the reference PL signal (Figure 11b). Interestingly, the PL signal under high level of excitation shows a partial time-dependent recovery beginning after 1.5 h of stress. Materials 2018, 11, 1552 13 of 15 If we consider that thermally treated samples showed PL recovery during thermal treatment up to 300 ◦C, this behavior can be related to the thermally induced annealing of the material surrounding the excitation spot. By comparing the experimental results outlined in this section, we can conclude that stress under high levels of optical excitation (i) triggers a very fast degradation process, which induces most of the non-recoverable PL decay during the first second of stress. Additionally, (ii) a recoverable PL decay is also present, which can be ascribed to the thermal quenching experienced by the luminescence material; (iii) below the critical excitation intensity, long-term exposition to optical excitation does not trigger any further degradation process. The strong dependence of the onset of permanent PL decay on the excitation intensity also highlighted the major role of power dissipation, i.e., temperature, in the degradation process. From an engineering perspective, this means that while the maximum operating temperature of the material is an intrinsic characteristic of the phosphor, and thus can only be changed by improving either its composition or its manufacturing process, the excitation intensity threshold can be easily increased by improving the thermal management capabilities of the system. In particular, this goal can be achieved by lowering the thermal resistance from the phosphors grains to substrate, for example by incorporating the luminescent material in a highly thermally conductive encapsulant, or by increasing the thermal conductivity of the substrate, by making use of ceramic plates instead of the sapphire supports employed in this case of study. 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Kawaguchi, M.; Imafuji, O.; Nozaki, S.; Hagino, H.; Takigawa, S.; Katayama, T.; Tanaka, T. Optical-loss suppressed InGaN laser diodes using undoped thick waveguide structure. In Gallium Nitride Materials and Devices XI; Chyi, J.-I., Fujioka, H., Morkoç, H., Nanishi, Y., Schwarz, U.T., Shim, J.-I., Eds.; International Society for Optics and Photonics: San Francisco, CA, USA, 2016; Volume 9748, p. 974818. [CrossRef] pp g p g Devices XI; Chyi, J.-I., Fujioka, H., Morkoç, H., Nanishi, Y., Schwarz, U.T., Shim, J.-I., Eds.; International Society for Optics and Photonics: San Francisco, CA, USA, 2016; Volume 9748, p. 974818. [CrossRef] 5. Kim, Y.; Kang, S. Investigation of the degradation mechanisms in BaMgAl10O17: Eu2+ phosphor: On the influence of thermal process on operational durability. Appl. Phys. B 2010, 98, 429–434. [CrossRef] 6. Ma, M.; Zhu, D.; Zhao, C.; Han, T.; Cao, S.; Lei, Y.; Tu, M. Thermal degradation of BaAl2Si2O8: Eu2+ phosphor excited by near ultraviolet light. Chin. Sci. Bull. 2012, 57, 1728–1732. [CrossRef] 7. Lacanilao, A.; Wallez, G.; Mazerolles, L.; Dubot, P.; Binet, L.; Pavageau, B.; Servant, L.; Buissette, V.; Le Mercier, T. Structural analysis of thermal degradation and regeneration in blue phosphor BaMgAl10O17: Eu2+ based upon cation diffusion. Solid State Ion. 2013, 253, 32–38. [CrossRef] 8. Yeh, C.-W.; Chen, W.-T.; Liu, R.-S.; Hu, S.-F.; Sheu, H.-S.; Chen, J.-M.; Hintzen, H.T. 5. Conclusions In conclusion, with this work we investigated the robustness and the degradation mechanisms of blue-emitting Sr5 (PO 4)3Cl : Eu2+ phosphors for near-UV laser excitation. By means of a series of long-term aging and short-term stress experiments under optical excitation and/or high-temperature environment, the limits for continuous operation of the luminescent material were found. In particular, for temperatures equal to or greater than 450 ◦C the material exhibited a time-dependent drop in the PL, which was attributed to the thermally induced auto-ionization of the Eu2+ optically active centers. By means of different material characterization techniques, evidence of this degradation process were also found on samples stressed under a relatively low 3.2 W/mm2 optical excitation density. This indicated that the optically (and thermally) induced ionization of the Eu2+ species is the most critical degradation process for this family of phosphorescent material. In addition to that, short-term stress under 405 nm optical excitation revealed that a threshold excitation intensity for continuous pumping of Eu-doped halophosphates luminescent pigments exists. The threshold value was found to be in the 1.5 W/mm2 to 2 W/mm2 range for the given deposition condition: this excitation threshold may depend on the specific morphology of the area under analysis, as well as on the thickness of the deposited material and on the type of substrate employed. Above threshold, decay of the steady-state PL intensity and/or degradation of the material occur with respect to lower excitation intensity, which suggests that the material is being operated in a not optimal excitation regime. Author Contributions: All the authors have equally contributed to the development of this work. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. 14 of 15 14 of 15 Materials 2018, 11, 1552 References Kim, D.; Kim, S.-C.; Bae, J.-S.; Kim, S.; Kim, S.-J.; Park, J.-C. Eu2+-activated alkaline-earth halophosphates, M5(PO4)3X: Eu2+ (M = Ca, Sr, Ba; X = F, Cl, Br) for NUV-LEDs: Site-selective crystal field effect. Inorg. Chem. 2016, 55, 8359–8370. [CrossRef] [PubMed] 19. Zheng, J.; Cheng, Q.; Wu, S.; Guo, Z.; Zhuang, Y.; Lu, Y.; Li, Y.; Chen, C. An efficient blue-emitting Sr5(PO4)3Cl: Eu2+ phosphor for application in near-UV white light-emitting diodes. J. Mater. Chem. C 2015, 3, 11219–11227. [CrossRef] 20. Deressa, G.; Park, K.W.; Jeong, H.S.; Lim, S.G.; Kim, H.J.; Jeong, Y.S.; Kim, J.S. Spectral broadening of blue (Sr, Ba)5(PO4)3Cl: Eu2+ phosphors by changing Ba2+/Sr2+composition ratio for high color rendering index. J. Lumin. 2015, 161, 347–351. [CrossRef] 21. Seah, M.P. Background subtraction: I. General behaviour of Tougaard-style backgrounds in AES and XPS. Surf. Sci. 1999, 420, 285–294. [CrossRef] 15 of 15 Materials 2018, 11, 1552 22. Binnemans, K. Interpretation of europium(III) spectra. Coord. Chem. Rev. 2015, 295, 1–45. [CrossRef] 23. Liu, Q.; Yuan, Y.; Li, J.; Liu, J.; Hu, C.; Chen, M.; Lin, L.; Kou, H.; Shi, Y.; Liu, W.; et al. Preparation and properties of transparent Eu: YAG fluorescent ceramics with different doping concentrations. Ceram. Int. 2014, 40, 8539–8545. [CrossRef] 24. Zhang, J.; Zhou, M.; Liu, B.; Wen, Y.; Wang, Y. The ultraviolet irradiation degradation of fluorescent lamp used BaMgAl10O17: Eu2+, Mn2+ phosphor. J. Lumin. 2012, 132, 1949–1952. [CrossRef] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Building better brains: the pleiotropic function of neurotrophic factors in postnatal cerebellar development
Frontiers in molecular neuroscience
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OPEN ACCESS OPEN ACCESS EDITED BY Tetsushi Sadakata, Gunma University, Japan REVIEWED BY Ruben Deogracias, University of Salamanca, Spain Sonia Canterini, Sapienza University of Rome, Italy *CORRESPONDENCE Lilian Kisiswa liki@biomed.au.dk RECEIVED 07 March 2023 ACCEPTED 26 April 2023 PUBLISHED 12 May 2023 CITATION Boxy P, Nykjær A and Kisiswa L (2023) Building better brains: the pleiotropic function of neurotrophic factors in postnatal cerebellar development. Front. Mol. Neurosci. 16:1181397. doi: 10.3389/fnmol.2023.1181397 OPEN ACCESS EDITED BY Tetsushi Sadakata, Gunma University, Japan REVIEWED BY Ruben Deogracias, University of Salamanca, Spain Sonia Canterini, Sapienza University of Rome, Italy *CORRESPONDENCE Lilian Kisiswa liki@biomed.au.dk RECEIVED 07 March 2023 ACCEPTED 26 April 2023 PUBLISHED 12 May 2023 CITATION Boxy P, Nykjær A and Kisiswa L (2023) Building better brains: the pleiotropic function of neurotrophic factors in postnatal cerebellar development. Front. Mol. Neurosci. 16:1181397. doi: 10.3389/fnmol.2023.1181397 Pia Boxy 1,2,3, Anders Nykjær 1,2,3 and Lilian Kisiswa 1,2,3* 1 Department of Biomedicine, Aarhus University, Aarhus, Denmark, 2 Danish Research Institute of Translational Neuroscience (DANDRITE)–Nordic EMBL Partnership for Molecular Medicine, Aarhus University, Aarhus, Denmark, 3 The Danish National Research Foundation Center, PROMEMO, Aarhus University, Aarhus, Denmark The cerebellum is a multifunctional brain region that controls diverse motor and non-motor behaviors. As a result, impairments in the cerebellar architecture and circuitry lead to a vast array of neuropsychiatric and neurodevelopmental disorders. Neurotrophins and neurotrophic growth factors play essential roles in the development as well as maintenance of the central and peripheral nervous system which is crucial for normal brain function. Their timely expression throughout embryonic and postnatal stages is important for promoting growth and survival of both neurons and glial cells. During postnatal development, the cerebellum undergoes changes in its cellular organization, which is regulated by a variety of molecular factors, including neurotrophic factors. Studies have shown that these factors and their receptors promote proper formation of the cerebellar cytoarchitecture as well as maintenance of the cerebellar circuits. In this review, we will summarize what is known on the neurotrophic factors’ role in cerebellar postnatal development and how their dysregulation assists in developing various neurological disorders. Understanding the expression patterns and signaling mechanisms of these factors and their receptors is crucial for elucidating their function within the cerebellum and for developing therapeutic strategies for cerebellar-related disorders. Front. Mol. Neurosci. 16:1181397. doi: 10.3389/fnmol.2023.1181397 © 2023 Boxy, Nykjær and Kisiswa. OPEN ACCESS This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. cerebellum, neurotrophic factors, signaling/signaling pathways, development, developmental disorder TYPE  Review PUBLISHED  12 May 2023 DOI  10.3389/fnmol.2023.1181397 TYPE  Review PUBLISHED  12 May 2023 DOI  10.3389/fnmol.2023.1181397 TYPE  Review PUBLISHED  12 May 2023 DOI  10.3389/fnmol.2023.1181397 Frontiers in Molecular Neuroscience Introduction Granule cell progenitors (GCPs), on the one hand, form the temporary external granule layer (EGL) in which they actively proliferate and expand. Consequently, the immature GCPs exit the cell cycle, commence differentiation, after which they migrate radially until they reach their destination within the internal granule layer (IGL) and become mature cerebellar granule neurons (CGNs). During this process, there is both extension of the CGN axons, namely the parallel fibers, and growth of the CGN dendrites (Consalez et al., 2021). The inhibitory interneurons (IN), on the other hand, originate from a pool of progenitors located in the cerebellar white matter (WM) that give rise to a variety of glial cells, including astrocytes, oligodendrocytes, and Bergmann glia (BG), as well as several types of interneurons. These progenitors migrate through the WM into the cerebellar cortex and subsequently differentiate into mature glia and INs (Zhang and Goldman, 1996). Lastly, synaptogenesis and target innervation of the PC axons occurs followed by synaptic and dendritic pruning (Leto et al., 2016). In this review, we will summarize what is currently known about the spatiotemporal expression of the different neurotrophic factors and their receptors within the cerebellar system during postnatal development (Figure 2; Table 1). Additionally, we will outline the neurotrophic factors’ diverse cellular functionalities and some of the downstream signaling mechanisms that require neurotrophic expression and activity. Lastly, we will highlight how aberrations in both neurotrophic expression and function affect the cerebellar cytoarchitecture and what it implicates for several neurodevelopmental disorders (Table 2). Cerebellar development is a complex process that is regulated by a variety of signaling molecules and growth factors, both in a cell- autonomous and non-cell-autonomous manner. One category of growth factors that play a crucial role in cerebellar development and circuit formation is the neurotrophic factors (Figure 1). This includes the classical neurotrophins brain-derived neurotrophic factor (BNDF), nerve growth factor (NGF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4), that are widely and timely expressed in different regions of the central and peripheral nervous system. Neurotrophins are key players during nervous system development where they regulate neurogenesis, morphogenesis, synaptogenesis, and cell maintenance, as well as during adulthood, in processes of cellular survival or death and, synaptic plasticity (Huang and Reichardt, 2001). The neurotrophins occur as both their secreted precursor state and their cleaved mature form (Lee et  al., 2001). Introduction CNTF binds the CNTF receptor (CNTFR), ephrins bind to the Eph tyrosine kinase receptors, EGF binds to the EGF receptor (EGFR) or receptor homologs, GDNF binds predominantly GFRα1 in complex with the RET receptor, neuregulins bind the ErbB family of receptors, PGRN binds TNF receptors as well as the sortilin receptor, and lastly, TGF-β isoforms binds to the TGF-β type I, II, and III receptors (TβRI, TβRII, and TβRIII) (Cheifetz et al., 1988; Treanor et al., 1996; Chang et al., 1997; Doré et al., 1998; Wieduwilt and Moasser, 2008; Hu et al., 2010; Tang et al., 2011; Fantone et al., 2020). Binding of these growth factors to their respective receptors activates various signaling pathways including the p38 and JNK-MAPK, Jak–STAT, Ras-MAPK, and PI3K-Akt signaling pathways (Bonni et al., 1993; Oh et al., 1998; Wong and Guillaud, 2004; Murphy and Bielby-Clarke, 2008; Paratcha and Ledda, 2008; Wee and Wang, 2017; Fantone et al., 2020; Wang et al., 2022). implicated in numerous neuropsychiatric disorders, such as schizophrenia and bipolar disorder, and neurodevelopmental disorders, including attention deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) (Allen et al., 2004; Peter et al., 2016; Stoodley, 2016; Sathyanesan et al., 2019). This is mainly because cerebellar learning, complementing its role in motor control, also has a bottom-up influence on cognitive functions through extensive interconnections between the deep cerebellar nuclei and limbic brain structures (Snider et al., 1976; Carta et al., 2019; Frontera et al., 2020). Hence, if genetically predisposed, environmental perturbations throughout most of the cerebellar development may impair neuronal maturation and synapse formation, and lead to incorrect circuit wiring.h y p g The cerebellum is a highly conserved brain structure that has an extensive and elaborate development, which starts in humans at gestational week 7 (E13  in rodents) and ends around 12 months postnatal (P21 in rodents) (Larramendi and Victor, 1967; Altman, 1972; Sathyanesan et al., 2019). For this review we will focus on the postnatal development of the cerebellum since the embryonic development has been covered in plenum by other reviews (Leto et al., 2016; Sathyanesan et al., 2019; Amore et al., 2021). During early postnatal stages, the dendritic complexity of Purkinje cells (PCs) develops extensively in terms of both branching and arbor length, resulting in the thickening of the molecular layer (Leto et al., 2016). Frontiers in Molecular Neuroscience Introduction Consequently, the immature GCPs exit the cell cycle, commence differentiation, after which they migrate radially until they reach their destination within the internal granule layer (IGL) and become mature cerebellar granule neurons (CGNs). During this process, there is both extension of the CGN axons, namely the parallel fibers, and growth of the CGN dendrites (Consalez et al., 2021). The inhibitory interneurons (IN), on the other hand, originate from a pool of progenitors located in the cerebellar white matter (WM) that give rise to a variety of glial cells, including astrocytes, oligodendrocytes, and Bergmann glia (BG), as well as several types of interneurons. These progenitors migrate through the WM into the cerebellar cortex and subsequently differentiate into mature glia and INs (Zhang and Goldman, 1996). Lastly, synaptogenesis and target innervation of the PC axons occurs followed by synaptic and dendritic pruning (Leto et al., 2016). Cerebellar development is a complex process that is regulated by a variety of signaling molecules and growth factors, both in a cell- t d ll t O t f Trks initiates several signaling cascades, including the mitogen- activated protein kinase (MAPK) cascade, the phosphatidylinositol- 3-kinase (PI3K) cascade, and the protein kinase C (PKC)- phospholipase-Cγ (PLC-γ) cascade (Klesse and Parada, 1999; Reichardt, 2006). Proneurotrophins, however, preferentially interact with the low-affinity and nonselective p75 neurotrophin receptor (p75NTR) of the tumor necrosis factor (TNF) receptor superfamily in a complex with members of the sortilin receptor family to regulate cell death via the c-Jun N-terminal kinase (JNK) or caspase-3 pathway (Yoon et al., 1998; Friedman, 2000; Hempstead and Salzer, 2002; Nykjaer et al., 2004). NGF binds TrkA, BDNF and NT-4 bind TrkB, and NT-3 binds primarily TrkC (Chao and Hempstead, 1995). In addition to the prototypical neurotrophins, there are numerous growth factors that also play a major role in proper brain development (Figure 1). These include ciliary neurotrophic factor (CNTF), ephrins, epidermal growth factor (EGF), glial cell line-derived neurotrophic factor (GDNF), neuregulins, progranulin (PGRN) and transforming growth factor (TGF-β). By binding to their respective receptors, they provide survival, differentiation, migration, maturation, and circuit formation signals to the developing nervous system (Abe et al., 1991; Lärkfors et al., 1994; Mount et al., 1995; Ozaki et al., 2000; Rodger et al., 2012; Araujo et al., 2016; Uesaka et al., 2018). Introduction CNTF binds the CNTF receptor (CNTFR), ephrins bind to the Eph tyrosine kinase receptors, EGF binds to the EGF receptor (EGFR) or receptor homologs, GDNF binds predominantly GFRα1 in complex with the RET receptor, neuregulins bind the ErbB family of receptors, PGRN binds TNF receptors as well as the sortilin receptor, and lastly, TGF-β isoforms binds to the TGF-β type I, II, and III receptors (TβRI, TβRII, and TβRIII) (Cheifetz et al., 1988; Treanor et al., 1996; Chang et al., 1997; Doré et al., 1998; Wieduwilt and Moasser, 2008; Hu et al., 2010; Tang et al., 2011; Fantone et al., 2020). Binding of these growth factors to their respective receptors activates various signaling pathways including the p38 and JNK-MAPK, Jak–STAT, Ras-MAPK, and PI3K-Akt signaling pathways (Bonni et al., 1993; Oh et al., 1998; Wong and Guillaud, 2004; Murphy and Bielby-Clarke, 2008; Paratcha and Ledda, 2008; Wee and Wang, 2017; Fantone et al., 2020; Wang et al., 2022). Trks initiates several signaling cascades, including the mitogen- activated protein kinase (MAPK) cascade, the phosphatidylinositol- 3-kinase (PI3K) cascade, and the protein kinase C (PKC)- phospholipase-Cγ (PLC-γ) cascade (Klesse and Parada, 1999; Reichardt, 2006). Proneurotrophins, however, preferentially interact with the low-affinity and nonselective p75 neurotrophin receptor (p75NTR) of the tumor necrosis factor (TNF) receptor superfamily in a complex with members of the sortilin receptor family to regulate cell death via the c-Jun N-terminal kinase (JNK) or caspase-3 pathway (Yoon et al., 1998; Friedman, 2000; Hempstead and Salzer, 2002; Nykjaer et al., 2004). NGF binds TrkA, BDNF and NT-4 bind TrkB, and NT-3 binds primarily TrkC (Chao and Hempstead, 1995). In addition to the prototypical neurotrophins, there are numerous growth factors that also play a major role in proper brain development (Figure 1). These include ciliary neurotrophic factor (CNTF), ephrins, epidermal growth factor (EGF), glial cell line-derived neurotrophic factor (GDNF), neuregulins, progranulin (PGRN) and transforming growth factor (TGF-β). By binding to their respective receptors, they provide survival, differentiation, migration, maturation, and circuit formation signals to the developing nervous system (Abe et al., 1991; Lärkfors et al., 1994; Mount et al., 1995; Ozaki et al., 2000; Rodger et al., 2012; Araujo et al., 2016; Uesaka et al., 2018). Introduction Mature neurotrophins bind preferentially to the high-affinity Trk receptors, a family of transmembrane tyrosine kinases, to regulate neuronal survival and differentiation (Lu et al., 2005). Activation of Introduction The cerebellum, or ¨little brain¨, is well-known for its sensorimotor function and its role in movement coordination (Gao et al., 1996; Strick et al., 2009). Nevertheless, growing evidence indicates that the little brain is also involved in higher-order cognitive processing, including spatial learning, attention, language, reward, emotion, social behavior and memory (Ito, 2006; Schmahmann and Caplan, 2006; Ito, 2008; Stoodley, 2012; Koziol et al., 2014; Adamaszek et al., 2017; Wagner et al., 2017; Carta et al., 2019; Kostadinov et al., 2019). Several neuroimaging and lesion studies have shown cerebellar aberrations to account for changes in affective and cognitive behavior, collectively termed the “cerebellar cognitive affective syndrome” with deficits in executive function, emotion regulation, and working memory (Schmahmann and Sherman, 1998). Aberrant cerebellar functionality and cerebellar-only genetic alterations have been Frontiers in Molecular Neuroscience Frontiers in Molecular Neuroscience 01 frontiersin.org Boxy et al. 10.3389/fnmol.2023.1181397 10.3389/fnmol.2023.1181397 implicated in numerous neuropsychiatric disorders, such as schizophrenia and bipolar disorder, and neurodevelopmental disorders, including attention deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) (Allen et al., 2004; Peter et al., 2016; Stoodley, 2016; Sathyanesan et al., 2019). This is mainly because cerebellar learning, complementing its role in motor control, also has a bottom-up influence on cognitive functions through extensive interconnections between the deep cerebellar nuclei and limbic brain structures (Snider et al., 1976; Carta et al., 2019; Frontera et al., 2020). Hence, if genetically predisposed, environmental perturbations throughout most of the cerebellar development may impair neuronal maturation and synapse formation, and lead to incorrect circuit wiring. The cerebellum is a highly conserved brain structure that has an extensive and elaborate development, which starts in humans at gestational week 7 (E13  in rodents) and ends around 12 months postnatal (P21 in rodents) (Larramendi and Victor, 1967; Altman, 1972; Sathyanesan et al., 2019). For this review we will focus on the postnatal development of the cerebellum since the embryonic development has been covered in plenum by other reviews (Leto et al., 2016; Sathyanesan et al., 2019; Amore et al., 2021). During early postnatal stages, the dendritic complexity of Purkinje cells (PCs) develops extensively in terms of both branching and arbor length, resulting in the thickening of the molecular layer (Leto et al., 2016). Granule cell progenitors (GCPs), on the one hand, form the temporary external granule layer (EGL) in which they actively proliferate and expand. frontiersin.org BDNF BDNF is abundantly expressed within the developing cerebellum, both embryonically and postnatally in humans and rodents (Menshanov et al., 2015; Camuso et al., 2022). While produced in both CGNs and PCs, BDNF is mostly expressed in the axons of mature CGNs of the IGL, mossy fibers (MFs), and the deep cerebellar nuclei (DCN) (Schwartz et al., 1997; Rico et al., 2002). The release of BDNF from CGN axonal terminals is facilitated by calcium-dependent 02 frontiersin.org Boxy et al. 10.3389/fnmol.2023.1181397 FIGURE 1 Neurotrophic factors regulate postnatal cerebellar development. This schematic diagram depicts the families of neurotrophic factors that regulate survival, differentiation, and migration of cerebellar neurons as well as neurite circuit formation and maintenance. BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; EGF, epidermal growth factor; GDNF, glial-derived neurotrophic factor; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; PGRN, progranulin; TGF-b, transforming growth factor beta. Figure produced in BioRender. FIGURE 1 Neurotrophic factors regulate postnatal cerebellar development. This schematic diagram depicts the families of neurotrophic factors that regulate survival, differentiation, and migration of cerebellar neurons as well as neurite circuit formation and maintenance. BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; EGF, epidermal growth factor; GDNF, glial-derived neurotrophic factor; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; PGRN, progranulin; TGF-b, transforming growth factor beta. Figure produced in BioRender. activator protein for secretion 2 (CAPS2), which is a granule- associated protein (Sadakata et al., 2007; Kokubo et al., 2009; Shinoda et  al., 2019). BDNF binds TrkB on both postsynaptic PCs and presynaptic CGNs, thereby leading to Trk signaling in both a paracrine and autocrine manner, respectively (Lindholm et al., 1997). Both BDNF and its immature form, proBDNF can act as a mitogenic and chemotactic factor in cerebellar development. While BDNF exerts cell survival effects through TrkB activation, proBDNF is known to be a proapoptotic mediator through activation of p75NTR, resulting in axon pruning and cell death (Glass et al., 1991; Ghosh et al., 1994; Singh et al., 2008). In murine CGN cultures, BDNF–TrkB signaling promotes neurite extension and survival of differentiated mature CGNs (Gao et al., 1995; Nonomura et al., 1996; Tanaka et al., 2000). Frontiers in Molecular Neuroscience frontiersin.org BDNF For instance, BDNF secreted by both excitatory MFs and 03 frontiersin.org Boxy et al. 10.3389/fnmol.2023.1181397 FIGURE 2 Different processes during postnatal cerebellar development require neurotrophic action. This schematic illustration depicts several stages of postnatal cerebellar development that involve the neurotrophic factors. EGL, external granule layer; oEGL, outer EGL; iEGL, inner EGL; PCL; Purkinje cell layer; ML, molecular layer; (I)GL, (internal) granule layer; WM, white matter; DCN, deep cerebellar nuclei; PC, Purkinje cell; CGN; cerebellar granule cell; MLI, molecular layer interneuron; BG, Bergmann glia; BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; EGF, epidermal growth factor; GDNF, glial-derived neurotrophic factor; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; PGRN, progranulin; TGF-b, transforming growth factor beta. Figure produced in BioRender. FIGURE 2 Different processes during postnatal cerebellar development require neurotrophic action. This schematic illustration depicts several stages of postnatal cerebellar development that involve the neurotrophic factors. EGL, external granule layer; oEGL, outer EGL; iEGL, inner EGL; PCL; Purkinje cell layer; ML, molecular layer; (I)GL, (internal) granule layer; WM, white matter; DCN, deep cerebellar nuclei; PC, Purkinje cell; CGN; cerebellar granule cell; MLI, molecular layer interneuron; BG, Bergmann glia; BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; EGF, epidermal growth factor; GDNF, glial-derived neurotrophic factor; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; PGRN, progranulin; TGF-b, transforming growth factor beta. Figure produced in BioRender. (mGluR1) activation by parallel fiber (PF) signal transduction, the latter being a key player in CF synapse elimination (Kano et al., 1997; Ichise et al., 2000). The maturation of the cerebellar circuitry requires de novo synthesis of BDNF followed by activation of the TrkB-MAPK signaling pathway and phosphorylation of transcription factor ETS translocation variant 1 (Etv1) which upregulates the expression of several maturation genes with a role in dendritic development and functional synaptic assembly of the cerebellar circuit (Abe et al., 2012). Activation of Etv1 is also necessary for CaMKK2/CaMKIV- dependent phosphorylation of cAMP response element-binding protein (CREB) which drives BDNF autoregulation (Kokubo et al., 2009; Ding et al., 2018). Together, these findings demonstrate the importance of BDNF in cerebellar postnatal development as an imbalance in BDNF expression or signaling results in altered cerebellar architecture and functionality, leading to several CGNs aids in inhibitory synaptogenesis by regulating gephyrin, a postsynaptic scaffolding protein, clustering on CGN and PC dendrites, respectively, via the PLCy calcium-dependent and the PI3K-Akt signaling pathway (Chen et al., 2016). BDNF Accordingly, BDNF not only has pro-survival but also anti-apoptotic capacities in CGNs that are cultured in either serum-free media, low K+ media, or media with high glutamate concentrations (Lindholm et al., 1993; Kubo et al., 1995; Zirrgiebel et al., 1995; Nonomura et al., 1996; Shimoke et al., 1997; Skaper et al., 1998; Tong and Perez-Polo, 1998; Bulleit and Hsieh, 2000; Leeds et al., 2005; Sanchez-Perez et al., 2005; Ortega et al., 2010). ProBDNF, on the other hand, does not exert a pro-survival effect on CGNs. Instead, proBDNF binds to p75NTR, which in turn leads to the activation of the JNK signaling pathway and cell death (Koshimizu et al., 2010). Both the pro and mature form of BDNF affect the migration of CGNs in vivo. While endogenous BDNF promotes GCPs to exit the cell cycle and initiate migration in an autocrine manner, proBDNF acts as a negative regulator, an effect which is mediated by binding p75NTR and its co-receptor sortilin (Borghesani et al., 2002; Zhou et al., 2007; Kokubo et al., 2009). As BDNF and TrkB are expressed in cerebellar PCs, they play a role in PC dendritogenesis and spine formation both in vitro and in vivo (Schwartz et al., 1997; Shimada et al., 1998; Yamashita et al., 2011). Lärkfors et al. (1996) found that survival of PCs increases after in vitro treatment with BDNF. Additionally, Morrison and Mason (1998) found that BDNF improves PC survival in isolated cultures, but decreases when co-cultured with CGNs, indicating that the neurotrophic action is context- and activity-dependent. However, recent findings suggest that BDNF does not exert a survival effect on naïve PCs in vivo but promotes survival in damaged PCs (Rakotomamonjy and Goumari, 2019). These data are supported by several other findings which show that BDNF does not affect survival in other neuronal populations such as cortical, hippocampal, and striatal neurons (Gorski et al., 2003; Baquet et al., 2004; Rauskolb et al., 2010). Synaptogenesis is a crucial developmental step that promotes normal brain function. Improper synapse formation is, therefore, associated with neuronal dysfunction. BDNF–TrkB signaling is involved in correct circuit wiring, synaptogenesis, and establishing a balance between inhibitory and excitatory synapses within the cerebellar system (Minichiello, 1996; Schwartz et al., 1997; Carter et al., 2002; Rico et al., 2002; Bosman et al., 2006; Shinoda et al., 2019). Frontiers in Molecular Neuroscience frontiersin.org FIGURE 2 Different processes during postnatal cerebellar development require neurotrophic action. This schematic illustration depicts several stages of postnatal cerebellar development that involve the neurotrophic factors. EGL, external granule layer; oEGL, outer EGL; iEGL, inner EGL; PCL; Purkinje cell layer; ML, molecular layer; (I)GL, (internal) granule layer; WM, white matter; DCN, deep cerebellar nuclei; PC, Purkinje cell; CGN; cerebellar granule cell; MLI, molecular layer interneuron; BG, Bergmann glia; BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; EGF, epidermal growth factor; GDNF, glial-derived neurotrophic factor; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; PGRN, progranulin; TGF-b, transforming growth factor beta. Figure produced in BioRender. BDNF This is consistent with reports that BDNF–TrkB signaling promote gamma amino butyric acid (GABA)ergic synaptogenesis (Bao et al., 1999; Seil, 1999; Seil and Drake-Baumann, 2000). Moreover, BDNF contributes to the development of the two major afferent systems in the cerebellar cortex. Rabacchi et al. (1999) found that CGN-derived BDNF acts in a retrograde manner to promote the growth and maturation of innervating basilar pontine MFs. In addition, BDNF from PCs acts retrogradely on TrkB located within climbing fibers (CF) in facilitating late-phase CF synapse elimination from PC soma (Bosman et al., 2006; Choo et al., 2017). Secretion of BDNF from PCs is most likely triggered following metabotropic glutamate receptor 04 frontiersin.org Boxy et al. 10.3389/fnmol.2023.1181397 TABLE 1  Function of neurotrophic factors during postnatal cerebellar development. TABLE 1  Function of neurotrophic factors during postnatal cerebellar development. TABLE 1  Function of neurotrophic factors during postnatal cerebellar development. Neurotrophic factors Cerebellar function References BDNF CGN survival Bulleit and Hsieh (2000), Kubo et al. (1995), Leeds et al. (2005), Lindholm et al. (1993), Nonomura et al. (1996), Ortega et al. (2010), Sanchez-Perez et al. (2005), Shimoke et al. (1997), Skaper et al. (1998), Tong and Perez-Polo (1998), Zirrgiebel et al. (1995), and Koshimizu et al. (2010) CGN migration Borghesani et al. (2002), Kokubo et al. (2009), and Zhou et al. (2007) CGN neurite outgrowth Gao et al. (1995), Nonomura et al. (1996), and Tanaka et al. (2000) PC survival Lärkfors et al. (1996), Rakotomamonjy and Goumari (2019), and Morrison and Mason (1998) PC neurite outgrowth Schwartz et al. (1997), Shimada et al. (1998), and Yamashita et al. (2011) Circuit wiring Bosman et al. (2006), Carter et al. (2002), Minichiello (1996), Rico et al. (2002), Schwartz et al. (1997), Shinoda et al. (2019), Chen et al. (2016), Bao et al. (1999), Seil (1999), Seil and Drake-Baumann (2000), Rabacchi et al. (1999), Choo et al. (2017), Ichise et al. (2000), Kano et al. (1997), and Abe et al. (2012) CNTF CGN survival de Luca et al. (1996a) PC survival Lärkfors et al. (1994) Astrocyte differentiation Okano-Uchida et al. (2013) Ephrins CGN survival Karam et al. (2000) and Sentürk et al. (2011) CGN migration Yacubova and Komuro (2002) CGN neurite outgrowth Karam et al. (2000), Sentürk et al. (2011), Moreno-Flores et al. (2002), and Wang et al. (2007) PC neurite outgrowth Karam et al. (2000), Heintz et al. (2016), and Saywell et al. CNTF Impairment in BDNF signaling within the cerebellum has been implicated in several cerebellar-related disorders. Ataxia is a group of neurological disorders mainly characterized by a lack of voluntary movement coordination (Schmahmann, 2004). Post-mortem studies of patients with spinocerebellar ataxia type 6 (SCA6) show reduced expression of BDNF which was also revealed in a SCA6 mouse model, as well as a spinocerebellar ataxia type 1 (SCA1) mouse model. Both mice models display PC pathology, abnormal firing rates and changes in motor behavior. Extrinsic BDNF delivery and subsequent activation of the TrkB-Akt signaling pathway improves the PC firing rate and delays the onset of the observed motor deficits (Mellesmoen et al., 2019; Cook et al., 2022). CNTF is a cytokine that has a multifunctional role in CNS development, for instance in neurite outgrowth and neuronal survival, as well as after injury (Oyesiku and Wigston, 1996). The expression of CNTF in cerebellum is relatively low during early postnatal weeks, however, it increases significantly during adulthood (Ohta et  al., 1996). Due to this low expression of CNTF in the developing cerebellum, there are limited studies on the effects of CNTF on cerebellar cells and only reports on the role of CNTF in cultured cerebellar cells. Although the expression is low, it is not absent, suggesting that CNTF might play a role during development and, therefore, warrant further studies. Friedreich’s ataxia (FA) is a predominantly neurodegenerative disease caused by recessive mutations that produce a deficiency of frataxin (FXN). FXN triggers apoptosis in CGNs, pathological changes in PCs as well as loss of motor coordination. In primary granule cultures of FXN-deficient mice, it was evidenced that BDNF can be used as a therapeutic agent that effectively prevents CGN apoptosis and PC pathogenesis (Katsu-Jiménez et  al., 2016). However, this remains to be tested in vivo. One report showed that CNTF improves PC and other cerebellar GABAergic neuron survival in vitro as showcased in rat primary PC cultures (Lärkfors et  al., 1994). Furthermore, CNTF exercises a neuroprotective effect in immature cerebellar granule cultures that are maintained in physiological low concentrations of potassium in vitro, which under normal circumstances, leads to apoptosis. CNTF can also prolong their survival in such non-depolarizing conditions (de Luca et  al., 1996b). However, the physiological relevance of CNTF in immature CGNs remains unclear. On the other hand, CNTF might act as a differentiation factor during development. BDNF (2014), Shinoda et al. (2019), and Sherrard and Bower (2002) PGRN PC survival Wang et al. (2022) PC neurite outgrowth Matsuwaki et al. (2015) Circuit wiring Uesaka et al. (2018) TGF-b CGN survival Wang et al. (2011), Elvers et al. (2005), de Luca et al. (1996b), Brown (1999), Constam et al. (1994), and Kane et al. (1996) CGN migration Wang et al. (2011) PC survival Zhou et al. (2003) and Wang et al. (2011) PC neurite outgrowth Wang et al. (2011) Circuit wiring Araujo et al. (2016), Ondáčová et al. (2017) Cerebellar foliation Wang et al. (2011) CGN, cerebellar granule neuron; PC, Purkinje cell; MLI, molecular layer interneuron; NSC, neuronal stem cell; BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; EGF, epidermal growth factor; GDNF, glial cell line-derived neurotrophic factor; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; PGRN, progranulin; TGF-β, transforming growth factor beta. TABLE 1  (Continued) Neurotrophic factors Cerebellar function References NT-4 CGN survival Proenca et al. (2016), Gao et al. (1995), Kubo et al. (1995), Shimoke et al. (1997), and Skaper et al. (1998) CGN neurite outgrowth Gao et al. (1995) PC survival Proenca et al. (2016), Lärkfors et al. (1996), and Morrison et al. (1988) Circuit wiring Sadakata et al. (2014), Shinoda et al. (2019), and Sherrard and Bower (2002) PGRN PC survival Wang et al. (2022) PC neurite outgrowth Matsuwaki et al. (2015) Circuit wiring Uesaka et al. (2018) TGF-b CGN survival Wang et al. (2011), Elvers et al. (2005), de Luca et al. (1996b), Brown (1999), Constam et al. (1994), and Kane et al. (1996) CGN migration Wang et al. (2011) PC survival Zhou et al. (2003) and Wang et al. (2011) PC neurite outgrowth Wang et al. (2011) Circuit wiring Araujo et al. (2016), Ondáčová et al. (2017) Cerebellar foliation Wang et al. (2011) CGN, cerebellar granule neuron; PC, Purkinje cell; MLI, molecular layer interneuron; NSC, neuronal stem cell; BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; EGF, epidermal growth factor; GDNF, glial cell line-derived neurotrophic factor; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; PGRN, progranulin; TGF-β, transforming growth factor beta. CGN, cerebellar granule neuron; PC, Purkinje cell; MLI, molecular layer interneuron; NSC, neuronal stem cell; BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; EGF, epidermal growth factor; GDNF, glial cell line-derived neurotrophic factor; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; PGRN, progranulin; TGF-β, transforming growth factor beta. PC dendritogenesis. BDNF This leads to poor circuit connectivity and failed paired-pulse facilitation at PF-PC synapses. This is in line with changes observed in ASD patients who display cellular disturbances, as well as hypoplasia around the vermis (Sadakata and Furuichi, 2009; Sadakata et al., 2014). cerebellar-related neurodevelopmental disorders which are discussed in the paragraph below. BDNF (2014) Circuit wiring Lackey and Sillitoe (2020) Cerebellar foliation Karam et al. (2000) and Rogers et al. (1999) EGF CGN survival Abe et al. (1991, 1992), Morrison et al. (1988), Yamada et al. (1997), Gunn-Moore and Tavaré (1998) and Leutz and Schachner (1981) CGN migration Carrasco et al. (2003), and Martinez et al. (2011) CGN neurite development Abe et al. (1991, 1992), Morrison et al. (1988), and Yamada et al. (1997) NSC proliferation Okano-Uchida et al. (2013) and Leutz and Schachner (1981) GDNF CGN survival Subramaniam et al. (2008) PC survival Mount et al. (1995) PC neurite outgrowth Mount et al. (1995) MLI survival Sergaki and Ibáñez (2017) NGF CGN survival Legrand and Clos (1991), Muller et al. (1994), Khursigara et al. (2001), Kisiswa et al. (2018), and Vicario et al. (2015) PC survival Cohen-Cory et al. (1991), Florez-McClure et al. (2004), Legrand and Clos (1991), and Mount et al. (1998) PC neurite outgrowth Cohen-Cory et al. (1991), Legrand and Clos (1991), and Mount et al. (1998) Circuit wiring Numakawa et al. (2003) Neuregulins Circuit wiring Rieff and Corfas (2006), Ozaki (2001), Ozaki et al. (2000), Xie et al. (2004), Xie et al. (2007), Rieff et al. (1999), Ozaki et al. (2004), Gajendran et al. (2009), and Fenster et al. (2012) NT-3 CGN survival Katoh-Semba et al. (2000), Bates et al. (1999), Joo et al. (2014), Bates et al. (1999), Joo et al. (2014), Katoh-Semba et al. (2000), Kubo et al. (1995), and Shimoke et al. (1997) CGN differentiation Doughty et al. (1998), Neveu and Arenas (1996), Takumi et al. (2005), Minichiello (1996), Zanin et al. (2016), Zanin and Friedman (2022), Zanin et al. (2019), and Segal et al. (1992) CGN migration Neveu and Arenas (1996) PC survival Lärkfors et al. (1996) and Mount et al. (1998) PC neurite outgrowth Joo et al. (2014), Neveu and Arenas (1996), and Tepper et al. (2020) Circuit wiring Sadakata et al. (2014), Shinoda et al. (2019), and Sherrard and Bower (2002) (Continued) 05 05 Frontiers in Molecular Neuroscience frontiersin.org frontiersin.org Boxy et al. 10.3389/fnmol.2023.1181397 TABLE 1  (Continued) Neurotrophic factors Cerebellar function References NT-4 CGN survival Proenca et al. (2016), Gao et al. (1995), Kubo et al. (1995), Shimoke et al. (1997), and Skaper et al. (1998) CGN neurite outgrowth Gao et al. (1995) PC survival Proenca et al. (2016), Lärkfors et al. (1996), and Morrison et al. (1988) Circuit wiring Sadakata et al. Frontiers in Molecular Neuroscience frontiersin.org CNTF The postnatal cerebellum contains neuronal stem cells (NSCs) which derive from the white matter and CNTF has been shown to facilitate NSCs differentiation into astrocytes (Okano-Uchida et al., 2013). However, it remains unclear whether CNTF is responsible for their differentiation in vivo. Furthermore, expression levels of BDNF, proBDNF, and its intrinsic receptor, TrkB, are reduced in the cerebella of patients with neuropsychiatric disorders, including schizophrenia, bipolar disorder (BPD), and major depressive disorder (MDD) as well as in a rodent model for ASD (Soontornniyomkij et al., 2011; Yang et al., 2017; Alò et al., 2021). Additionally, CAPS2-deficient mice that show reduced secretion of BDNF from CGNs exhibit developmental deficits around the cerebellar vermis, such as increased CGN apoptosis and impaired 06 frontiersin.org Boxy et al. 10.3389/fnmol.2023.1181397 TABLE 2  Involvement of neurotrophic factors in cerebellar-related neurodevelopmental disorders. TABLE 2  Involvement of neurotrophic factors in cerebellar-related neurodevelopmental disorders. Neurotrophic factors Cerebellar-associated neurodevelopmental disorder References BDNF ASD Sadakata and Furuichi (2009), Sadakata et al. (2014), and Alò et al. (2021) MDD Yang et al. (2017) BPD Soontornniyomkij et al. (2011) and Yang et al. (2017) Schizophrenia Yang et al. (2017) FA Katsu-Jiménez et al. (2016) SCA Cook et al. (2022) and Mellesmoen et al. (2019) CNTF GLD Lin et al. (2015) Eprins MB Bhatia et al. (2015) EGF MB Schönholzer et al. (2020) and Neve et al. (2017) GDNF ADHD Bilgiç et al. (2017) and Shim et al. (2015) Schizophrenia Tunca et al. (2015) CMD Sakuma et al. (2002) NGF ADHD Bilgiç et al. (2017), Clemow et al. (2000), Guney et al. (2014), Syed et al. (2007), and Tiveron et al. (2013) MS Damarjian et al. (2004) Neuregulins MB Di Marcotullio et al. (2006) and Gilbertson et al. (1998) Schizophrenia Kircher et al. (2009), Nickl-Jockschat et al. (2014), Schmitt et al. (2010), Yeganeh-Doost et al. (2011), and Barros et al. (2009) NT-3 ASD Sajdel-Sulkowska et al. (2009), Sajdel-Sulkowska et al. (2011), and Sadakata et al. (2014) NT-4 CMD Sakuma et al. (2002) PGRN ASD Matsuwaki et al. (2015), Uesaka et al. (2018), and Wang et al. (2022) TGF-b MB Marino (2005), Roussel and Hatten (2011), Santhana Kumar et al. (2018), and Aref et al. (2013) ASD Ferretti and Hollander (2015) and Xu et al. (2017) CA Cook et al. (2022) and Mellesmoen et al. CNTF (2019) MB, medulloblastoma; ASD, autism spectrum disorder; ADHD, attention deficit hyperactivity disorder; MDD, major depressive disorder; BPD, bipolar disorder; MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; CMD, congenital muscular dystrophy; FA, Friedreich’s ataxia; CA, cerebellar ataxia; SCA, spinocerebellar ataxia; GLD, globoid cell leukodystrophy; BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; EGF, epidermal growth factor; GDNF, glial cell line-derived neurotrophic factor; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; PGRN, progranulin; TGF-β, transforming growth factor beta. MB, medulloblastoma; ASD, autism spectrum disorder; ADHD, attention deficit hyperactivity disorder; MDD, major depressive disorder; BPD, bipolar disorder; MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; CMD, congenital muscular dystrophy; FA, Friedreich’s ataxia; CA, cerebellar ataxia; SCA, spinocerebellar ataxia; GLD, globoid cell leukodystrophy; BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; EGF, epidermal growth factor; GDNF, glial cell line-derived neurotrophic factor; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; PGRN, progranulin; TGF-β, transforming growth factor beta. Frontiers in Molecular Neuroscience CNTF in cerebellar-related neurodevelopmental disorders are mainly type-specific and consist of two subfamilies, EphA and EphB (Chang et al., 1997). Their signaling is bidirectional, meaning it occurs via both phosphorylation of intracellular proteins via the Eph receptors or by intracellular signal transmission via the ephrin ligands itself upon receptor binding, a process known as reverse signaling (Rodger et  al., 2012). In the chicken cerebellum, ephrin-A4 and ephrin-A5 are expressed at the earliest during embryonic stages, followed by expression of ephrin-A2 and ephrin-A3. Expression of ephrin-B1 and ephrin-B2, however, is mostly found during postnatal stages in migrating CGNs (Karam et al., 2000). Ephrin-B1 is expressed in both CGNs and PCs, while expression of its receptor, EphB, is mainly constricted to CGNs during postnatal development (Moreno- Flores et al., 2002). Globoid cell leukodystrophy (GLD) is a lysosomal storage disease that is characterized by demyelination and astrogliosis. Such neuropathy leads to neurobehavioral changes, including cerebellar ataxia. In a murine model of GLD, cerebellar neurons as well as Bergmann glia undergo degeneration, an effect which is accompanied by the altered expression of several neurotrophic factors. For example, CNTF expression is markedly increased in GLD cerebella, which could possibly mitigate remyelination of demyelinated neurons (Lin et al., 2015). The family of ephrins is involved in the formation and maintenance of PC compartments (Karam et al., 2000). For example, ephrin/Eph signaling regulates both the type and density of spines on PCs, a process which is required for defining either CFs or PFs that innervate different parts of PC dendrites. As a result, dendritogenesis on PCs is subject to competition between these fibers; CFs occupy the more proximal dendrites of PCs by suppressing the formation of frontiersin.org Ephrins Ephrins are membrane-bound proteins that are expressed in many regions of the developing brain. They consist of two subclasses, the A-type (ephrin-A1-5) and the B-type (ephrin-B1-3) and bind to their respective tyrosine kinase receptors, namely the Eph receptors which 07 frontiersin.org Boxy et al. 10.3389/fnmol.2023.1181397 10.3389/fnmol.2023.1181397 receptor (EGFR, also called ErbB1) or receptor homologs ErbB2, ErbB3, or ErbB4 (Wieduwilt and Moasser, 2008). EGF and its receptor, EGFR, are expressed during all stages of life depending on the cell type, however, during postnatal development, they can be  found within the CGNs, PCs and astrocytes of the cerebellar cortex as well as the DCN (Gómez-Pinilla et  al., 1988; Seroogy et  al., 1995; Scalabrino, 2022). EGF binding to EGFR results in activation of several signaling pathways including the PLC, PI3-K, and the Ras-MAPK signaling pathway (Wong and Guillaud, 2004). smaller spines that are typically associated with PFs. Instead, CFs make room for a few larger spines to form contact with PCs. In an in vitro cerebellar model, it was shown that the ephrin/Eph signaling pathway affects the more proximal dendrites on PCs by inactivating integrin downstream signaling (Heintz et al., 2016). Additionally, ephrins and their receptors effectively function as PC axon guidance and growth molecules in a spatiotemporal manner (Saywell et al., 2014). One study found that both ephrin-A2 and ephrin-A5 by binding to their respective receptor control PC-MF communication during circuit formation. This is necessary for the proper patterning of mossy fiber afferents into discrete zones located within the granule layer (Lackey and Sillitoe, 2020).f g g p y g EGF has the ability to support neuronal growth and survival in cultured rodent CGNs (Morrison et al., 1988; Abe et al., 1991, 1992; Yamada et al., 1997). This indicates that EGF can act as a neurotrophic factor that promotes the elongation and maintenance of neurites in cerebellar neurons which is most likely achieved through the activation of protein kinases (Morrison et al., 1988; Abe et al., 1991, 1992; Yamada et  al., 1997). Moreover, EGF effectively reduces glutamate-associated apoptosis in primary CGN cultures and has thus a neuroprotective effect against glutamate-induced neurotoxicity (Abe and Saito, 1992; Gunn-Moore and Tavaré, 1998). In addition, it also enhances survival of serum-deprived cerebellar cultures (Leutz and Schachner, 1981). Ephrins Concomitant to its function in CGN survival and maturation, EGF has been suggested to be involved in CGN migration, largely due to expression of EGFR found in premigratory post-mitotic CGNs and its function in facilitating Bergmann glia elongation (Carrasco et al., 2003; Martinez et al., 2011). NSCs which derive from the cerebellar white matter require EGF to keep their proliferative ability (Okano-Uchida et al., 2013). Once these NSCs differentiate into astrocytes they express EGFR. It has been reported that EGF-EGFR signaling in these astrocytes stimulates DNA synthesis increasing their proliferation (Leutz and Schachner, 1981). While the different types of ephrin-A are crucial for PC development, ephrin-Bs regulate CGN development. For example, ephrin-B1 facilitates CGN survival, migration, dendritogenesis, as well as axonal extension (Karam et al., 2000; Sentürk et al., 2011). It also mitigates the expression of certain cell adhesion and microtubule- associated proteins which are necessary for axonal extension and guidance, as well as dendritogenesis of CGNs (Moreno-Flores et al., 2002; Wang et al., 2007). Both ephrin-B2 and its receptor EphB2 are strongly expressed in the EGL at postnatal day 3 in mice, a timepoint prior to the initiation of postmitotic CGN migration. Their concerted action is thought to inhibit the effect of certain chemokines which control the migration GCPs, consequently leading to the initiation of migration (Yacubova and Komuro, 2002). On a macroscopic scale, the ephrins and Eph receptors are thought to play an important role in cerebellar foliation as they have the ability to demarcate the cerebellar anlage (Rogers et al., 1999; Karam et al., 2000). Frontiers in Molecular Neuroscience GDNF Medulloblastoma (MB) is an aggressive tumor that arises from GCPs in the cerebellum. Proper formation and migration of these precursors require ephrin-A5 and its receptors. In a mouse model of MB, it was found that deletion of ephrin-A5 inhibits tumor growth, providing a platform for development of ephrin-based pharmacological interventions of medulloblastoma (Bhatia et al., 2015). GDNF is expressed in several different types of neurons, including PCs, and plays a crucial role in regulating various processes in the developing nervous system, such as neuron survival, cell migration, axon growth, and synapse formation (Mount et al., 1995; McAlhany et al., 1997, 1999; Paratcha and Ledda, 2008). It acts in a paracrine manner by pairing with the GDNF family receptor α1 (GFRα1) (Treanor et al., 1996). The GDNF/GFRa1 complex subsequently binds with the “rearranged during transfection” (RET) tyrosine kinase receptor or the neural cell adhesion molecule (NCAM), though with lower affinity to activate either the MAPK or P13K–Akt pathway (Treanor et al., 1996; Trupp et al., 1996; Paratcha and Ledda, 2008; Sergaki and Ibáñez, 2017). EGF cerebellar-related neurodevelopmental disorders Ephrins have also been implicated in developmental disorders, in particular schizophrenia and medulloblastoma. The drug olanzapine is effective in treating schizophrenia, but its precise mechanism remains unclear. One study found that olanzapine treatment may regulate the DNA methylation of certain genes in the cerebellum, including the ephrin/Eph receptor family. This family plays a crucial role in axon guidance during development and synaptic plasticity in adulthood, including long-term potentiation, which has been linked to psychosis. Therefore, the epigenetic changes in these genes may account for the therapeutic effects of olanzapine observed in a rat model of schizophrenia (Melka et al., 2014). Similar to ephrins, EGF signaling is involved in MB as it rapidly increases nuclear activation of the ERK1/2-MAPK pathway in MB cells which speeds the invasion of these cells (Schönholzer et al., 2020). Neve et al. (2017) found that in organotypic cerebellar slices, EGF effectively enhances tumor growth and infiltration, indicating its tumor progressing capabilities. EGF EGF is part of the large EGF superfamily which also contains the transforming growth factor alpha (TGF-α) and the neuregulins (Wieduwilt and Moasser, 2008). It binds the epidermal growth factor 08 frontiersin.org Boxy et al. 10.3389/fnmol.2023.1181397 10.3389/fnmol.2023.1181397 10.3389/fnmol.2023.1181397 GDNF, expressed by PCs, has been shown to be crucial for the survival of molecular layer interneurons (MLIs) during postnatal cerebellar development (Sergaki and Ibáñez, 2017). It binds the GFRa1-RET receptor complex on MLIs in a cell-autonomous manner to stimulate IN survival. The absence of either receptor leads to the loss of MLIs, decreased GABAergic inputs to PC dendrites, and an increase in PC firing rate, subsequently resulting in compromised motor learning as well as eyeblink conditioning (Sergaki and Ibáñez, 2017). Additionally, GDNF has neurotrophic capacities in cultured PCs as it aids spine formation and thickening of the dendritic tree as well as in CGNs as it increases survival of these neurons (Mount et al., 1995; Subramaniam et al., 2008). NGF has been implicated as a differentiation and a survival factor for PCs in vitro in the presence of BDNF/TrkB signaling support (Cohen-Cory et al., 1991; Legrand and Clos, 1991; Mount et al., 1998; Florez-McClure et al., 2004). Not only in PCs, NGF also promotes the proliferation of immature granule cells and the postmitotic survival of newly differentiated CGNs (Legrand and Clos, 1991; Muller et al., 1994). The in vitro and in vivo survival effect of NGF on CGNs is RIP2-dependent and leads to an increase in NF-κB activity (Khursigara et al., 2001). In the absence of RIP2, however, NGF can induce JNK-dependent apoptosis (Kisiswa et al., 2018). The ability of the NGF-p75NTR complex to induce cell death in CGNs is normally suppressed or masked by concurrent activation of NF-kB signaling (Vicario et al., 2015). proNGF, on the other hand, displays proapoptotic activity through the increase of c-Jun phosphorylation via the JNK-dependent pathway. Therefore, NGF and proNGF function in an antagonistic manner, and their balance is key deterministic in CGN survival during postnatal cerebellar development. It is worth noting that immature granule neuron-derived NGF can induce an increase in intracellular calcium through the ryanodine receptor, which is followed by a rapid release of glutamate via the p75NTR-dependent pathway. Such release of glutamate from PF terminals is important for the strengthening of PF-PC synapses (Numakawa et al., 2003). GDNF in cerebellar-related neurodevelopmental disorders GDNF has been implicated in several cerebellar-related neurodevelopmental disorders including schizophrenia and ADHD. For example, serum of schizophrenia patients shows reduced levels of GDNF compared to healthy controls (Tunca et al., 2015). Conversely to schizophrenia, plasma levels of GDNF in children with ADHD are markedly increased (Shim et al., 2015; Bilgiç et al., 2017). Although the levels of GDNF are altered in both schizophrenia and ADHD children, both studies measured circulation levels of GDNF suggesting that this alteration might not be restricted to the cerebellum but a global nervous system impairment. NGF NGF expression has been shown in many brain regions, most prominently in the cerebellum (Shelton and Reichardt, 1986). Studies from our and other groups have reported expression of NGF in CGNs (Matsui et al., 1990; Cohen-Cory et al., 1993; Kisiswa et al., 2018). NGF can bind two distinct receptors, TrkA and p75NTR, where activation of TrkA induces multiple signaling pathways such as PI3K-Akt and MAPK that regulate cellular survival, differentiation, and neurite outgrowth (Crowder and Freeman, 1998; Kaplan and Miller, 2000). The proform of NGF, proNGF is thought to induce apoptosis when bound to p75NTR in the presence of sortilin, but also acts as a growth factor and induces neurite outgrowth when bound to TrkA in the absence of p75NTR (Nykjaer et al., 2004; Buttigieg et al., 2007). In the cerebellum, p75NTR is expressed in PCs and CGNs (Pioro and Claudio Cuello, 1988; Kisiswa et al., 2018). TrkA, on the other hand, is not expressed in the healthy cerebellum, indicating that NGF signals through p75NTR to exert its neurotrophic capacities. Frontiers in Molecular Neuroscience NGF in cerebellar-related neurodevelopmental disorders ADHD has recently been associated with the cerebellum, although the degree of cerebellar contribution to ADHD pathophysiology requires further studies (Mackie et al., 2007; Sathyanesan et al., 2019). Nevertheless, the levels of NGF in ADHD animal models and in children with ADHD is significantly increased in blood samples (Clemow et al., 2000; Syed et al., 2007; Tiveron et al., 2013; Guney et al., 2014; Bilgiç et al., 2017). However, like GDNF, the current data indicate that the alteration of NGF in these patients is a global effect and not cerebellar-specific. Considering the beneficial effect of NGF on cerebellar neurons, we speculate that the increase of NGF could be  a compensatory mechanism to protect the cerebellum from impairment caused by ADHD. Furthermore, GDNF might also play a role in congenital muscular dystrophy (CMD). CMD encompasses a group of genetic muscle diseases, characterized by muscle weakness, hypotonia, and muscle atrophy and often accompanied by respiratory complications as well as intellectual disability (Bertini et al., 2011). In a mouse model of CMD, it was found that GDNF expression was markedly enhanced in both PCs and CGNs (Sakuma et al., 2002). Since GDNF is a potent PC survival agent, such elevated expression levels may be the result of a compensatory mechanism due to PC degeneration. Conversely, exuberant levels of GDNF may be  neurotoxic to the PCs and contribute to cerebellar degeneration. Patients with multiple sclerosis (MS) and animal models of experimental autoimmune encephalomyelitis (EAE) often display cerebellar ataxia. This pathophysiological phenomenon is partly caused by abnormal PC firing as a result of an imbalance in sodium channel expression. NGF acting via p75NTR has the ability to modulate the expression of sodium channel Nav1.8  in PCs and, therefore, contribute to the regular PC firing rate. In a murine EAE model, it was revealed that levels of NGF and p75NTR are increased which leads to the upregulation of Nav1.8 channels (Damarjian et al., 2004). Neuregulins in cerebellar-related neurodevelopmental disorders Multiple studies have suggested that the neuregulin-1 gene (NRG1) serves as an important risk gene for schizophrenia that is thought to be  characterized by deficits in glutamatergic neurotransmission (Kircher et al., 2009; Nickl-Jockschat et al., 2014). In patients with schizophrenia, it was found that gene expression of the NMDA receptor subunit 2D (NMDAR2D) was significantly increased in the cerebellum, which results in a hyperexcitability of the NMDA receptor, and which may be a secondary upregulation due to a dysfunctional receptor. In patients with the NRG1 risk variant, they found that expression of the NMDAR2C subunit was significantly reduced which could lead to hypofunctionality of the NMDA receptor, that in turn may lead to dysfunction of the GABA system (Schmitt et  al., 2010). Accordingly, from post-mortem studies, there is accumulating evidence that GABAergic signaling is decreased in the cerebellum of schizophrenia patients (Yeganeh-Doost et al., 2011). Not only NRG1, but also its receptors, ErbB2 and ErbB4 are candidate susceptibility genes for schizophrenia. While deletion of ErbB2 and ErbB4 does not affect brain anatomy on a macroscopic scale, it can lead to impaired spine maturation as well impaired interactions with postsynaptic scaffold proteins, such as PSD95, with glutamate receptors which in turn leads to behavioral abnormalities (Barros et al., 2009). One in vivo study found that neuregulin–ErbB signaling initiates dendritogenesis and maturation of postsynaptic compartments in the developing murine cerebellum (Rieff and Corfas, 2006). Neuregulins have the ability to serve as cell adhesion molecules on CGNs for synaptic recognition of MFs, leading to the formation of the cerebellar MF system (Ozaki et al., 2000; Ozaki, 2001). These growth factors are not only essential for glutamatergic circuit wiring as they have also been ascribed a role in the GABA system as well. More specifically, neuregulin-1 through activation of the ErbB4 receptor tyrosine kinase effectively induces expression of the GABAA receptor β2 subunit via the MAPK, PI3K, and the cyclin-dependent kinase-5 (cdk5) pathway in primary CGN cultures (Xie et al., 2004). Activation of cdk5 leads to recruitment of PSD95 which in turn facilitates the effects of neuregulin through its interaction with ErbB4. As a result, this mechanism functions as a positive feedback system to neuregulin signaling and consequent expression of the GABAA β2 subunit (Xie et al., 2007). Neuregulins Neuregulins are a group of trophic factors that are part of the large EGF superfamily and have an essential role in both the developing brain and during synaptic plasticity in the adult brain (Wong and Guillaud, 2004). The neuregulins which are composed of neuregulin-1 (or heregulin), neuregulin-2, neuregulin-3, and neuregulin-4 interact with the ErbB family of receptors (Chang et al., 1997). Both the 09 frontiersin.org Boxy et al. 10.3389/fnmol.2023.1181397 10.3389/fnmol.2023.1181397 neuregulins and their receptors are expressed in a spatiotemporal manner within the developing cerebellum. Neuregulins and ErbB2, ErbB3 and ErbB4 are all expressed in CGNs while ErbB4 is also expressed in radial glial cells such as the Bergmann glia (Rio et al., 1997; Ozaki et  al., 1998; Rahman et  al., 2019). In the maturing cerebellum, neuregulins are concentrated in glutamatergic MFs that innervate CGNs located in the IGL (Ozaki et al., 1997). Several studies found that in murine primary cultures of CGN, neuregulin-1 signals through the ErbB4 receptor to regulate its interaction with PSD95 which is crucial for CGN differentiation. The C-terminal part of the ErbB4 receptor associates with PSD95 leading to the assembly of the nitric oxide synthase (NOS)-1 complex, a process which is thought to be mediated via the MAPK pathway (Krainock and Murphy, 2001a,b; Murphy and Bielby-Clarke, 2008). Neuregulin-1 also promotes differentiation and morphogenesis of Bergmann glia which in turn, enhances migration of CGNs (Rio et al., 1997; Yacubova and Komuro, 2002; Buffo and Rossi, 2013). MFs that innervate CGNs located in the IGL, one study found that cultured cerebellar slices stimulated with a neuregulin isoform dramatically increase the expression of NR2C messenger RNAs. This mechanism is mediated by the binding of neuregulins onto its receptors ErbB2 and ErbB4 located on CGNs. In conclusion, cell- autonomous signaling of NRG1/ErbB can modulate both glutamatergic and GABAergic neurotransmitter receptor composition during development and regulate synaptic plasticity (Ozaki et al., 1997; Fenster et al., 2012). Frontiers in Molecular Neuroscience Neuregulins in cerebellar-related neurodevelopmental disorders Extrinsic delivery of neuregulin causes an increase in the GABAA receptor β2 subunit expression of CGN cultures in vitro which is paralleled by an increase in functional GABAA receptors (Rieff et al., 1999). MB is associated with decreased activity of the mitogen sonic hedghehog (shh). Under normal conditions, shh downregulates the expression of ErbB4, while in MB subsets, there is accumulation of both ErbB2 and ErbB4. This leads to both anti-apoptotic and loss of cell growth arrest signaling in neuronal progenitors of the cerebellum (Gilbertson et al., 1998; Di Marcotullio et al., 2006). It has been reported that neuregulin can mediate synaptogenesis via two distinctive mechanisms. First, the soluble form of neuregulin can be proteolytically cleaved from the membrane-associated form as a result of protein kinase activation. This soluble form is then able to transsynaptically and in a paracrine manner act as a neurotrophic factor and regulate the expression of the NMDA receptor subunit NR2C (Ozaki et al., 2000). The soluble form of neuregulin-1 can be  shedded in a frequency-dependent manner due to electrical stimulation in both CGN and pontine nucleus neurons that form MF afferents and synapse onto CGN. Such cleaved neuregulin-1 is thus important for synaptic transmission across MF-CGN synapses (Ozaki et al., 2004). Second, the membrane-anchored form in both CGNs and MF terminals can serve as a cell-recognition molecule to stimulate MF-CGN synapse formation (Ozaki et al., 2000). However, there is some debate on neuregulins’ role in synaptogenesis as one study found that neuregulin/ErbB signaling to CGNs is dispensable for the normal development of their synaptic inputs as compared to previous in vitro experiments (Gajendran et al., 2009). Nevertheless, expression of NR2C is specifically induced during synaptogenesis of CGNs within the IGL, leading to dramatic changes in the NMDA receptor composition during development. As neuregulins are expressed in the frontiersin.org NT-3 In the developing cerebellum, NT-3 and its high-affinity tyrosine kinase receptor TrkC are expressed by both the differentiated CGNs of the IGL and their precursors in the EGL as well as in PCs (Neveu and Arenas, 1996; Doughty et al., 1998). In the rodent cerebellum, levels of NT-3 markedly decrease after the first 10 postnatal days (Katoh-Semba et al., 2000). Formation of the PC arbor and competitive dendritogenesis in the mouse cerebellum is regulated by NT-3, expressed in CGNs, a process which is TrkC-dependent (Neveu and Arenas, 1996; Joo et al., 2014; Tepper et al., 2020). In cultured PCs, NT-3 effectively increases cell numbers via the PKC-dependent pathway while enhancing their survival and phenotypic differentiation (Lärkfors et al., 1996; Mount 10 frontiersin.org Boxy et al. 10.3389/fnmol.2023.1181397 10.3389/fnmol.2023.1181397 (Proenca et al., 2016). On the one hand, NT-4 improves the survival of isolated PCs (Morrison et al., 1988) as well as their phenotypic differentiation in vitro (Morrison et al., 1988; Lärkfors et al., 1996). In CGNs, on the other hand, NT-4 promotes survival as well as neurite extension and dendritic arborization in a similar manner to BDNF, via the TrkB-dependent pathway (Gao et al., 1995). In murine CGN cultures, NT-4 has a cytoprotective capacity and can circumvent glutamate-induced oxidative death via activation of the PI3K or MAPK pathway (Kubo et al., 1995; Shimoke et al., 1997; Skaper et al., 1998). NT-4 has similar capacities to BDNF and NT-3 in promoting correct circuit wiring of the cerebellar cortex and, concomitant to BDNF, NT-4 aids inhibitory synaptogenesis (Seil, 1999). Additionally, NT-4 promotes initial olivary axonal growth to the cerebellar plate and CF synaptogenesis as well as axonal outgrowth and survival of pontocerebellar MF neurons (Rabacchi et al., 1999; Sherrard and Bower, 2002). et al., 1998). Concomitant to BDNF, CAPS2-mediated NT-3 release is known to be involved in the development and maturation of synapses and the balance between inhibitory and excitatory synapses (Sadakata et al., 2014; Shinoda et al., 2019). In addition, NT-3 promotes initial olivary axonal outgrowth to the cerebellar cortex and early CF synaptogenesis onto PCs (Sherrard and Bower, 2002). NT-3 also has an important role in CGN development. In the murine cerebellum, NT-3 promotes the differentiation of premigratory granule cells, accelerating the cell cycle exit (Neveu and Arenas, 1996; Doughty et al., 1998; Takumi et al., 2005). PGRN PGRN is the precursor protein for granulin, expressed in both the periphery and central nervous system (Townley et al., 2018). The propeptide is mainly delivered into lysosomes as it plays a role in regulating protein homeostasis via the lysosomal pathway (Paushter et  al., 2018). It has also been hypothesized to have neurotrophic capacities and function as an autocrine neuronal growth factor (Van Damme et al., 2008; Paushter et al., 2018). The expression levels of PGRN are thought to be regulated by the sortilin receptor which mediates its lysosomal endocytosis (Kao et al., 2017). However, PGRN can also exert its neurotrophic properties in a sortilin-independent manner, via prosaposin which carries PGRN into the lysosomes and regulates its expression (Zhou et al., 2015). PGRN is highly expressed in the cerebellar PCs during the late stages of postnatal development (Matsuwaki et al., 2015). NT-3 It does this via either direct autocrine signaling, indirect via PCs, or by a combination of both and synergistically to BDNF (Minichiello, 1996). More recently, it was found that proNT-3, and not mature NT-3, affects GCP proliferation and differentiation. ProNT-3 binds to p75NTR and the co-receptor SorCS2, which is a member of the sortilin receptor family, to antagonize the shh-induced proliferation of GCPs and initiate cell cycle exit (Zanin et al., 2016, 2019; Zanin and Friedman, 2022). NT-3, on the other hand, aids the migration of newly differentiated GCPs from the EGL in vivo, an effect which is antagonized by p75NTR (Neveu and Arenas, 1996). Since GCPs express p75NTR during their proliferative but not their migratory state, p75NTR effectively prevents GCPs from migrating by maintaining elevated levels of active RhoA, a member of the Rho-GTPase family that plays a role in neuronal migration (Zanin and Friedman, 2022). Once CGPs have differentiated to CGNs and completed proliferation, NT-3 provides maturation support to these neurons (Segal et al., 1992). In vitro, NT-3 is known to support the survival of mature CGNs via TrkC (Bates et al., 1999; Katoh-Semba et al., 2000; Joo et al., 2014) and provide neuroprotective capacities in low K+ cultured CGNs (Kubo et al., 1995; Shimoke et al., 1997; Bates et al., 1999; Katoh-Semba et al., 2000; Joo et al., 2014). Frontiers in Molecular Neuroscience NT-3 in cerebellar-related neurodevelopmental disorders Abnormalities in the expression of NT-3 have been associated with autism spectrum disorders. Exorbitant levels of NT-3 affect normal axonal targeting and synapse formation, and result in a decrease in PC numbers, all of which are effects seen in ASD pathology (Sajdel-Sulkowska et al., 2009, 2011). CAPS2 is essential for the release of NT-3 from CGNs, however, in patients with ASD, an alternative splice variant of CAPS2 that lacks exon 3, namely dex3, alters the release of NT-3. In a representative mouse model, NT-3 is markedly reduced in the axons of CGNs, an effect that results in reduced PC arborization and GCP proliferation. This leads to both a smaller vermal volume, as well as impaired paired-pulse facilitation at PF-PC synapses which is in line with autistic phenotypes (Sadakata et al., 2014). Matsuwaki et al. (2015) found that in PGRN-deficient mice, the PC dendritic density is significantly increased, possibly due to a lack of synaptic pruning, while no changes occur in the number of PCs. Taken together, this suggests that PGRN affects PC dendritogenesis but not neurogenesis and/or survival. However, in a study by Wang et al. (2022), it was found that PGRN aids neuronal survival and synaptic development via activation of the PI3K-Akt signaling pathway. In accordance with its role in synapse formation, PGRN also plays a role in defining CFs as a PC-derived regulator, by both counteracting redundant CFs and reinforcing the strongest CF inputs via the sortilin-dependent pathway. This retrograde mechanism is driven by voltage-gated calcium channels and the mGLuR1 signaling cascade (Uesaka et al., 2018). NT-4 in cerebellar-related neurodevelopmental disorders In a murine model for CMD, expression of NT-4 is markedly reduced in the cerebellum, in the spinal cord, and hindlimb muscles. Such a marked decrease may contribute to the progressive degeneration of muscle fibers and, due to its role in CGN and PC survival, cerebellar hypoplasia (Sakuma et al., 2002). TGF-β in cerebellar-related neurodevelopmental disorders TGF-β is a multipotent cytokine which is generally induced by acute or chronic brain injury, however, it also has cell differentiation, proliferation and apoptotic capacities during development (Dobolyi and Palkovits, 2008). Nonetheless, TGF- β does not only exert a neuroprotective function, as it can also induce neuronal and glial degeneration after injury (Wang et al., 1995; Yamashita et al., 1999). TGF-β exists as three isoforms in mammals, namely TGF-β1, TGF-β2, and TGF-β3 (Voisin et al., 2020). Under normal conditions, TGF-β is scarcely expressed within the cerebellum, however, TGF-β2 expression can be found in GCPs of the EGL and post-mitotic CGNs located in the IGL of the developing cerebellar cortex until postnatal day 10. It does remain expressed in PCs during both development and adulthood (Constam et al., 1994; Kane et al., 1996). TGF-β1 expression, on the other hand, is low during early postnatal stages but increases after postnatal day 12 and remains high until postnatal day 30 (Araujo et al., 2016). TGF- β signaling is initiated by the binding of extracellular TGF-β ligands to their respective receptors forming a complex. This complex formation allows phosphorylation of Smad proteins which then translocate to the cell nucleus to regulate the expression of multiple early target genes, including those that have a role in cell proliferation and differentiation, for example ID1-3, CDKN1A, OVOL1 and JUNB (Kowanetz et al., 2004; Zhang et al., 2016). However, their effects on cerebellar neurons are yet to be unraveled. We, therefore, propose that more studies are warranted for delineating cellular mechanisms regarding cerebellar-related neurodevelopmental disorders. MB is thought to arise from disruptions in cerebellar development and growth factors, such as TGF-β, are thought to play a role in its progression (Marino, 2005; Roussel and Hatten, 2011; Santhana Kumar et al., 2018). The canonical TGF-β signaling pathway involves activation of Smad3 in a subset of GCPs that possibly represents the putative cells of origin for MB (Aref et al., 2013). In a murine model of autism, TGF-β1 expression is significantly decreased within the cortex, hippocampus, and cerebellum. This is in line with a previous study which found reduced levels in patients with ASD (Ferretti and Hollander, 2015; Xu et al., 2017). Cerebellar ataxia (CA) is usually accompanied by microglia- mediated neuroinflammation, yet how it contributes to cerebellar pathogenesis remains unsolved. NT-4 NT-4 has similar properties to BDNF within the developing cerebellum, although its expression levels peak higher during the first postnatal week (Proenca et al., 2016). NT-4 signals predominantly through the TrkB receptor and has a functional role in both CGN and PC maturation as well as survival and inhibitory synaptogenesis 11 frontiersin.org Boxy et al. 10.3389/fnmol.2023.1181397 10.3389/fnmol.2023.1181397 TGF-β in cerebellar-related neurodevelopmental disorders In one study of CA model rats, it was found that exogenous administration of anti-inflammatory TGF-β1 reduces neuronal loss and microglial activation in both brain stem and cerebellum, and consequently ameliorates motor deficits as seen in CA (Cao et al., 2020). In another study of cerebellar ataxia, it was found that TGF-β1 is significantly upregulated, likely as a result of increased neuroinflammation (Jiang et al., 2015). frontiersin.org PGRN in cerebellar-related neurodevelopmental disorders capacity to change the electrophysiological properties and voltage- dependent ion currents of CGNs after injury which leads to functional changes in the CNS (Ondáčová et al., 2017). However, TGF-β can also serve as a pro-apoptotic agent in low K+ cultured CGNs (de Luca et al., 1996a). More specifically, TGF-β1 has a neurotoxic effect on mixed neuronal and astrocytic cultures as the CGNs become dependent on astrocytes for survival. TGF-β1 acts as a cytokine and inhibits the ability of astrocytes to clear glutamate, which leads to an increase in the glutamate concentration within the media that is toxic to the CGNs and eventually decreases their survival (Brown, 1999). TGF-β2, on the other hand, differently regulates proliferation and survival of CGNs, depending on the media conditions. TGF-β2 functions as a proliferative agent in serum-treated media, while it inhibits proliferation in serum-free media, indicating that as it requires exogenous regulatory factors (Constam et al., 1994; Kane et al., 1996). PGRN has previously been implicated in cerebellar-associated degenerative but also in neurodevelopmental disorders (Matsuwaki et al., 2015; Simonati and Williams, 2022). It has been shown that PGRN is essential for PC dendritogenesis and CF-PC synaptogenesis and that abnormalities in PGRN expression may lead to synaptic disturbances as well as behavioral deficits, such as impaired motor function and coordination, reduced social preference, and increased repetitive behaviors (Matsuwaki et al., 2015; Uesaka et al., 2018; Wang et al., 2022). These behavioral phenotypes are all characteristic of those seen in ASD patients. Wang et al. (2022) found that abnormal spatiotemporal expression of PGRN is related to neurodevelopmental impairments in an ASD murine model. Frontiers in Molecular Neuroscience References Requirement of TrkB for synapse elimination in developing cerebellar Purkinje cells. 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R248-2017-431 and PROMEMO grant DNRF133. Conclusion Neurotrophic factors and their receptors exert different cellular functions, and their spatiotemporal expression is crucial for the normal development of the cerebellar cytoarchitecture. In this review, we mainly focused on the purpose of these factors during the postnatal development of cells within the cerebellar cortex. However, several are also expressed during adulthood and play a role in both short-term and long-term synaptic plasticity. Because of their multifaceted features in neuronal differentiation, survival, synaptogenesis, and circuit wiring, it is inferred that the ablation of these factors can lead to serious defects on the tissue, cellular and molecular levels. A range of neurological disorders with a cerebellar component and abnormalities in either neurotrophic factor expression and/or activity have been discussed. It is important to note that this is not restricted to motor disorders, which are known to involve the cerebellar system, but also non-motor disorders. This indicates that the cerebellum, supplementing its role in motor performance, also plays a crucial role in cognitive and emotional Deletion of Smad4, a critical mediator of TGF-β, results in Purkinje and GABAergic interneuron cell loss which leads to neurobehavioral deficits, including motor dysfunction (Zhou et al., 2003). Another study found that Smad2, another mediator of TGF-β signaling and highly expressed in the mouse brain during early postnatal development, is necessary for proper cerebellar foliation. Absence of Smad2 results in aberrant PC dendritic arborization and cell loss as well as other cerebellar deficits such as increased apoptosis and defect migration of CGNs which leads to motor dyscoordination (Wang et al., 2011). TGF-β has a pro-survival and pro-growth effect on GCPs (Elvers et al., 2005). TGF-β1 effectively increases the number of glutamatergic synapses in CGN cultures, an effect which is dependent on binding to its receptor, TβRII. TGF-β1 thus mediates excitatory synapse formation in CGNs (Araujo et al., 2016). Moreover, TGF-β1 has the 12 Boxy et al. 10.3389/fnmol.2023.1181397 Conflict of interest development. Further studies regarding neurotrophic factors and the effector downstream signaling mechanisms within the cerebellar system could illuminate whether they might serve as pharmacological agents to moderate certain disease models. development. Further studies regarding neurotrophic factors and the effector downstream signaling mechanisms within the cerebellar system could illuminate whether they might serve as pharmacological agents to moderate certain disease models. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 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Transcultural Adaptation and Validation of the Spanish Bristol Foot Score (BFS-S)
Aging and disease
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Volume 9, Number 5; 861-868, October 2018 Original Article Original Article Emmanuel Navarro-Flores1, Marta Elena Losa-Iglesias2, Ricardo Becerro-de-Bengoa-Vallejo3, Daniel Lopez-Lopez4, *, Juan Manuel Vilar-Fernandez5, Patricia Palomo-Lopez6, Cesar Calvo- Lobo7 1Faculty of Medicine, Universidad Miguel Hernandez de Elche, and Department of Nursing and Podiatry, University of Valencia, Spain 2Faculty of Health Sciences, Universidad Rey Juan Carlos, Spain y p ing, Physiotherapy and Podiatry, Universidad Complutense de Madrid, Spain 3School of Nursing, Physiotherapy and Podiatry, Universidad Complutense de Madrid, S 4Research, Health and Podiatry Unit, Department of Health Sciences, Faculty of Nursing and Podiatry, Universidade da Coruna, Spain 4Research, Health and Podiatry Unit, Department of Health Sciences, Faculty of Nursing and Podiatry, Universidade da Coruna, Spain 5Modeling, Optimization and Statistical Inference Research Group, Universidade da Coruna, Spain 6University Center of Plasencia, Universidad de Extremadura, Spain 5Modeling, Optimization and Statistical Inference Research Group, Universidade da Coruna, Spain 6University Center of Plasencia, Universidad de Extremadura, Spain 7Nursing and Physical Therapy Department, Institute of Biomedicine (IBIOMED), Universidad de Leon, Ponferrada, Leon, Spain Copyright: © 2017 Navarro-Flores E et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Transcultural Adaptation and Validation of the Spanish Bristol Foot Score (BFS-S) Emmanuel Navarro-Flores1, Marta Elena Losa-Iglesias2, Ricardo Becerro-de-Bengoa-Vallejo3, Daniel Lopez-Lopez4, *, Juan Manuel Vilar-Fernandez5, Patricia Palomo-Lopez6, Cesar Calvo- Lobo7 *Correspondence should be addressed to: E. Navarro-Flores (manu.navarroflores@gmail.com), ME Losa-Iglesias (marta.losa@urjc.es), R. Becerro-de-Bengoa-Vallejo (ribebeva@ucm.es), D. Lopez-Lopez (daniellopez@udc.es), JM. Vilar-Fernandez (juan.vilar@udc.es), P. Palomo-Lopez (patibiom@unex.es), C. Calvo-Lobo (cecalvo19@hotmail.com). These authors contributed equally to this work. Correspondence should be addressed to: E. Navarro Flores (manu.navarroflores@gmail.com), ME Losa Iglesias (marta.losa@urjc.es), R. Becerro-de-Bengoa-Vallejo (ribebeva@ucm.es), D. Lopez-Lopez (daniellopez@udc.es), JM. Vilar-Fernandez (juan.vilar@udc.es), P. Palomo-Lopez (patibiom@unex.es), C. Calvo-Lobo (cecalvo19@hotmail.com). These authors contributed equally to this work. Copyright: © 2017 Navarro-Flores E et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Translation procedure The recommended forward/backward translation protocol was applied for the procedure of translation, transcultural adaptation and validation from United Kingdom to Spain [2,3,12–14]. The translation procedure was conducted according to the recommended international guidelines [12,17]. Considering the BFS domains, 3 underlying factors were considered. First factor, concerns about feet and pain was shown to be the most powerful to predict the 50% of the set of 15 responses. Second and third factors, footwear and general foot health as well as mobility were reported to predict the 10% and 9% of the variance, respectively [10]. Nevertheless, transcultural adaptation, contruct validity and reliability should be carried out following guideliness in order to preservate the crosscultural measurement properties [3,12-14]. To date, the BFS has not been adapted or validated to Spanish language [10,15]. Therefore, this study aim was to perform the transcultural adaptation and validation of the Spanish BFS version (BFS-S). First, the author of the original questionnaire (SB) was contacted in order to carried out this translation [10]. Second, forward translation was performed by two independent bilingual Spanish translators. Third, the reconciliation in the forward translations was performed and written with each translator separately. Fourth, the reconciled forward translated version of the BFS-S was translated back to Spanish by 4 authors (ENF, DLL, PPL and CCL), 3 podiatrists and 1 physiotherapist PhD university professors. Fifth, the translated version was compared with the original version to be sure about conceptual equivalence of the translation, discrepancy or unclear terms. Sixth, the harmonization was carried out by an expert panel formed by 6 authors (ENF, DLL, PPL, CCL, MELI and RBBV), 5 podiatrists and 1 physiotherpist PhD university professors, in order to be agreeing about the translation. Seventh, cognitive interviews were carried out in physiotherapy and podiatry centers in order to provide validity and avoid potential errors [17]. Finally, the proofread version of the BFS-S was composed by a Likert scale to improve administration and psychometric properties [2,10]. Participants The Bristol Foot Score (BFS) may be considered as a self- reported health questionnaire with 15 items for measuring the impact of foot problems such as concern and pain (7 items), footwear and general foot health (4 items), and patient mobility (3 items). The BFS was developed and validated in the United Kingdom with a high reliability (Cronbach α = 0.90) [10]. This questionnaire is sensitive to change after toenail surgery. Nevertheless, a poor level of concordance was reported between the BFS and the Chiropody Assessment Criteria Score [10,11]. Consequently, the BFS may reflect patients’ perceptions of their own foot health and may be useful for assessing the efficacy after interventions and establishing foot health within populations [10]. Despite the domains of the FHSQ (foot pain, foot function, footwear, and general foot health), FFI (pain, disability and activity limitation) and MFPDI (foot pain and function) may be considered similar tools validated and translated into Spanish [1-4], specifically the BFS adds new domains such as the patient mobility [10]. A total sample of 53 participants with a mean ± SD (range) of 49.55 ± 16.17 (23-78) years, 69.26 ± 11.92 (47- 98) kg, 168 ± 0.08 (151-189) cm and 24.20±3.37 (17.68- 35.54) kg/cm2 was recruited from podiatry and physiotherapy clinical centers. Inclusion criteria comprised participants with foot pain for at least the past 3 months. Exclusion criteria included psychiatric or cognitive disorders in the medical record, refusal to give consent form and the inability to following the instructions necessary to carry out the present investigation [1-4,10]. [Received October 28, 2017; Revised December 14, 2017; Accepted December 15, 2017] [Received October 28, 2017; Revised December 14, 2017; Accepted December 15, 2017] ABSTRACT: The Bristol Foot Score is considered an instrument for measuring the impact of foot problems and pain. It was developed and validated in United Kingdom. Therefore, this aim was to perform the transcultural adaptation and validation of the Spanish version. The recommended forward/backward translation protocol was applied for the procedure of translation, transcultural adaptation and validation to Spain. Considering each domain and question, internal consistency and reliability were analyzed through the Crombach alpha (α) and intraclass correlation coefficient (ICC) with a 95% confidence interval (95% CI). A very good internal consistency was shown for the 3 domains: concern and pain showed a Cronbach of 0.896, footwear and general foot health of 0.790, mobility 0.887. Each question had a very good test-retest reliability, ranged from 0.721 to 0.963 with no systematic differences (P>0.05) in each question of the Spanish Bristol Foot Score (BFS-S) questionnaire. The test-retest reliability was excellent (ICC 95%): concern and foot pain 0.950 (0.913-0971); footwear and general foot health 0.914 (0.851-0.950), mobility 0.973 (0.953-0.984) and there were no sistematic differences in any domain (P > 0.05). The BFS-S was shown to be a valid and reliable tool with an acceptable use in the Spanish population. Key words: foot, quality of life, health impact assessment, validation studies Worldwide, clinimetric tools such as the Foot Health Status Questionnaire (FHSQ), Foot Function index (FFI) as well as Manchester Foot Pain and Disability Index (MFPDI) were validated and translated for assessing the quality of life related to patient´s foot health [1-4]. Approximately, foot pain and disorders were presented in 25% of the adult population [5]. Up to 8% of musculoskeletal pain consultations by general practitioners were related to foot and ankle conditions [6]. Indeed, foot pain may increase this prevalence in older 861 ISSN: 2152-5250 Bristol Foot Score (BFS-S): Spanish version Navarro-Flores E., et al was obtained from all subjects. The Helsinki Declaration, Organic Low of Protection Data (15/1999) and ethical standards in human experimentation were respected. adults with specific foot conditions being associated to higher disability [7]. In addition, the worst quality of life related to the foot health may be associated to the risk increase of fall [8,9]. Study design A cross-sectional descriptive study was carried out between june and september 2017, following The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement and checklist [16]. Transcultural adaptation and validation was performed using the BFS as a clinimetric tool [10]. Aging and Disease • Volume 9, Number 5, October 2018 Ethical statements Test-retest was performed by the following link: https://docs.google.com/forms/d/e/1FAIpQLSfMG yHjbZf75C23562HVZlfoUhpPA_1SozoN_UvzU9p6dZ The Ethics Committee approval was obtained from the University of La Coruña. Furthermore, informed consent Aging and Disease • Volume 9, Number 5, October 2018 862 Bristol Foot Score (BFS-S): Spanish version Navarro-Flores E., et al gHw/viewform. Furthermore, the sociodemographic data (age, sex, profession and study degree), comorbidities (diabetes, peripheral vascular disease, rheumatism, psoriasis, and osteoarthritis), lifestyle (sedentary or active) and foot conditions were self-reported in this link. Participants with foot conditions were recruited from podiatry and physiotherapy clinical centers where universitary students carried out their practices. A pilot study was conducted in order to establish the linguistic comprehension of the BFS-S. Considering a correlation with an ICC of 0.40 and a 95% confidence interval (CI) for a two-tailed test, an error α of 0.05 and a desired analysis power of 80% (error β = 20%), a final sample size of 53 paticipants was obtained. The sample was heterogeneous in order to test this questionnaire for multiple and variated foot conditions [2]. The questions and domains (concern and pain; footwear and general foot health; and mobility) scores of the BFS-S were collected [10]. All patients were able to complete the questionnaire by themselves and the time employed in filling it out was approximately about 5 minutes. Table 1. Socio-demographic characteristics of the sample population. Total group Mean ± SD Range N = 53 Men Mean ± SD Range N = 23 Women Mean ± SD Range N = 30 P Value Age, years 49.55±16.17 (23-78) 54.33±15.32 (47.86-60.79) 45.58±16.01 (39.49- 51.66) 0.004 Weight (kg) 69.26±11.92 (47-98) 72.20±9.11 (73.35-81.04) 62.68±9.84 (58.93-66.42) 0.747 Height (cm) 168±0.08 (151-189) 1.74±0.7 (1.70-1.77) 164±0.05 (161-166) 0.756 BMI (kg/m2) 24.20±3.37 (17.68-35.54) 25.42±0.07 (25.39-25.44) 23.18±3.69 (21.77-24.58) 0.082 Abbreviations: BMI, body mass index; SD, standard deviation. In all the analyses, P < .01 (with a 99-confidence interval) was considered statistically significant. P-values are from Independent student t-test. Table 1. Socio-demographic characteristics of the sample population. Abbreviations: BMI, body mass index; SD, standard deviation. In all the analyses, P < .01 (with a 99-confidence interval) was considered statistically significant. P-values are from Independent student t-test. Aging and Disease • Volume 9, Number 5, October 2018 Statistical analysis use of coefficient of variation (CV) values has been the most common approach previously to examine variability between tests, and in the current study, a %CV for method error was calculated as follows: CV = 100 × (2 × (SDd /√2)/(X1 + X2) [19]. SDd represents the standard deviation of the differences between the two tests, and X1 and X2 represent the two-test means, respectively. The 95% limits of agreement statistics (LoA) were also calculated for the absolute comparison of parameters and express the degree of error proportional to the mean; the statistics were calculated using the methods described by Bland and Altman [20] and if the differences between the measurements tend to agree, the result will be close to zero. In addition, standard errors of measurement (SEM) were calculated to measure the range of error of each gait parameter. SEM is a quantitative expression of the range of error that can occur whenever the same participant repeats certain tests. In addition, SEM values were calculated from the ICCs and SDs for each session, using the higher of the 2 SD measurements to determine the range of error attributed between sessions. SEM were calculated according to the formula SEM = SD × sqrt (1 - ICC). Similarly, and for convenience of interpretation, the use of coefficient of variation (CV) values has been the most common approach previously to examine variability between tests, and in the current study, a %CV for method error was calculated as follows: CV = 100 × (2 × (SDd /√2)/(X1 + X2) [19]. SDd represents the standard deviation of the differences between the two tests, and X1 and X2 represent the two-test means, respectively. The 95% limits of agreement statistics (LoA) were also calculated for the absolute comparison of parameters and express the degree of error proportional to the mean; the statistics were calculated using the methods described by Bland and Altman [20] and if the differences between the measurements tend to agree, the result will be close to zero. In addition, standard errors of measurement (SEM) were calculated to measure the range of error of each gait parameter. SEM is a quantitative expression of the range of error that can occur whenever the same participant repeats certain tests. Statistical analysis In addition, to determine the smallest amount of change that is real and beyond the bound of measurement error, minimum detectable changes (MDC) were calculated at a confidence level of 95%: MDC values, which reflect the magnitude of change necessary to provide confidence that a change is not be the result of random variation or Aging and Disease • Volume 9 Number 5 October 2018 864 Table 2. Results of reliability, test-retest of the Spanish Bristol Foot Score (BFS-S) questionnaire according to each question. TEST (n=53) Mean ± SD (CI 95%) RETEST (n=53) Mean ± SD (CI 95%) ICC (CI 95%) P- value SEM %CV SEM% LoA Mean diference (limits) MDC P-value Breusch- Pagan Question 1. Do problems with your feet affect whether you go out of the house to visit family or friends? 1.60±0.92 (1.34-1.85) 1.56±0.86 (1.32-1.80) 0.963 (0.936-0.79) 0.419 0.005 1.684 0.321 0.038 (-0.324-0.999) 0.014 0.103 Question 2. Do problems with your feet affect whether you walk to the shops? 1.62±0.90 (1.37-1.87) 1.67±0.91 (1.42- 11.93) 0.959 (0.928-0.976) 0.261 0.008 2.424 0.494 -0.057 (-0.348-1.073) 0.023 0.239 Question 3. Do problems with your feet affect you when standing still? 1.67±0.97 (1.41-1.94) 1.73 ±.092 (1.48-1.99) 0.962 (0.935-0.978) 0.261 0.008 2.344 0.457 -0.057 (-0.348-1.073) 0.022 0.001 Question 4. Do problems with your feet affect you when walking on bumpy or stony ground? 2.18±1.05 (1.98-2.48) 2.15 ±.1.02 (1.86-2.43) 0.944 (0.903-0.968) 0.569 0.006 1.230 0.289 0.038 (-0.460-1.417) 0.017 0.007 Question 5. Over the last two weeks how painful have your feet been? 2.16±1.29 (1.81-2.52) 2.13±1.27 (1.78-2.48) 0.897 (0.822-0.941) 0.727 0.024 2.417 1.107 -0.075 (-1.095-3.377) 0.068 0.002 Question 6. Over the last two weeks, how often have you felt this way about your feet? "I have felt conscious of my feet". 2.81±1.75 (2.32-3.29) 2.64±1.71 (2.16-3.11) 0.918 (0.857-0.953) 0.201 0.026 3.402 0.949 0.132 (-0.884-2.725) 0.072 0.091 Question 7. Over the last two weeks, how often have you felt this way about your feet? "I have felt fed up about my feet". 2.45±1.68 (1.98-2.91) 2.35±1.69 (1.89-2.82) 0.950 (0.913-0.971) 0.358 0.015 2.773 0.622 0.094 (-0.711-2.192) 0.041 0.010 Question 8. Over the last two weeks, how often have you felt this way about your feet? "I have felt worried that my feet will get worse in the future". 2.54±1.61 (1.79-2.69) 2.13±1.56 (1.69-2.56) 0.933 (0.883-0.961) 0.308 0.021 3.657 0.950 0.113 (-0.768-2.369) 0.058 0.926 Question 9. Over the last two weeks, have you felt this way about your feet? Statistical analysis In addition, SEM values were calculated from the ICCs and SDs for each session, using the higher of the 2 SD measurements to determine the range of error attributed between sessions. SEM were calculated according to the formula SEM = SD × sqrt (1 - ICC). Similarly, and for convenience of interpretation, the All variables were examined for normality of distribution using the Kolmogorov-Smirnov test, and data were considered normally distributed if P > 0.05. Independent Student t-tests were performed to find if differences are statistically significative when showing a normal distribution. Measurements which were not normally distributed were tested using non-parametric Wilcoxon signed-rank test. Considering each domain and question, internal consistency and reliability were analyzed through the Crombach alpha (α) with 0 indicating no internal consistency and 1 corresponding to perfect internal consistency and intraclass correlation coefficient (ICC) with a 95% confidence interval (95% CI). To interpret ICC values, we used benchmarks as proposed by Landis and Koch [18]: 0.20 or less, slight agreement; 0.21 to 0.40, fair; 0.41 to 0.60, moderate; 0.61 to 0.80, substantial; and 0.81 or greater, almost perfect. For the statistical analysis, a two-way random effects model (2.1), single measures, absolute agreement, and ICC were used to express reliability. In addition, paired samples t-test was applied to test systematic differences between test and retest. The Aging and Disease • Volume 9, Number 5, October 2018 863 Bristol Foot Score (BFS-S): Spanish version Navarro-Flores E., et al measurement error, were calculated as follows [21]: MDC = √2 × 1.96 × SEM. Furthermore, Bland and Altman plots were analyzed to evaluate agreement and heteroscedasticity [20]. Each measure was evaluated for homoscedasticity with Breusch–Pagan test for heteroscedasticity (P < 0.05) in a linear regression model [22]. A P value < 0.05 with a confidence interval of 95% was considered statistically significant for all tests (SPSS for Windows, version 20.0; SPSS Inc., Chicago, Illinois). percent error of the SEM (SEM%) was calculated as the SEM divided by the mean per 100 and provided an estimate of the inherent error or variability normalized to the mean (SEM % = SEM/mean*100 %) [20]. Statistical analysis DOMAIN Test Mean ± SD (CI 95%) Retest Mean ± SD (CI 95%) ICC (CI 95%) P- value SEM %CV SEM % LoA Mean diference (limits) MDC P-value Breusch- Pagan Concern and pain 13.69±7.81 (11.54-15.85) 13.56±7.14 (11.59-15.53) 0.950 (0.913-0971) 0.945 0.021 0.685 0.153 0.132 (-3.151-9.714) 0.058 0.020 Footwear and general foot health 8.35±3.51 (7.38-9.32) 8.64±3.63 (7.63-9.64) 0.914 (0.851-0.950) 0.487 0.059 2.354 0.691 -0.283 (-1.933-5.959) 0.163 0.002 Mobility 5.43 ±2.64 (4.70-6.16) 5.56±2.59 (4.85-6.28) 0.973 (0.953- 0.984) 0.357 0.015 1.698 0.282 -0.132 (-0.821-2.533) 0.043 0.041 Abbreviations: SD, Standard Desviation; CI 95%, confidence interval 95%; ICC, Intraclas Correlation Index. P value from Wilcoxon Signed-Rank Test; SEM, standard error of measurement; %CV, coefficient of variation; SEM%, percent error of the SEM; LoA, 95% limits of agreement statistics; MDC = minimum detectable change; P value from Breusch–Pagan test for heteroskedasticity Validation and reliability The sociodemographic data, such as age, weight, height, and BMI, were shown in table 1. All of the demographic variables presented a normal distribution (P > 0.05) and all items and domains presented a no normal distribution (P < 0.05). Tables 2 and 3 show the test and retest means, ICC, P-value for non-parametric test, SEM, %CV, SEM%, MDC and P-values from Breusch–Pagan test for heteroscedasticity. Wilcoxon Signed-Rank test demonstrated no systematic differences between test and retest for any ítem and domain (P > 0.05), shown in table Bristol Foot Score (BFS-S): Spanish version Navarro-Flores E., et al Navarro-Flores E., et al Question 10. Because of your feet have you had problems sleeping, in the last two weeks? 1.24±0.75 (1.03-1.45) 1.28±0.76 (1.07-1.49) 0.984 (0.972-0.991) 0.159 0.003 2.111 0.271 -0.038 (-0.185-0.569) 0.009 0.055 Question 11. In the last two weeks have you been able to put your everyday shoes on easily. 1.60±0.83 (1.37-1.83) 1.79±1.02 (1.50-2.07) 0.701 (0.481-0.827) 0.133 0.074 7.857 4.336 -0.189 (-0.864-2.664) 0.204 0.001 Question 12. Over the last two weeks how often have you been able to wear any shoes you liked. 1.94±1.44 (1.15-2.34) 2.15±1.59 (1.71-2.59) 0.888 (0.806-0.935) 0.125 0.050 7.169 2.429 -0.208 (-0.929-2.865) 0.138 0.001 Question 13. If you could afford any shoes you wanted, how easily could you find new shoes that fit comfortably? 2.16±0.87 (1.92-2.40) 2.11±0.84 (1.87-2.34) 0.860 (0.758-0.919) 0.497 0.015 1.869 0.696 0.057 (-0.578-1.781) 0.041 0.072 Question 14. In general, would you say your foot health is: 2.64±1.19 (2.37-2.97) 2.58±1.18 (2.25-2.91) 0.939 (0.895-0.965) 0.472 0.010 1.532 0.376 0.057 (-0.546-1.684) 0.027 0.917 Question 15. Translation The forward translations were performed with only minor discrepancies and a good agreement was observed Table 3. Results of reliability, test-retest of the Spanish Bristol Foot Score (BFS-S) questionnaire according to each domain. DOMAIN Test Mean ± SD (CI 95%) Retest Mean ± SD (CI 95%) ICC (CI 95%) P- value SEM %CV SEM % LoA Mean diference (limits) MDC P-value Breusch- Pagan Concern and pain 13.69±7.81 (11.54-15.85) 13.56±7.14 (11.59-15.53) 0.950 (0.913-0971) 0.945 0.021 0.685 0.153 0.132 (-3.151-9.714) 0.058 0.020 Footwear and general foot health 8.35±3.51 (7.38-9.32) 8.64±3.63 (7.63-9.64) 0.914 (0.851-0.950) 0.487 0.059 2.354 0.691 -0.283 (-1.933-5.959) 0.163 0.002 Mobility 5.43 ±2.64 (4.70-6.16) 5.56±2.59 (4.85-6.28) 0.973 (0.953- 0.984) 0.357 0.015 1.698 0.282 -0.132 (-0.821-2.533) 0.043 0.041 Abbreviations: SD, Standard Desviation; CI 95%, confidence interval 95%; ICC, Intraclas Correlation Index. P value from Wilcoxon Signed-Rank Test; SEM, standard error of measurement; %CV, coefficient of variation; SEM%, percent error of the SEM; LoA, 95% limits of agreement statistics; MDC = minimum detectable change; P value from Breusch–Pagan test for heteroskedasticity bility, test-retest of the Spanish Bristol Foot Score (BFS-S) questionnaire according to each domain. Table 3. Results of reliability, test-retest of the Spanish Bristol Foot Score (BFS-S) questionnaire acc Statistical analysis Would you say your general health is: 2.62±0.94 (2.36-2.88) 2.64±0.85 (2.40-2.87) 0.957 (0.925-0.975) 0.709 0.003 0.507 0.104 -0.019 (-0.352-1.085) 0.008 0.635 Abbreviations: SD, Standard Desviation; CI 95%, confidence interval 95%; ICC, Intraclas Correlation Index. P value from Wilcoxon Signed-Rank Test; SEM, standard error of measurement; %CV, coefficient of variation; SEM%, percent error of the SEM; LoA, 95% limits of agreement statistics; MDC = minimum detectable change; P value from Breusch–Pagan test for heteroskedasticity between the 2 versions. The back translations between BFS and BFS-S were similar in many of the items. Cognitive interviews showed good understanding and comprehension of the BFS-S. between the 2 versions. The back translations between BFS and BFS-S were similar in many of the items. Cognitive interviews showed good understanding and comprehension of the BFS-S. Aging and Disease • Volume 9, Number 5, October 2018 Statistical analysis "I have felt my feet are not really part of me". 1.37±0.62 (1.20-1.55) 1.30±0.57 (1.14-1.46) 0.886 (0.803-0.934) 0.159 0.018 3.984 1.356 0.075 (-0.369-1.139) 0.050 0.163 Table 2. Results of reliability, test-retest of the Spanish Bristol Foot Score (BFS-S) questionnaire a question. reliability, test-retest of the Spanish Bristol Foot Score (BFS-S) questionnaire according to each 864 Navarro-Flores E., et al Bristol Foot Score (BFS-S): Spanish version Aging and Disease • Volume 9, Number 5, October 2018 865 Question 10. Because of your feet have you had problems sleeping, in the last two weeks? 1.24±0.75 (1.03-1.45) 1.28±0.76 (1.07-1.49) 0.984 (0.972-0.991) 0.159 0.003 2.111 0.271 -0.038 (-0.185-0.569) 0.009 0.055 Question 11. In the last two weeks have you been able to put your everyday shoes on easily. 1.60±0.83 (1.37-1.83) 1.79±1.02 (1.50-2.07) 0.701 (0.481-0.827) 0.133 0.074 7.857 4.336 -0.189 (-0.864-2.664) 0.204 0.001 Question 12. Over the last two weeks how often have you been able to wear any shoes you liked. 1.94±1.44 (1.15-2.34) 2.15±1.59 (1.71-2.59) 0.888 (0.806-0.935) 0.125 0.050 7.169 2.429 -0.208 (-0.929-2.865) 0.138 0.001 Question 13. If you could afford any shoes you wanted, how easily could you find new shoes that fit comfortably? 2.16±0.87 (1.92-2.40) 2.11±0.84 (1.87-2.34) 0.860 (0.758-0.919) 0.497 0.015 1.869 0.696 0.057 (-0.578-1.781) 0.041 0.072 Question 14. In general, would you say your foot health is: 2.64±1.19 (2.37-2.97) 2.58±1.18 (2.25-2.91) 0.939 (0.895-0.965) 0.472 0.010 1.532 0.376 0.057 (-0.546-1.684) 0.027 0.917 Question 15. Would you say your general health is: 2.62±0.94 (2.36-2.88) 2.64±0.85 (2.40-2.87) 0.957 (0.925-0.975) 0.709 0.003 0.507 0.104 -0.019 (-0.352-1.085) 0.008 0.635 Abbreviations: SD, Standard Desviation; CI 95%, confidence interval 95%; ICC, Intraclas Correlation Index. P value from Wilcoxon Signed-Rank Test; SEM, standard error of measurement; %CV, coefficient of variation; SEM%, percent error of the SEM; LoA, 95% limits of agreement statistics; MDC = minimum detectable change; P value from Breusch–Pagan test for heteroskedasticity RESULTS Translation The forward translations were performed with only minor discrepancies and a good agreement was observed between the 2 versions. The back translations between BFS and BFS-S were similar in many of the items. Cognitive interviews showed good understanding and comprehension of the BFS-S. Table 3. Results of reliability, test-retest of the Spanish Bristol Foot Score (BFS-S) questionnaire according to each domain. Validation and reliability (P < 0.05). Tables 2 and 3 show the test and retest means, ICC, P-value for non-parametric test, SEM, %CV, SEM%, MDC and P-values from Breusch–Pagan test for heteroscedasticity. Wilcoxon Signed-Rank test demonstrated no systematic differences between test and retest for any ítem and domain (P > 0.05), shown in table The sociodemographic data, such as age, weight, height, and BMI, were shown in table 1. All of the demographic variables presented a normal distribution (P > 0.05) and all items and domains presented a no normal distribution 865 Navarro-Flores E., et al Bristol Foot Score (BFS-S): Spanish version 2 and 3, respectively. Calculated between-test variabilities (%CV) for each ítem are shown in table 2 ranged from 1.230 to 3.984, except for ítem 11 and 12 with a %CV of 7.857 and 7.161, respectivley. %CV for each domain is presented in table 3, ranged from 0.685 to 2.354. The MDC values for each item, shown in table 2, ranged from 0.008 to 0.204 and each domain, table 3, ranged from 0.043 to 0.101. The SEM% values for each item, shown in table 2, ranged from 0.104 to 4.366 and each domain, table 3, ranged from 0.132 to 0.691. Results of reliability, test-retest and systematic differences of the BFS-S questionnaire by questions and domains are shown in table 2 and 3, respectively. A very good internal consistency was shown for the three domains: concern and pain showed a Cronbach of 0.896, the domain footwear and general foot health of 0.790 and domain mobility 0.887; and retest reliability was shown for each domain: concern and pain (α = 0.896; ICC = 0.950 [95% CI = 0.913 - 0971]), footwear and general foot health (α = 0.790; ICC = 0.914 [95% CI = 0.851-0950]), and mobility (α = 0.887; ICC = 0.953 [95% CI = 0.953- 0.984]). The test-retest reliability was excellent (ICC 95%): concern and foot pain 0.950 (0.913-0971); footwear and general foot health 0.914 (0.851-0.950) and mobility 0.973 (0.953-0.984) and there were no sistematic differences in any domain (P > 0.05). For total score, statistically significant differences were not shown for the mean (SD) difference between test and retest (27.49 ± 13.18 [95% CI = 23.85-21.12] points; 27.77 ± 12.37 [95% CI = 24.36-31.17] points; P = 0.658). Bland and Altman plots visual distributions did not show statistically significant or clinically relevant differences from test to retest (Fig. 1). Figure 1. Validation and reliability Bland–Altman plot showing the agreem between test and retest for the mobility (A), concern pain (B), and footwear and general health (C) domains. DISCUSSION p y y The result generalizations of this study should be interpreted with caution due to a non-randomized consecutive sampling method was used. This study weakness may influence the participants´ behavior and the procedure results in a biased sample of the domains under study [25]. The major strengths of this study comprised the first novel validation and transcultural adaptation of the BFS, as well as the possibility to evaluate the quality of life related to patient´s foot health and mobility into Spanish [10]. Furthermore, the clinical application of this questionnaire comprised the quality of life related to foot health evaluation through a new validated and reliable tool in the Spanish adult and older adult populations regarding the most common foot conditions such as metatarsalgia, hallux valgus, hallux rigidus, lesser toe deformities, hyperkeratosis, nails disorders or plantar heel pain [26]. [4] [4] Paez-Moguer J, Budiman-Mak E, Cuesta-Vargas AI (2014). Cross-cultural adaptation and validation of the Foot Function Index to Spanish. Foot Ankle Surg, 20:34- 39. [5] Hawke F, Burns J (2009). Understanding the nature and mechanism of foot pain. J Foot Ankle Res, 2:1. [6] [6] Menz HB, Jordan KP, Roddy E, Croft PR (2010). Characteristics of primary care consultations for musculoskeletal foot and ankle problems in the UK. Rheumatology (Oxford), 49:1391-1398. [7] gy [7] Benvenuti F, Ferrucci L, Guralnik JM, Gangemi S, Baroni A (1995). Foot pain and disability in older persons: an epidemiologic survey. J Am Geriatr Soc, 43:479-484. [8] [8] Mickle KJ, Munro BJ, Lord SR, Menz HB, Steele JR (2011). Cross-sectional analysis of foot function, functional ability, and health-related quality of life in older people with disabling foot pain. Arthritis Care Res (Hoboken), 63:1592-1598. Finally, possible limitations should be considered regarding this study. First, the BFS-S was carried out from podiatry and physiotherapy clinical centers where universitary students carried out their practices, while the original BFS was developed from a podiatry department of the healthcare national service [10]. Second, test-retest was performed through a link in the present study, while the original BFS and other Spanish validated scales were developed by face to face self-reporting of the patient [3,4,10]. Finally, age distributions such as children were not considered in this version validation, while other scales such as the Oxford ankle foot questionnaire (OxAFQ) translation was validated from 5 to 16 years old [27]. DISCUSSION Considering international recommended guidelines [12,17], The BFS-S may be used as a valid and reliable tool for measuring the self-reported health impact of foot problems such as concern and pain, footwear and general foot health, and patient mobility in the Spanish population. The original BFS was validated in the Podiatry Department at the United Bristol Healthcare National Health Service Trust with a high reliability and sensitivity to change after clinical interventions [10,11]. Previously, Spanish transcultural adaptation and validation of foot health related questionnaires were carried out with similar results [3,4]. The Spanish version of the FFI (FFI-Sp) was valid and reliable tool with a very good internal consistency for evaluating pain (0.95) and disability (0.96) of the foot [4]. Furthermore, the Spanish MFPDI version was a robust measurement tool with 3 domains such as foot pain, function and appearance due to an adequate Rasch model, excellent reliability and unidimensionality were provided [3]. To the authors´ knowledge, this Spanish version may be considered as the first validation and transcultural adaptation of the original BFS. Furthermore, the BFS-S provided similar psychometric properties compared to the Spanish version of the FHSQ. An appropriated construct validity with moderate-to-high domains correlations was shown for the Spanish FHSQ (α = ≥0.739) and BFS-S (α = ≥0.790). Test-retest reliability was shown to be satisfactory for both Spanish FHSQ (ICC > 0.932) and Figure 1. Bland–Altman plot showing the agreement between test and retest for the mobility (A), concern and pain (B), and footwear and general health (C) domains. Aging and Disease • Volume 9, Number 5, October 2018 866 Bristol Foot Score (BFS-S): Spanish version Navarro-Flores E., et al BFS-S (ICC > 0.914) [23]. Comparing the domains from the section one of the FHSQ and the BFS, similar subscales were evaluated [1,10]. Nevertheless, the section two of the FHSQ assessed general health, physical activity, social capacity and vigour [1,24], while the BFS provided a new key domain evaluation for mobility [10]. to measure foot-health status. J Am Podiatr Med Assoc, 88:419-428. [2] [2] Jorgensen JE, Andreasen J, Rathleff MS (2015). Translation and validation of the Danish Foot Function Index (FFI-DK). Scand J Med Sci Sports, 25:e408-13. [3] [3] Gijon-Nogueron G, Ndosi M, Luque-Suarez A, Alcacer- Pitarch B, Munuera PV, Garrow A, et al (2014). Cross- cultural adaptation and validation of the Manchester Foot Pain and Disability Index into Spanish. Qual Life Res, 23:571-579. DISCUSSION Despite it may not influence the results of transcultural adaptation and validation, there were statistically significant age differences between men and women. [9] Kaoulla P, Frescos N, Menz HB (2011). A survey of foot problems in community-dwelling older Greek Australians. J Foot Ankle Res, 4:23. [10] Barnett S, Campbell R, Harvey I. The Bristol Foot Score: developing a patient-based foot-health measure. J Am Podiatr Med Assoc, 95:264-272. [11] Riskowski JL, Hagedorn TJ, Hannan MT (2011). Measures of foot function, foot health, and foot pain: American Academy of Orthopedic Surgeons Lower Limb Outcomes Assessment: Foot and Ankle Module (AAOS-FAM), Bristol Foot Score (BFS), Revised Foot Function Index (FFI-R), Foot Health Status Questionnair. Arthritis Care Res (Hoboken), 63:S229- S239. [12] Beaton DE, Bombardier C, Guillemin F, Ferraz MB (2000). Guidelines for the process of cross-cultural adaptation of self-report measures. Spine (Phila Pa 1976), 25:3186-3191. Conclusion [13] Scott NW, Fayers PM, Aaronson NK, Bottomley A, de Graeff A, Groenvold M, et al (2009). The practical impact of differential item functioning analyses in a health-related quality of life instrument. Qual Life Res, 18:1125-1130. The BFS-S was shown to be a valid and reliable tool with an acceptable use in the Spanish population and may be used for total or each domain scores, such as concern and pain, footwear and general foot health, and patient mobility. [14] [14] Tennant A, Penta M, Tesio L, Grimby G, Thonnard JL, Slade A, et al (2004). Assessing and adjusting for cross- cultural validity of impairment and activity limitation scales through differential item functioning within the framework of the Rasch model: the PRO-ESOR project. Med Care, 42:I37-48. [1] Bennett PJ, Patterson C, Wearing S, Baglioni T (1998). Development and validation of a questionnaire designed Aging and Disease • Volume 9, Number 5, October 2018 References [15] [15] Walmsley S, Williams AE, Ravey M, Graham A (2010). The rheumatoid foot: a systematic literature review of [15] Walmsley S, Williams AE, Ravey M, Graham A (2010). The rheumatoid foot: a systematic literature review of Aging and Disease • Volume 9, Number 5, October 2018 867 Bristol Foot Score (BFS-S): Spanish version Navarro-Flores E., et al psychotherapy research. J Consult Clin Psychol, 59:12– 19. patient-reported outcome measures. J Foot Ankle Res, 3:12. [22] Breusch T, Pagan A (1979). A simple test for heteroscedasticity and random coefficient variation. Econometrica, 47:1287–1294. [16] V Vandenbroucke JP, von Elm E, Altman DG, Gøtzsche PC, Mulrow CD, Pocock SJ, et al (2014). Strengthening the Reporting of Observational Studies in Epidemiology (STROBE): explanation and elaboration. Int J Surg, 12:1500-1524. [23] Cuesta-Vargas A, Bennett P, Jimenez-Cebrian AM, Labajos-Manzanares MT (2013). The psychometric properties of the Spanish version of the Foot Health Status Questionnaire. Qual Life Res, 22:1739-1743. [17] Wild D, Grove A, Martin M, Eremenco S, McElroy S, Verjee-Lorenz A, et al (2005). Principles of Good Practice for the Translation and Cultural Adaptation Process for Patient-Reported Outcomes (PRO) Measures: Report of the ISPOR Task Force for Translation and Cultural Adaptation. Value Heal, 8:94- 104. [24] Landorf KB, Radford JA (2008). Minimal important difference: Values for the Foot Health Status Questionnaire, Foot Function Index and Visual Analogue Scale. Foot, 18:15-19. [25] Altmann J (1974). Observational Study of Behavior: Sampling Methods. Behaviour, 49:227-266. [18] Landis JR, Koch GG (1977). The measurement of observer agreement for categorical data. Biometrics, 33:159-174. [26] Rodríguez-Sanz D, Tovaruela-Carrión N, López-López D, Palomo-López P, Romero-Morales C, Navarro-Flores E, et al (2018). Foot disorders in the elderly: A mini- review. Dis Mon, 64(3):64-91 [19] Portney L, Watkins M. Foundations of Clinical Research: Applications to Practice. 3rd ed. (Hall PP, ed.). New Jersey, 2009. [27] Martinkevich P, Moller-Madsen B, Gottliebsen M, Kjeldgaard Pedersen L, Rahbek O (2015). Validation of the translated Oxford ankle foot questionnaire in 82 Danish children aged between five and 16 years. Bone Joint J, 97-B:420-426. [20] Bland JM, Altman DG (1986). Statistical methods for assessing agreement between two methods of clinical measurement. Lancet (London, England), 1:307-310. [21] Jacobson N, Truax P (1991). Clinical significance: A statistical approach to defining meaningful change in Aging and Disease • Volume 9, Number 5, October 2018 868
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English
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BLOOD CULTURE IN TYPHOID FEVER.
Lancet
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To the Editors of THE LANCET. SiRs,-Mr. J. D. Malcolm in his paper published in THE LANCET of Feb. 23rd deals with a subject in which it is more important that correct theoretical views should be entertained than in some other spheres in which there is a legitimate difference of opinion. This is so because an apparently logical inference drawn from certain premisses may very well guide treatment which may have unfortunate consequences, and it is little satisfaction to the individual to suffer in the wake of an unimpeachable syllogism. One cannot but admire the careful consideration Mr. Malcolm has given to the important subject with which he deals nor the tenacity with which he defends his thesis that the blood- vessels are actually contracted in shock. His argument, how- ever, fails to carry conviction to my mind for several reasons, of which the following it may suffice to mention. My apology for again drawing attention to this subject, Sirs, is the importance of the question raised in Mr. Malcolm’s paper, his own standing as an earnest and observant surgeon, and the admirable boldness with which he has, in my view, shivered his lance on the tough shield of accepted opinion in this matter. I am, Sirs, yours faithfully, I am, Sirs, yours faithfully, y, ALEXANDER MORISOX. Upper Berkeley-street, W., March 2nd, 1907. ALEXANDER MORISON. y ALEXANDER MORISOX. Upper Berkeley-street, W., March 2nd, 1907. ALEXANDER MORISON. of which the following it may suffice to mention. In the first place I hope I do not misrepresent Mr. Malcolm when I say that he appears to me to fix his atten- tion too exclusively on the peripheral circulation. An active narrowing of the vessels, the blood-supply remaining the same and the heart itself not depressed in action, would necessarily result in the more forcible jetting of severed vessels, and Mr. Malcolm states that he is convinced from clinical observation that in shock this is so. His thesis would be successfully defended if these central and peripheral con- ditions could be accepted as normally occurring in shock. Indeed, one would be justified in concluding, were these things so, that any appreciable depression of vitality should be absent. But the experience of physicians certainly is that the cardio-vascular state in shock is one of abased central force accompanied by a simultaneous depression in the neuro-muscular activity of the peripheral circulation. 686 T 686 T 686 diseases are very limited in number, the employers, in order to protect themselves, will have to have all their employees medically examined before taking them on. This would lead to many thousands of employees who are now able to get a good wage being rejected. As far as the rejected ones are concerned they would be thrown upon the workhouses and the rates would go up. As regards the unrejected ones, their labour would be at a premium, they would command very high wages, prices would go up, and therefore foreign competition would be increased. present argument. In Mr. Malcolm’s argument from the river in nature to the circulation in shock there is the important difference that the force, volume, and direction of the water are assumed to be the same although displaced in their channels. For these reasons then, if for no others, in Mr. Malcolm’s closely reasoned argument, I confess that to my mind arterial depletion and venous repletion, in accord- ance with the long-entertained view, is the rule in shock, and any narrowing of arteries due to a comparative emptiness of blood in passively retracting elastic vessels. If these are also Dr. Crile’s views, whose work I regret I have not yet read, they corroborate opinions long and generally held. ou d I am, Sirs, yours faithfully, I am, Sirs, yours faithfully, ARNOLD J. GREENE, M.R.O.S. Eng., L.R.C.P. Lond. Wigan, March 4th, 1907. ARNOLD J. GREENE, M.R.C.S. Eng., L.R.C.P. Lond. I am, Sirs, yours faithfully, ARNOLD J. GREENE, M.R.O.S. Eng., L.R.C.P. Lond. Wigan, March 4th, 1907. ARNOLD J. GREENE, M.R.C.S. Eng., L.R.C.P. Lond. read, they opinions long generally As touching Mr. Malcolm’s argument from therapeutics based upon the beneficial effects of heat in shock, does not the good result flow from the arrest of arterial depletion and venous stagnation due to stimulation by way of the nervous system of the force of cardiac action, aspirative and propulsive ? Given unabated central force the appli- cation of cold in shock might not do great harm, but if, as is generally believed, the reverse be the case, all experience teaches that the road to fatal syncope would lie through the depression of the surface temperature by cold no less cer- tainly in medical contingencies than in such an exposure by shipwreck as the country has recently had to lament off the sullen coast of Holland. To the Editors of THE LANCET. SIRS,&mdash;Dr. Spriggs attempts to cover his retreat from an untenable position by bringing into prominence side issues which have little bearing on the matter in dispute. I am conversant with the methods which he describes in the paper to which he refers me, and with all deference to Dr. Spriggs I consider that, however suitable for the purposes of a class, it would be impossible to find methods more unsuited to the carrying out of an extensive research. I know that it is almost impossible to prepare good sections of human teeth by the paraffin method, but rats’ teeth are so small that with care it is quite possible to obtain a large percentage of success, and as thousands of serial sections were cut and examined for the purposes of this investigation I maintain that, for anyone who has anything else to do in life, the paraffin method, with all its drawbacks, was the only possible one to adopt adopt. In conclusion, may I suggest to Dr. Spriggs that he should not give too much credence to the appearances of photo- micrograms, however excellent he may consider them, especially in regard to such minute details as the appear- ances of individual cells, for, as a great histologist said many years ago, and as every tyro knows now, it is only with one’s finger on the fine adjustment that the intimate structure of a tissue can be unravelled. I am, Sirs, yours faithfully, Edinburgh, Feb. 23rd, 1907. J. H. GIBBS. J. H. GIBBS. I am, Sirs, yours faithfully, Edinburgh, Feb. 23rd, 1907. J. H. GIBBS. J. H. GIBBS. I am, Sirs, yours faithfully, Edinburgh, Feb. 23rd, 1907. J. H. GIBBS. J. H. GIBBS. lly, J. H. GIBBS. J. H. GIBBS. ON THE CONDITION OF THE BLOOD- VESSELS DURING SHOCK. To the Editors of THE LANCET. To the Editors of THE LANCET. The highest degree of shock as observed by physicians is associated with cardiac still-stand in diastole, both ventricles containing blood on necropsy and the venous system being engorged. There is nothing o show that this is not so, also, during life at the moment of such profound shock. The more gradual yet progressive depression of the cardio-vascular system in less sudden failure from shock is associated with diminished left ventricular and increased right ventricular repletion. This is observed after death and clinical signs indicate its presence during life under the circumstances mentioned. In the report of my remarks at the discussion on Mr. Malcolm’s paper I observe that by a trivial error in your usually accu- rate account of the transactions of medical societies I am made to represent both ventricles as containing little blood under these circumstances. My experience, however, is as above stated. To the Editors of THE LANCET. SIRS,-There are some points in the circular of the principal medical officer of the Local Government Board (quoted in your issue of Feb. 23rd) that seem to me to call for special comment and consideration. After remark- ing that the disease sometimes occurs in mild and anomalous forms that render identification difficult, the circular cites in illustration I the prevalence of what would seem to have been cerebro-spinal fever in Northamptonshire in 1890-91, where the malady was, for the most part, diagnosed as pneumonia or as sore throat, and by the occurrence of cerebro-spinal fever in Irthlingborough in the present year, where many of the persons attacked were regarded as suffer- ing from influenza." SIRS,-From the letter which appears in THE LANCET of Feb. 23rd Dr. C. C. Douglas seems to think that we have not accurately represented the facts in re- lation to the case of meth&aelig;moglobin&aelig;mia which was recorded by him and Dr. G. A. Gibson in your columns a few months ago. We should be very sorry if this were so. In that article they report only one bacterio- logical observation, but on the strength of it they suggest the term " microbic cyanosis " to take the place of methasmo- globinasmia. At the same time they say : "It seems to us very desirable that these observations should not be taken as final, and if opportunity presents itself they will be repeated, especially those of a bacteriological nature." What we said in referring to their paper was: "These findings (i.e., the bacteriological findings) were not con- firmed and the authors expressly state that they should not be taken as final." There does not seem to be any real differ- ence in meaning between these two passages, for we are not aware that anything has been published in relation to their case since the original article in July, from which the words quoted are taken. If the bacteriological observation has been confirmed it is important that that should be reported. ing This ignores entirely the possibility of mixed infections, which I have often witnessed since the year 1890, though very seldom before. The cases that were diagnosed as pneu- monia and sore-throat respectively were, in all probability, quite correctly so named, and it is just as likely that cerebro- spinal fever was also present either simultaneously or con- secutively. To the Editors of THE LANCET. SIRS,-I am a regular reader of your valuable journal and I have followed with special interest the articles that have appeared about the cerebro-spinal meningitis epidemic in Belfast and Glasgow. I see that no mention has yet been made of the anti-meningococcic serum which is prepared by the" Institut zur Erforschung der Infektions-Krankheiten " in Bern. Although this serum is practically new I under- stand that it has proved successful in many cases, and I think it my duty to mention it to your readers, who could get fuller information by writing to the above-mentioned institute in Bern. I am, Sirs, yours faithfully, portant I am, Sirs, yours faithfully, rtant I am, Sirs, yours faithfully, am, Sirs, yours faithfully, J. C. HOLDICH LEICESTER, M.D. Lond. J. C. HOLDICH LEICESTER, M.D. Lond. rs, yours faithfully, J. C. HOLDICH LEICESTER, M.D. Lond. J. C. HOLDICH LEICESTER, M.D. Lond. , yours faithfully, J. C. HOLDICH LEICESTER, M.D. Lond. J. C. HOLDICH LEICESTER, M.D. Lond. , Medical Officers’ Quarters, Presidency General Hospital, Bhowanipur, Calcutta, Feb. 5th, 1907. C S , , Medical Officers’ Quarters, Presidency General Hospital, Bhowanipur, Calcutta, Feb. 5th, 1907. , To send proofs to Captain Holdich Leicester in Calcutta would have caused great delay in the publication of his paper. We had to take on ourselves the responsibility of passing his tables for press and in so doing the errors occurred. In respect of the second slip we plead guilty, but in the first case Captain Holdich Leicester’s 8 very closely resembles a "3."-ED. L. am, Sirs, yours faithfully, Dr. R. DE LA HARPE, L.M. Dr. R. DE LA HARPE, L.M Vevey, Switzerland, Feb. 27th, 1907. Vevey, Switzerland, Feb. 27th, 1907. , Vevey, Switzerland, Feb. 27th, 1907. , To the Editors of THE LANCET. And in those other cases " where many of the patients attacked were regarded as suffering from influenza," I find no difficulty in believing that they did so suffer, seeing especially that the influenza throat appears to be nowadays the usual precursor of an attack of meningitis. That an attack of influenza which has broken down the normal defences of the body and has caused a lesion in the throat should thus pave the way to the invasion of other pathogenic microbes is surely what we must expect if we reason about the causation at all. Nor is it a matter of theory only, for the conclusion so arrived at is abundantly confirmed by the results of experience. I am sure that friction frequently arises between practitioners on this point, and that much un- deserved annoyance and even damage may be caused in this way to him who recognises cases of mixed infection when his diagnosis is openly contradicted by one who does not. important We are, Sirs, yours faithfully, important We are, Sirs, yours faithfully, faithfully, SAMUEL WEST. SAMUEL WEST. faithfully, SAMUEL WEST. SAMUEL WEST. T. WOOD CLARKE. T. WOOD CLARKE. March 2nd, 1907. To the Editors of THE LANCET. SIRS,&mdash;I write to point out two errors in my paper on this subject published in THE LANCET of Jan. 19th. In Table VI., p. 152, "Bengalis," length of the maximum diameter (last column), in the sixth line of figures from the top, for 4-737 read 4’787. In Table VII., p. 152, "Pelves of 4-125" inches conjugate, under the column headed " Bi-parietal diameter," second line from top of this series, for 2’397 read 3-397. This latter is, of course, much the more important correction. ly by I am, Sirs, yours faithfully, ly by I am, Sirs, yours faithfully, am, Sirs, yours faithfully, W. FLEMING PHILLIPS, M.B. Glasg. W. FLEMING PHILLIPS, M.B. Glasg. , Sirs, yours faithfully, W. FLEMING PHILLIPS, M.B. Glasg. Fairlie, N.B., Feb. 25th, 1907. W. FLEMING PHILLIPS, M.B. Glasg. Fairlie, N.B., Feb. 25th, 1907. BLOOD CULTURE IN TYPHOID FEVER. This venous stasis in shock appears to me to account suffi- ciently for some of Dr. Crile’s observations, in the interpreta- tion of which Mr. Malcolm differs from that author, and to which I need not now refer at greater length. Again, Mr. Malcolm’s argument drawn from the changes in the volume of one channel of a hypothetical river as compared with another which is supposed to be impeded, appears to me to be vitiated by the same failure to recognise the simultaneous depression of centre and periphery during shock. He quotes Dr. Crile as disputing this central fact but unless the mechanics of surgical shock are very different from those observed in the domain of the physician there is always an abasement of systolic cardiac force under these circumstances. The only circumstances under which I have observed peripheral loss of tension with for a time normal central impulse are those in which accidental haemorrhage occurs in an otherwise healthy organism, conditions which in no way bear upon our o )j-jc,&mdash;&plusmn;. ’. jr. -Ljusujuc.ij. au zu vvuauauuatnuavu UFVLL nnc value of blood culture in typhoid fever in THE LANCET of Feb. 16th, p. 425, records a case which he considers to "com- pare" with one reported by myself to the Pathological Society on Jan. 15th last. Interesting as Dr. Bushnell’s case is it contrasts rather than compares with my own case. 1. The clinical condition in my case did not suggest typhoid fever, so that the isolation of the bacillus (upon the fifth day) did not "confirm the clinical diagnosis," as it did in Dr. Bushnell’s case (in which the bacillus was isolated during the third week, when rose-spots, palpable spleen, diarrhoea, abdominal distension, &c., were present). 2. In my case the organism was isolated at a date in the disease when agglutinins are known often to be absent and the Widal test therefore negative. 3. But the point of chief pathological 687 687 interest in my case was the fact that the bacillus grew well without any dilution of the blood, refusing, therefore, to obey Dr. Bushnell’s dictum that " it is imperative to dilute the blood well." I am aware, as I stated in reporting my case, that this dictum is usually held to be true, but I think we do ill to copy it from continental authorities without verification for ourselves. BLOOD CULTURE IN TYPHOID FEVER. Since reporting my case I have again grown the typhoid bacillus from the blood with no more dilution than is given by the usual condensation fluid in an agar tube. That dilution is never needed I am not prepared to say, but that it is sometimes quite unnecessary I am certain. fact that the colder seasons of the year produce an unfavour- able effect upon diabetes and I was also the first to give the causation thereof. It goes without saying that the con- clusions I drew from the above fact are necessarily new as well, as there is no statement in the whole of the literature to the effect that a diet which suited the patient during the summer does not suit him in the winter. I do not ascribe too much importance to my observations, but I hardly think there is anybody who would not attach some scientific and therapeutic value to the symptom I described and I think I am fully justified in asking you not to deprive me of the right of priority which undoubtedly belongs to uite unnecessary I am, Sirs, yours faithfully, quite unnecessary I am, Sirs, yours faithfully, quite unnecessary I am, Sirs, yours faithfully, ty which undoubtedly belongs to Your obedient servant, am, Sirs, yours faithfully, Harley-street, W., Feb. 26th, 1907. THOMAS J. HORDER. THOMAS J. HORDER. , , yours y, Harley-street, W., Feb. 26th, 1907. THOMAS J. HORDER. THOMAS J. HORDER. rvant, S. A. ARANY. servant, Carlsbad, March 1st, 1907. S. A. ARANY. servant, Carlsbad, March 1st, 1907. S. A. ARANY. Carlsbad, March 1st, 1907. SOME POINTS IN DIABETES. To the Editors of THE LANCET. , S s, yours y, Vankaner, Feb. 7th, 1907. K. A. NANAVUTTY. K. A. N&Aacute;N&Aacute;VUTTY. To the Editors of THE LANCET. SIRS,-I have to thank you for having published an extract of my paper on diabetes. The extract contains the chief points ot my treatise and enables the reader to get familiar with the facts which are closely associated with myself. This, however, you would not admit in your resume, but you come to the conclusion that, "There is nothing particularly new in Dr. Arany’s observations," &c. As far as this latter remark is concerned, I beg to say that if you go through the whole of the literature you will find that I was the first who called attention to the SIRS,-I read with interest your annotation under the above heading in THE LANCET of Jan. 19th. I shall be much obliged if any of your readers will inform me through the medium of your journal the effects of taking sodium bicarbonate for a very long period on metabolism and what changes occur or lesions are found on the skin and hair during its long-continued internal use. during long continued I am, Sirs, yours faithfully, ng continued I am, Sirs, yours faithfully, am, Sirs, yours faithfully, Vankaner, Feb. 7th, 1907. K. A. NANAVUTTY. K. A. N&Aacute;N&Aacute;VUTTY. faithfully, K. A. NANAVUTTY. K. A. N&Aacute;N&Aacute;VUTTY.
https://openalex.org/W2915004205
https://figshare.com/articles/journal_contribution/A_test_for_deterministic_dynamics_in_spatial_processes/8427059/1/files/15679541.pdf
English
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A test for deterministic dynamics in spatial processes
Spatial economic analysis
2,019
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spatial processes” Jose A. Garc´ıa-C´ordoba, Mariano Matilla-Garc´ıa Manuel Ruiz Mar´ın. Jose A. Garc´ıa-C´ordoba, Mariano Matilla-Garc´ıa Manuel Ruiz Mar´ın. Abstract We propose a statistical procedure to determine if a spatial structure that is ob- served in the data is generated by a deterministic (even chaotic) spatial process, rather than by a stochastic process. This procedure can be used as a specification test. It is robust against nonlinearity and nonstationarity and can complete the toolbox for testing diagnosis as well. The advantages of the presented methods are high power, simplicity, and ease and ample applicability for tests to be conducted, provided that weak conditions are required. Herein, we conduct several simulations to evaluate the performance of our procedure on well-known spatial processes and in situations where standard tests for spatial autocorrelation fail to detect spatial dependence. Guidelines for using the technique are also provided herein. r s is a rate of variances: variance of the stochastic part on variance of the deterministic Appendix 1 Table A1: Pr(Reject determinism/s −deterministic) for fixed R and increasing m s 0,1 0,5 1 1,5 2 0,1 0,5 1 1,5 2 Model 1 Model 5 m = 5 0,18 0,17 0,21 0,17 0,15 0,25 0,24 0,3 0,25 0,24 m = 6 0,04 0,02 0,1 0,06 0,08 0,06 0,16 0,13 0,15 0,26 m = 7 0 0 0 0 0 0 0 0,02 0 0 m = 8 0 0 0 0 0 0 0 0 0 0 Model 2 Model 6 m = 5 0,19 0,24 0,21 0,27 0,17 0,17 0,27 0,22 0,24 0,2 m = 6 0,06 0,05 0,05 0,02 0,04 0,06 0,11 0,1 0,09 0,11 m = 7 0 0,01 0 0 0 0 0 0 0 0 m = 8 0 0 0 0 0 0 0 0 0 0 Model 3 Model 7 m = 5 0,18 0,19 0,17 0,16 0,16 0,69 0,84 0,91 1 1 m = 6 0,04 0,09 0,1 0,08 0,04 0,59 0,94 0,99 1 1 m = 7 0 0,01 0 0,01 0 0,44 0,83 0,97 1 1 m = 8 0 0 0 0 0 0,2 0,67 0,93 0,99 1 Model 4 Model 8 m = 5 0,22 0,21 0,28 0,23 0,31 0,22 0,24 0,31 0,31 0,47 m = 6 0,06 0,08 0,13 0,15 0,16 0,03 0,12 0,13 0,22 0,44 m = 7 0,01 0 0,02 0 0 0,03 0 0 0,03 0,13 m = 8 0 0 0 0 0 0 0 0 0 0 =1000, parameter s is a rate of variances: variance of the stochastic part on variance of the determinist part part 2
https://openalex.org/W3208490113
https://jcmr-online.biomedcentral.com/track/pdf/10.1186/s12968-021-00813-5
English
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Endogenous T1ρ cardiovascular magnetic resonance in hypertrophic cardiomyopathy
Journal of cardiovascular magnetic resonance
2,021
cc-by
7,698
Abstract Background:  Hypertrophic cardiomyopathy (HCM) is characterized by increased left ventricular wall thickness, car- diomyocyte hypertrophy, and fibrosis. Adverse cardiac risk characterization has been performed using late gadolinium enhancement (LGE), native T1, and extracellular volume (ECV). Relaxation time constants are affected by background field inhomogeneity. T1ρ utilizes a spin-lock pulse to decrease the effect of unwanted relaxation. The objective of this study was to study T1ρ as compared to T1, ECV, and LGE in HCM patients. Methods:  HCM patients were recruited as part of the Novel Markers of Prognosis in Hypertrophic Cardiomyopathy study, and healthy controls were matched for comparison. In addition to cardiac functional imaging, subjects under- went T1 and T1ρ cardiovascular magnetic resonance imaging at short-axis positions at 1.5T. Subjects received gado- linium and underwent LGE imaging 15–20 min after injection covering the entire heart. Corresponding basal and mid short axis LGE slices were selected for comparison with T1 and T1ρ. Full-width half-maximum thresholding was used to determine the percent enhancement area in each LGE-positive slice by LGE, T1, and T1ρ. Two clinicians indepen- dently reviewed LGE images for presence or absence of enhancement. If in agreement, the image was labeled posi- tive (LGE + +) or negative (LGE −−); otherwise, the image was labeled equivocal (LGE + −). Results:  In 40 HCM patients and 10 controls, T1 percent enhancement area (Spearman’s rho = 0.61, p < 1e-5) and T1ρ percent enhancement area (Spearman’s rho = 0.48, p < 0.001e-3) correlated with LGE percent enhancement area. T1 and T1ρ percent enhancement areas were also correlated (Spearman’s rho = 0.28, p = 0.047). For both T1 and T1ρ, HCM patients demonstrated significantly longer relaxation times compared to controls in each LGE category (p < 0.001 for all). HCM patients also showed significantly higher ECV compared to controls in each LGE category (p < 0.01 for all), and LGE −− slices had lower ECV than LGE + + (p = 0.01). Conclusions:  Hyperenhancement areas as measured by T1ρ and LGE are moderately correlated. T1, T1ρ, and ECV were elevated in HCM patients compared to controls, irrespective of the presence of LGE. These findings warrant addi- tional studies to investigate the prognostic utility of T1ρ imaging in the evaluation of HCM patients. Keywords:  Hypertrophic cardiomyopathy, T1ρ, LGE, T1 Keywords:  Hypertrophic cardiomyopathy, T1ρ, LGE, T1 Open Access RESEARCH Endogenous T1ρ cardiovascular magnetic resonance in hypertrophic cardiomyopathy Elizabeth W. Thompson1,2  , Srikant Kamesh Iyer3, Michael P. Solomon1, Zhaohuan Li4,5, Qiang Zhang6, Stefan Piechnik6, Konrad Werys7, Sophia Swago1, Brianna F. Moon1, Zachary B. Rodgers4, Anya Hall1, Rishabh Kumar8, Nosheen Reza4, Jessica Kim4, Alisha Jamil9, Benoit Desjardins3, Harold Litt3, Anjali Owens4, Walter R. T. Witschey3 and Yuchi Han3,4,10*  Open Access Endogenous T1ρ cardiovascular magnetic resonance in hypertrophic cardiomyopathy Thompson et al. J Cardiovasc Magn Reson (2021) 23:120 https://doi.org/10.1186/s12968-021-00813-5 Thompson et al. J Cardiovasc Magn Reson (2021) 23:120 https://doi.org/10.1186/s12968-021-00813-5 Endogenous T1ρ cardiovascular magnetic resonance in hypertrophic cardiomyopathy Elizabeth W. Thompson1,2  , Srikant Kamesh Iyer3, Michael P. Solomon1, Zhaohuan Li4,5, Qiang Zhang6, Stefan Piechnik6, Konrad Werys7, Sophia Swago1, Brianna F. Moon1, Zachary B. Rodgers4, Anya Hall1, Rishabh Kumar8, Nosheen Reza4, Jessica Kim4, Alisha Jamil9, Benoit Desjardins3, Harold Litt3, Anjali Owens4, Walter R. T. Witschey3 and Yuchi Han3,4,10* Background Hypertrophic cardiomyopathy (HCM), characterized by an unexplained increase in left ventricular (LV) wall thickness, is the most common genetic cardiac disor- der, with a prevalence of approximately 1 in 500; this *Correspondence: yuchi.han@pennmedicine.upenn.edu 10 Perelman School of Medicine, University of Pennsylvania, 11‑135, South Pavilion, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA Full list of author information is available at the end of the article CMR imaging CMR CMR was performed using a 1.5 T CMR scanner (Avanto; Siemens Healthineers; Erlangen, Germany), equipped with 18 channel anterior and posterior array coils. Ret- rospectively gated, short axis, multi-slice cine CMR was performed with a temporal resolution = 34–40  ms, flip angle = 70°, bandwidth = 940  Hz/pixel, spatial resolu- tion = 1.8 × 1.8 ­mm2, slice thickness = 8 mm. T1ρ CMR is an endogenous contrast method for tis- sue characterization that does not require GBCAs and is distinct from both T1 and T2 contrast. It utilizes a low power radiofrequency pulse, also called a spin-lock pulse, to enable measurement of longitudinal relaxation in the rotating frame (T1ρ). The spin lock pulse mitigates the loss of transverse magnetization, suppressing contribu- tions to relaxation from chemical exchange and water diffusion through magnetic field gradients [11]. Its abil- ity to detect myocardial fibrosis has been validated in animal models of ischemia and reperfusion [12–14] as well as in explanted hearts from patients with dilated car- diomyopathy [15]. Despite its mechanistic relevance to HCM pathophysiology, few studies have investigated the value of T1ρ in this population. Thus, we sought to evalu- ate and characterize the role of T1ρ in HCM patients by comparing it to conventional LGE and native T1. 2D T1ρ breath-held single-shot balanced steady-state free precession (bSSFP) sequences were performed at 3 short axis slice positions for HCM patients (apical, mid, and basal) in systole and 2 short axis slice positions for controls (mid and basal) using a motion- and heart rate-corrected spin echo, spin lock (SL) T1ρ pulse clus- ter ­(90x—SLy—180y—SL-y—90-x) at end-systole [17–19]. T1ρ images were acquired with different SL times (TSL) using the following parameters: TSL = 2, 10, 18, 26, 34, 42, 50  ms, ­B1 = 400–500  Hz, spatial resolution = 1.4 × 1.4 ­mm2, slice thickness = 8  mm, flip angle = 70°, echo time (TE) = 1.45 ms, repetition time (TR) = 2.9 ms, number of segments ­(NSeg) = 55, bandwidth = 900  Hz/pixel, linear k-space phase encoding ordering, parallel imaging with acceleration factor = 2, 34 reference k-space lines obtained in a separate heartbeat, and allowing 1 additional heartbeat for T1 relaxation between shots. The T1ρ pulse amplitude was set at the highest available within scanner specific absorption rate (SAR) limits ­(B1 = 400–500  Hz). Motion correction was used to reduce residual cardiac and res- piratory motion between T1ρ images (Equation [1]). T1 mapping and extracellular vol- ume (ECV) quantification through CMR have also been correlated with increased risk of cardiovascular events [4, 5]. However, not all HCM patients will go on to have an event; LGE has a high prevalence (as high as 70%) in this population [6, 7] but a low specificity for the prediction of future cardiovascular events, limiting its negative pre- dictive value [8]. Additionally, gadolinium-based contrast agents (GBCAs) confer a risk of nephrogenic systemic fibrosis in patients with renal disease, and additionally are deposited in brain tissue [9, 10]. Accordingly, there is interest in the development and validation of more spe- cific and non-contrast methods for myocardial charac- terization in HCM patients. prevalence may be as high as 1 in 200 when accounting for both genotype-positive/phenotype-positive and gen- otype-negative/phenotype-positive individuals [1]. Typi- cal pathologic findings of HCM include cardiomyocyte hypertrophy and disarray, as well as focal or diffuse inter- stitial fibrosis [2]. In recent years, cardiovascular mag- netic resonance (CMR) has been used to characterize and quantify myocardial fibrosis. Increased fibrosis, seen as late gadolinium enhancement (LGE), has been identified as a risk factor for sudden cardiac death and heart failure in this population [3]. T1 mapping and extracellular vol- ume (ECV) quantification through CMR have also been correlated with increased risk of cardiovascular events [4, 5]. However, not all HCM patients will go on to have an event; LGE has a high prevalence (as high as 70%) in this population [6, 7] but a low specificity for the prediction of future cardiovascular events, limiting its negative pre- dictive value [8]. Additionally, gadolinium-based contrast agents (GBCAs) confer a risk of nephrogenic systemic fibrosis in patients with renal disease, and additionally are deposited in brain tissue [9, 10]. Accordingly, there is interest in the development and validation of more spe- cific and non-contrast methods for myocardial charac- terization in HCM patients. CMR imaging CMR The relaxation rate R1ρ = 1 T1ρ and intercept B were estimated by two-parameter fit © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Thompson et al. J Cardiovasc Magn Reson (2021) 23:120 Page 2 of 9 as hypertension and aortic stenosis, or other infiltrative cardiomyopathies such as amyloidosis and sarcoidosis. Additional exclusion criteria were: (1) prior septal myec- tomy or alcohol septal ablation, (2) prior myocardial infarc- tion or coronary artery disease, (3) incessant ventricular arrhythmias, (4) inability to lie flat, (5) contraindications to CMR including pacemakers, defibrillators, intraocular metal, certain types of intracranial aneurysm clips, severe claustrophobia, and stage IV/V chronic kidney disease with estimated glomerular filtration rate < 30 mL/min/1.73 ­m2, (6) diabetes mellitus with end organ damage, (7) pregnancy, and (8) inability to provide informed consent. In addition, we recruited 10 healthy subjects without cardiovascular risk factors or diseases and on no medications to serve as a control group. The study protocol was approved by the Institutional Review Board of the University of Pennsylva- nia and all subjects gave written informed consent prior to enrollment. prevalence may be as high as 1 in 200 when accounting for both genotype-positive/phenotype-positive and gen- otype-negative/phenotype-positive individuals [1]. Typi- cal pathologic findings of HCM include cardiomyocyte hypertrophy and disarray, as well as focal or diffuse inter- stitial fibrosis [2]. In recent years, cardiovascular mag- netic resonance (CMR) has been used to characterize and quantify myocardial fibrosis. Increased fibrosis, seen as late gadolinium enhancement (LGE), has been identified as a risk factor for sudden cardiac death and heart failure in this population [3]. (1) min R1ρ,B ln(Si) −B + R1ρ · TSLi 2 2 Determination of myocardial relaxation times, scar size, and ECV where Si is the magnitude signal at each spin lock dura- tion TSLi . Motion correction and parametric mapping (Eq [1]) were implemented using custom C + + software on the CMR scanner [17]. Relaxation times were measured in pre-contrast T1, post-contrast T1, and T1ρ images by manual contour- ing of the LV myocardium using QMass (Medis, Leiden, Netherlands). In LGE, T1, and T1ρ images, enhance- ment area was quantified using full width at half maxi- mum (FWHM) thresholding and reported as the ratio of enhanced to total LV area (%). ECV was calculated per Equation [2] using blood and entire myocardial T1 val- ues, and hematocrit (Hct) obtained within 24 h of CMR [24]. 2D T1 images were obtained with a breath-held shortened modified Look-Locker inversion recovery (ShMOLLI) [20] sequence at 3 short axis slice positions matched to T1ρ at mid-end-diastole [21]. Other param- eters were: spatial resolution = 1.4 × 1.4 ­mm2, slice thick- ness = 8  mm, flip angle = 35°, TE = 1.2, TR = 2.4  ms, ­NSeg = 57, bandwidth = 1080  Hz/pixel, linear k-space encoding, parallel imaging acceleration factor = 2, 34 ECV = 100%×(1 −Hct)×1/Myocardial T1post−contrast −1/Myocardial T1pre−contrast 1/Blood T1post−contrast −1/Blood T1pre−contrast ECV = 100%×(1 −Hct)×1/Myocardial T1post−contrast −1/Myocardial T1pre−contrast 1/Blood T1post−contrast −1/Blood T1pre−contrast ECV = 100%×(1 −Hct)×1/Myocardial T1post−contrast −1/Myocardial T1pre−contrast 1/Blood T1post−contrast −1/Blood T1pre−contrast (2) Statistical analysis reference k-space lines obtained in a separate heartbeat. These images were prospectively electrocardiogram gated. Statistical analysis was performed using R 3.6.1 (R Foun- dation for Statistical Computing, Vienna, Austria) and MATLAB R2019b (The MathWorks Inc., Natick, Mas- sachusetts, USA). Categorical variables are expressed as N (%); continuous variables are expressed as mean ± SD or median [interquartile range (IQR)] depending on the distribution of the data. Normality testing was performed using the Shapiro–Wilk test. If the data were normally distributed, parametric methods were used, otherwise non-parametric methods were used. Student’s t-test, Wilcoxon Signed Rank test, one-way analysis of variance (ANOVA), and Kruskal–Wallis test (with post-hoc Dunn test adjusted with the Benjamini–Hochberg method) were used as appropriate based upon the variables and data distribution. To compare proportions of categori- cal variables, Chi-square test and Fisher’s exact test were used, as appropriate. The correlation between T1ρ and other parameters was assessed using Pearson’s and Spearman’s correlation coefficients, as appropriate. p val- ues less than 0.05 were considered statistically significant. A 0.15 mmol/kg intravenous injection of gadolinium- based contrast was used for LGE imaging (Magnevist; Bayer Schering Pharma; Leverkusen, Germany). Imag- ing was performed 15–20  min after injection of con- trast agent using an inversion time (TI) scout sequence to determine the TI to null myocardial tissue signal. LGE CMR was obtained using a 2D segmented phase- sensitive inversion recovery (PSIR) sequence at spatial resolution = 1.2 × 1.2 ­mm2, flip angle = 50°, TE = 1.6 ms, TR = 3.2 ms, slice thickness = 8 mm, and parallel imaging acceleration factor = 2 [22]. Image analysis Cardiac function Cardiac volumes and functional data were analyzed on the short-axis cine images using a commercially avail- able software (Suiteheart, Neosoft, Pewaukee, Wis- consin, USA) The endocardium and epicardium were automatically traced at end-diastole and end-systole and manually adjusted following Society for Cardiovascular Magnetic Resonance guidelines [23]. Papillary muscles were included in the ventricular volume. Patient characteristics A total of 48 subjects were enrolled through the HCMR study [16]; 8 subjects were excluded for (1) having other diseases (n = 5), (2) no CMR performed (n = 2), and (3) withdrawal from the study (n = 1; Fig. 1). Baseline char- acteristics are presented in Table 1. Median age was 50 [IQR 35–57] years, 48% of patients were female, and median body surface area (BSA) was 2.0 ­m2. Controls had similar distributions of age, gender, and BSA. 30% of patients had a history of ventricular arrhythmia, and 15% had a history of syncope. Maximum LV wall thickness was 17.5 ± 3.3 mm. 35% of patients had obstruction seen Study population l We prospectively enrolled HCM patients between August 10, 2015 and July 10, 2017 as part of the Novel Markers of Prognosis in Hypertrophic Cardiomyopathy (HCMR) study. Detailed trial inclusion and exclusion criteria have been previously published [16]. In brief, key inclusion cri- teria were patients aged 18–65  years with an established HCM diagnosis defined as unexplained myocardial hyper- trophy of ≥ 15 mm without cavity dilation, etiologies such (1) min R1ρ,B ln(Si) −B + R1ρ · TSLi 2 2 min R1ρ,B ln(Si) −B + R1ρ · TSLi 2 2 (1) Thompson et al. J Cardiovasc Magn Reson (2021) 23:120 Thompson et al. J Cardiovasc Magn Reson (2021) 23:120 Page 3 of 9 Determination of myocardial relaxation times, scar size, and ECV Presence of enhancement on LGE All LGE images were anonymized, shuffled, and pre- sented to 2 blinded expert readers (B.D. and H.L., each with > 10  years of CMR experience), who labeled each slice as showing positive visible enhancement or not. Slices were labeled as showing positive (++) or negative enhancement (–) if both experts agreed, and otherwise were labeled equivocal (+ −). Thompson et al. J Cardiovasc Magn Reson (2021) 23:120 Page 4 of 9 Fig. 1  Study participant flow diagram. Disposition of hypertrophic cardiomyopathy (HCM) patients is shown; 48 subjects were enrolled, and after applying exclusionary criteria, 40 subjects were included in final analysis. HCM hypertrophic cardiomyopathy, CMR cardiovascular magnetic resonance LGE, alongside a control patient with no LGE. Gener- ally, areas of LGE were visibly associated with areas of elevated T1 and T1ρ. In LGE-positive slices, the median area of enhancement within the slice area as assessed by: (1) LGE at FWHM was 10.1% [6.0, 13.7%], (2) native T1 at FWHM was 17.1% [8.3, 22.6%], and (3) T1ρ at FWHM was 14.4% [11.0, 18.1%]. Native T1- and T1ρ- measured enhancement areas were each significantly larger than LGE-measured enhancement area (p < 0.01 for both), while T1ρ- and native T1-measured enhance- ment area were not significantly different from each other (p = 0.21). Both T1 percent enhancement area (Spearman’s rho = 0.61) and T1ρ percent enhancement area (Spearman’s rho = 0.48) were significantly correlated with LGE percent enhancement area (Fig. 3; p < 0.001 for both). T1 and T1ρ demonstrated a mild correlation (Spearman’s rho = 0.28, p = 0.047). Fig. 1  Study participant flow diagram. Disposition of hypertrophic cardiomyopathy (HCM) patients is shown; 48 subjects were enrolled, and after applying exclusionary criteria, 40 subjects were included in final analysis. HCM hypertrophic cardiomyopathy, CMR cardiovascular magnetic resonance CMR measurements and LGE ratings CMR measurements for both HCM patients and con- trols are shown in Table 2. Compared to controls, HCM patients had higher LV mass (148 g vs. 94 g, p < 0.001), LV mass index (74.8 vs. 48.6 g/m2, p < 0.001), and LV ejec- tion fraction (LVEF) (65.0% vs. 60.2%; p < 0.001). In the right ventricle (RV), HCM patients had lower indexed end diastolic volume (EDVI; 74.4 mL/m2 vs. 93.4 mL/m2, p = 0.011), end systolic volume (ESV; 52.6 mL vs. 81.4 mL; p = 0.001) and indexed RV ESV (RVESVI; 26.7 mL/m2 vs. 42.7  mL/m2; p = 0.001). HCM patients had higher RV ejection fraction (64.4% vs. 54.1%; p < 0.001). Comparisons of T1ρ, native and post‑contrast T1, and ECV between LGE categories To assess whether myocardial tissue characteristics dif- fered by LGE rating, we compared pre-contrast T1, T1ρ, post-contrast T1, and ECV across HCM LGE + +, LGE + −, LGE −−, and control short-axis slices (Fig. 4). For pre-contrast T1, T1ρ, and ECV, Kruskal Wallis test identified differences between groups (p < 0.001 for all); for post-contrast T1, no statistically significant differ- ences were identified. For pre-contrast T1, differences were seen between (1) control and LGE + +, (2) control and LGE + −, and (3) control and LGE −− (p < 0.001 for all). For T1ρ, differences were also seen between (1) con- trol and LGE + +, (2) control and LGE + −, and (3) con- trol and LGE −− (p < 0.001 for all). For ECV, differences were seen between (1) control and LGE + +, (2) control and LGE + −, and (3) control and LGE −− (p < 0.01 for all), as well as (4) LGE + + and LGE −− (p = 0.01). on echocardiogram, and the majority had mild mitral regurgitation. 32.5% of patients had NYHA Class II heart failure, 20% of patients had Class III heart failure, and no patients had Class IV heart failure. 25% of patients had a likely pathogenic or pathogenic genetic variant. Discussion In our study characterizing the role of endogenous T1ρ imaging in the assessment of patients with HCM, we found that (1) percent area enhancement as measured by T1 and T1ρ at FWHM were moderately correlated with LGE area enhancement, (2) HCM short-axis slices categorized as LGE + +, LGE + −, and LGE −− each demonstrated elevated pre-contrast T1, T1ρ, and ECV compared to controls, and (3) ECV was significantly dif- ferent between images rated LGE + + compared to LGE Overall, HCM patients also had higher pre-contrast T1 (986 ms vs. 923 ms; p < 0.001), T1ρ (72.2 ms vs. 65.4 ms; p < 0.001), and ECV (28.1% vs. 24.3%; p < 0.001) compared to controls; T1 post-contrast was not significantly differ- ent between HCM patients and controls (p = 0.618). 28 patients (70%) had at least one LGE + + slice. Both T1- and T2-weighted imaging have been used to demonstrate elevations in HCM patient myocardial relaxation times relative to normal patients [5, 25–29]. Cardiac T2 mapping may be sensitive to several differ- ent mechanisms of relaxation in  vivo. Some of these Associations between T1, T1ρ, and LGE enhancement in HCM patients Figure 2 shows T1, T1ρ, and LGE images from three dif- ferent HCM patients with patchy, focal, and negligible Thompson et al. J Cardiovasc Magn Reson (2021) 23:120 Page 5 of 9 Table 1  Characteristics of the HCM Patient and Control Cohorts Values are presented as Mean (Standard Deviation), Median [Interquartile Range], or N (%) depending on the distribution of the data BSA body surface area, ESC European Society of Cardiology, LV left ventricle, LVOT left ventricular outflow tract, NYHA New York Heart Association HCM patients (N = 40) Healthy Controls (N = 10) p-value Age (years) 50 [35, 57] 51 [38, 55] 1 Gender 0.724  Male 21 (52.5%) 4 (40.0%)  Female 19 (47.5%) 6 (60.0%) BSA ­(m2) 2.0 [1.8, 2.2] 1.8 [1.7, 2.1] 0.254 Hematocrit (%) 42.0 [39, 43.3] 39.5 [37.0, 41.0] 0.002 Medical history  Coronary artery disease 0 (0%)  Hypertension 16 (40.0%)  Diabetes mellitus 2 (5.0%)  Stroke or transient ischemic attack 0 (0%)  Hospitalization for heart failure 1 (2.5%)  Ventricular arrhythmia 12 (30.0%)  Syncope 6 (15.0%) Maximum LV wall thickness (mm) 17.5 (3.25) LVOT obstruction 14 (35.0%) Mitral regurgitation  None 5 (12.5%)  Mild/trace 27 (67.5%)  Moderate 5 (12.5%)  Severe 3 (7.5%) NYHA heart failure classification  I 19 (47.5%)  II 13 (32.5%)  III 8 (20.0%)  IV 0 (0%) ESC risk score (%) 2.19 (0.924) Genotype positive 10 (25.0%) Table 1  Characteristics of the HCM Patient and Control Cohorts Values are presented as Mean (Standard Deviation), Median [Interquartile Range], or N (%) depending on the distribution of the data BSA body surface area, ESC European Society of Cardiology, LV left ventricle, LVOT left ventricular outflow tract, NYHA New York Heart Ass mechanisms may be considered ‘undesired’ because they suppress ∆T2 between diseased and healthy myocar- dium. Since each mechanism of relaxation is additive to the overall relaxation rate (i.e., R2 = R2,a + R2,b + . . . , where a , b , and so on refer to a different relaxation mech- anism), eliminating these ‘undesired’ sources of relaxa- tion could increase the difference in the net transverse relaxation. While the ‘unwanted’ contributions to T2 in myocardium are not fully elucidated at present, their effect is to dephase magnetization irreversibly. Potential ‘undesired’ mechanisms of relaxation may include dif- fusion through background magnetic fields, chemical exchange, among others. By using a sufficiently strong SLk pulse, it is possible to prevent these unwanted mech- anisms of relaxation [11]. Associations between T1, T1ρ, and LGE enhancement in HCM patients Using a moderate amplitude (> 400 Hz) SL pulse, we have found that there is a signifi- cantly larger ∆T1ρ than ∆T2 in these regions [30]. The net effect of this is an increase in the contrast between normal and diseased myocardium.i mechanisms may be considered ‘undesired’ because they suppress ∆T2 between diseased and healthy myocar- dium. Since each mechanism of relaxation is additive to the overall relaxation rate (i.e., R2 = R2,a + R2,b + . . . , where a , b , and so on refer to a different relaxation mech- anism), eliminating these ‘undesired’ sources of relaxa- tion could increase the difference in the net transverse relaxation. While the ‘unwanted’ contributions to T2 in myocardium are not fully elucidated at present, their effect is to dephase magnetization irreversibly. Potential ‘undesired’ mechanisms of relaxation may include dif- fusion through background magnetic fields, chemical exchange, among others. By using a sufficiently strong SLk pulse, it is possible to prevent these unwanted mech- anisms of relaxation [11]. Using a moderate amplitude (> 400 Hz) SL pulse, we have found that there is a signifi- cantly larger ∆T1ρ than ∆T2 in these regions [30]. The y Patchy fibrosis occurs in the majority of HCM patients. This is observed primarily as replacement fibrosis, but may also take the form of interstitial fibro- sis, which can be imaged and quantified by T1 mapping and subsequent ECV calculation [31, 32]. Most studies of fibrosis in HCM patients have focused on LGE imag- ing, which allows visualization of replacement fibrosis and has demonstrated associations with adverse out- comes [3]. However, fibrosis accumulates throughout the course of HCM, and additionally, LGE has limited specificity for the prediction of events such as sud- den cardiac death and heart failure [8]. It is therefore of both clinical and research interest to investigate new contrast mechanisms such as T1ρ in the HCM population. Thompson et al. Associations between T1, T1ρ, and LGE enhancement in HCM patients J Cardiovasc Magn Reson (2021) 23:120 Page 6 of 9 Table 2  CMR imaging findings HCM patients (N = 40) Controls (N = 10) HCM patients (N = 40) Controls (N = 10) p-value Values are presented as Mean (Standard Deviation) p-values < 0.05 are bolded CMR cardiovascular magnetic resonance, ECV extracellular volume, EDV end diastolic volume, EDVI end diastolic volume index, ESV end systolic volume, ESVI end systolic volume index, HCM hypertrophic cardiomyopathy, LV left ventricle, LVEF left ventricular ejection fraction, RV right ventricle, RVEF right ventricular ejection fraction p ( ) ( ) p Left ventricle (LV)  LV mass (g) 148 (51) 94 (32) < 0.001  LV mass index (g/m2) 74.8 (22.8) 48.6 (11.8) < 0.001  LVEDV (mL) 167 (36.0) 163 (46.5) 0.797  LVEDVI (mL/m2) 85.1 (13.6) 85.3 (15.8) 0.961  LVESV (mL) 58.8 (17.2) 66.1 (20.6) 0.322  LVESVI (mL/m2) 30.0 (7.90) 34.6 (7.73) 0.114  LV stroke volume (mL) 109 (25.3) 97.8 (26.5) 0.263  LVEF (%) 65.0 (6.18) 60.2 (2.25) < 0.001 Right ventricle (RV)  RVEDV (mL) 147 (35.0) 177 (49.1) 0.091  RVEDVI (mL/m2) 74.4 (13.1) 93.4 (18.8) 0.011  RVESV (mL) 52.6 (18.0) 81.4 (26.0) 0.001  RVESVI (mL/m2) 26.7 (7.75) 42.7 (11.3) 0.001  RV stroke volume (mL) 94.2 (23.5) 95.8 (25.0) 0.855  RVEF (%) 64.4 (7.10) 54.1 (4.31) < 0.001 Tissue characterization  T1 pre-contrast (ms) 986 (41.0) 923 (30.0) < 0.001  T1ρ (ms) 72.2 (5.86) 65.4 (5.24) < 0.001  T1 post-contrast (ms) 471 (31.1) 476 (38.4) 0.618  ECV (%) 28.1 (3.28) 24.3 (2.24) < 0.001 p-values < 0.05 are bolded CMR cardiovascular magnetic resonance, ECV extracellular volume, EDV end diastolic volume, EDVI end diastolic volume index, ESV end systolic volume, ESVI end systolic volume index, HCM hypertrophic cardiomyopathy, LV left ventricle, LVEF left ventricular ejection fraction, RV right ventricle, RVEF right ventricular ejection fraction p values < 0.05 are bolded CMR cardiovascular magnetic resonance, ECV extracellular volume, EDV end diastolic volume, EDVI end diastolic volume index, ESV end systolic volume, ESVI end systolic volume index, HCM hypertrophic cardiomyopathy, LV left ventricle, LVEF left ventricular ejection fraction, RV right ventricle, RVEF right ventricular ejection f ti Fig. 2  Varying levels of LGE compared to T1⍴ and T1. Short axis LGE, T1, and T1⍴ images are shown for three HCM patients with patchy, focal, and no LGE, and one control patient with no LGE. Areas of elevated T1 and T1⍴ are visually associated with areas of LGE. Associations between T1, T1ρ, and LGE enhancement in HCM patients LGE late gadolinium enhancement Fig. 2  Varying levels of LGE compared to T1⍴ and T1. Short axis LGE, T1, and T1⍴ images are shown for three HCM patients with patchy, focal, and no LGE, and one control patient with no LGE. Areas of elevated T1 and T1⍴ are visually associated with areas of LGE. LGE late gadolinium enhancement Thompson et al. J Cardiovasc Magn Reson (2021) 23:120 Page 7 of 9 Fig. 3  Correlation of T1, T1⍴, and LGE. In LGE-positive slices, we analyzed the correlation of percent area enhancement in A T1 versus LGE (Spearman’s rho = 0.61, p < 1e-5), B T1⍴ versus LGE (Spearman’s rho = 0.48, p < 1e-3) and C T1⍴ versus T1 (Spearman’s rho = 0.28, p = 0.047) images using FWHM thresholding. FWHM full width half maximum, LGE late gadolinium enhancement Fig. 3  Correlation of T1, T1⍴, and LGE. In LGE-positive slices, we analyzed the correlation of percent area enhancement in A T1 versus LGE (Spearman’s rho = 0.61, p < 1e-5), B T1⍴ versus LGE (Spearman’s rho = 0.48, p < 1e-3) and C T1⍴ versus T1 (Spearman’s rho = 0.28, p = 0.047) images using FWHM thresholding. FWHM full width half maximum, LGE late gadolinium enhancement Fig. 4  Comparison of myocardial T1⍴, pre-contrast T1, post-contrast T1, and ECV. A Average myocardial pre-contrast T1, B T1ρ, C post-contrast T1, and D ECV were measured for HCM patients and controls as indicated. HCM patients were subcategorized by LGE rating: LGE + +, LGE + −, and LGE −−. Kruskal–Wallis test showed statistically significant differences between groups for pre-contrast T1, T1ρ, and ECV (p < 0.001 for all). For both T1 and T1ρ, a post-hoc Dunn test showed differences between controls and each LGE category (p < 0.001 for all). For ECV, a post-hoc Dunn test adjusted for multiple comparisons showed differences between controls and each LGE category (p < 0.01 for all), as well as LGE + + and LGE −− (p = 0.01). Statistically significant differences are indicated by * on the bar graphs our group applied FWHM thresholding to LGE images, rather than manual measurement of enhancement area, decreasing observer bias. The use of FWHM threshold- ing therefore increases the robustness of our measure- ments, allowing for direct comparison in future studies. Associations between T1, T1ρ, and LGE enhancement in HCM patients An additional study of T1ρ in a mouse model of cardiac hypertrophy [34] examined T1ρ at several timepoints after transverse aortic constriction and verified fibrosis ex vivo using Masson’s trichrome staining [34]. Similarly, their findings showed that T1ρ increased over time and was highly correlated with fibrotic areas [34].i i Our study brings to light several interesting find- ings. We show moderate correlations between LGE and T1 and T1ρ-assessed percent enhancement area, and mild correlation between T1 and T1ρ. Variations in the enhancement areas calculated by each method may reflect a physiologic difference in the way that LGE, T1, and T1ρ assess healthy and abnormal tissue. Our results indicate that LGE, T1, and T1ρ may each give different and additive information that one method alone cannot provide, a finding that warrants further study. Addition- ally, we demonstrate that HCM patients showed eleva- tions in non-contrast quantitative MR measurements (pre-contrast T1 and T1ρ) regardless of LGE status. The significance of T1ρ imaging and its added value will need to be prospectively evaluated. Fig. 4  Comparison of myocardial T1⍴, pre-contrast T1, post-contrast Fig. 4  Comparison of myocardial T1⍴, pre-contrast T1, post-contrast T1, and ECV. A Average myocardial pre-contrast T1, B T1ρ, C post-contrast T1, and D ECV were measured for HCM patients and controls as indicated. HCM patients were subcategorized by LGE rating: LGE + +, LGE + −, and LGE −−. Kruskal–Wallis test showed statistically significant differences between groups for pre-contrast T1, T1ρ, and ECV (p < 0.001 for all). For both T1 and T1ρ, a post-hoc Dunn test showed differences between controls and each LGE category (p < 0.001 for all). For ECV, a post-hoc Dunn test adjusted for multiple comparisons showed differences between controls and each LGE category (p < 0.01 for all), as well as LGE + + and LGE −− (p = 0.01). Statistically significant differences are indicated by * on the bar graphs To date, only one study has measured T1ρ in human patients with HCM; Wang et  al. compared visually- assessed LGE area with 2–6 standard deviation- thresholding of T1ρ in 18 HCM patients, finding high correlation (Pearson’s r ranging from 0.81 to 0.88) of percent fibrosis between these modalities [33]. In our cohort, we found a lower correlation of T1ρ with LGE- assessed enhancement area using Spearman’s rho, which may be due to several reasons. Associations between T1, T1ρ, and LGE enhancement in HCM patients Our cohort is larger with 40 HCM patients and is more heterogenous with both genotype-positive and -negative patients. Additionally, Abbreviations ANOVA O ANOVA: One-way analysis of variance; BSA: Body surface area; bSSFP: Balanced steady-state free procession; CMR: Cardiovascular magnetic resonance; ECV: Extracellular volume fraction; EDV: End-diastolic volume; EDVI: End-diastolic volume index; ESC: European Society of Cardiology; ESV: End-systolic volume; TE: Echo time; FWHM: Full width half maximum; GBCAs: Gadolinium-based contrast agents; HCT: Hematocrit; HCM: Hypertrophic cardiomyopathy; IQR: Interquartile range; LGE: Late gadolinium enhancement; LV: Left ventricle/left ventricular; LVEF: Left ventricular ejection fraction; LVEDV: Left ventricular end- diastolic volume; LVEF: Left ventricular ejection fraction; LVESV: Left ventricular end-systolic volume; LVOT: Left ventricular outflow tract; NYHA: New York Heart Association; Nseg: Number of segments; PSIR: Phase-sensitive inver- sion recovery; RV: Right ventricle/right ventricular; RVEDV: Right ventricular end-diastolic volume; RVEF: Right ventricular ejection fraction; RVESV: Right ventricular end-systolic volume; SAR: Specific absorption rate; ShMOLLI: Short- ened modified Look-Locker inversion recovery; SL: Spin lock; TE: Echo time; TI: Inversion time; TR: Repetition time; TSL: Spin lock time. Received: 31 July 2021 Accepted: 13 September 2021 Received: 31 July 2021 Accepted: 13 September 2021 Availability of data and materials 9. Ramalho M, Ramalho J, Burke LM, Semelka RC. Gadolinium retention and toxicity-an update. Adv Chronic Kidney Dis. 2017;24(3):138–46. 9. Ramalho M, Ramalho J, Burke LM, Semelka RC. Gadolinium retention and toxicity-an update. Adv Chronic Kidney Dis. 2017;24(3):138–46. The datasets generated and/or analyzed during the current study are not pub- licly available due to patient privacy but are available from the corresponding author on reasonable request. 10. Gulani V, Calamante F, Shellock FG, Kanal E, Reeder SB. International society for magnetic resonance in M. Gadolinium deposition in the brain: summary of evidence and recommendations. Lancet Neurol. 2017;16(7):564–70. Funding Funding for the study is from NIH U01 HL117006 (subcontract to AO), R01HL132130 (to YH), and R01HL137984, P41EB029460, T32EB009384, and T32EB020087 (to WRTW). NR is supported by the National Center for Advanc- ing Translational Sciences of the National Institutes of Health under award number KL2TR001879. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. EWT is supported by NIH Medical Scientist Training Program T32 GM07170. 7. Habib M, Adler A, Fardfini K, Hoss S, Hanneman K, Rowin EJ, et al. Progres- sion of myocardial fibrosis in hypertrophic cardiomyopathy: a cardiac magnetic resonance study. JACC Cardiovasc Imaging. 2020. 7. Habib M, Adler A, Fardfini K, Hoss S, Hanneman K, Rowin EJ, et al. Progres- sion of myocardial fibrosis in hypertrophic cardiomyopathy: a cardiac magnetic resonance study. JACC Cardiovasc Imaging. 2020.i 8. He D, Ye M, Zhang L, Jiang B. Prognostic significance of late gadolinium enhancement on cardiac magnetic resonance in patients with hyper- trophic cardiomyopathy. Heart Lung. 2018;47(2):122–6. Limitations Several limitations to our study should be acknowl- edged. Our cohort was small; thus our findings require validation and further investigation in larger groups of patients. Given the low annual cardiovascular event rate in patients with HCM, longer term follow-up will be needed to understand the utility of T1ρ in the assessment of patients with HCM. Thompson et al. J Cardiovasc Magn Reson (2021) 23:120 Thompson et al. J Cardiovasc Magn Reson (2021) 23:120 Page 8 of 9 Thompson et al. J Cardiovasc Magn Reson (2021) 23:120 Declarations 11. Han Y, Liimatainen T, Gorman RC, Witschey WR. Assessing myocardial disease using T1rho MRI. Curr Cardiovasc Imaging Rep. 2014;7(2):9248. Acknowledgements Not applicable. Dr. Robert Edelman served as a JCMR Guest Editor for this manuscript. 4. Raiker N, Vullaganti S, Collins JD, Allen BD, Choudhury L. Myocardial tissue characterization by gadolinium-enhanced cardiac magnetic resonance imaging for risk stratification of adverse events in hypertrophic cardio- myopathy. Int J Cardiovasc Imaging. 2020;36(6):1147–56. Consent for publication Not applicable. g g 14. Yin Q, Abendschein D, Muccigrosso D, O’Connor R, Goldstein T, Chen R, et al. A non-contrast CMR index for assessing myocardial fibrosis. Magn Reson Imaging. 2017;42:69–73. 14. Yin Q, Abendschein D, Muccigrosso D, O’Connor R, Goldstein T, Chen R, et al. A non-contrast CMR index for assessing myocardial fibrosis. Magn Reson Imaging. 2017;42:69–73. References 1. Semsarian C, Ingles J, Maron MS, Maron BJ. New perspectives on the prevalence of hypertrophic cardiomyopathy. J Am Coll Cardiol. 2015;65(12):1249–54. 2. Marian AJ, Braunwald E. Hypertrophic cardiomyopathy: genetics, pathogenesis, clinical manifestations, diagnosis, and therapy. Circ Res. 2017;121(7):749–70. 3. Weng Z, Yao J, Chan RH, He J, Yang X, Zhou Y, et al. Prognostic value of LGE-CMR in HCM: a meta-analysis. JACC Cardiovasc Imaging. 2016;9(12):1392–402. Authors’ contributions Study design: WRTW, YH. Data acquisition: YH, NR, JK, AJ, AO. Data analy- sis: EWT, SKI, MPS, ZL, QZ, SP, KW, SS, BFM, ZBR, AH, RK, YH, BD, HL, WRTW. Manuscript drafting and editing: EWT, MPS, YH, WRTW. All authors read and approved the final manuscript. 5. Xu J, Zhuang B, Sirajuddin A, Li S, Huang J, Yin G, et al. MRI T1 mapping in hypertrophic cardiomyopathy: evaluation in patients without late gadolinium enhancement and hemodynamic obstruction. Radiology. 2020;294(2):275–86. 5. Xu J, Zhuang B, Sirajuddin A, Li S, Huang J, Yin G, et al. MRI T1 mapping in hypertrophic cardiomyopathy: evaluation in patients without late gadolinium enhancement and hemodynamic obstruction. Radiology. 2020;294(2):275–86. 6. Kwon DH, Smedira NG, Rodriguez ER, Tan C, Setser R, Thamilarasan M, et al. Cardiac magnetic resonance detection of myocardial scarring in hypertrophic cardiomyopathy: correlation with histopathology and prevalence of ventricular tachycardia. J Am Coll Cardiol. 2009;54(3):242–9 6. Kwon DH, Smedira NG, Rodriguez ER, Tan C, Setser R, Thamilarasan M, et al. Cardiac magnetic resonance detection of myocardial scarring in hypertrophic cardiomyopathy: correlation with histopathology and prevalence of ventricular tachycardia. J Am Coll Cardiol. 2009;54(3):242–9. Author details 1 Department of Bioengineering, School of Engineering and Applied Sci- ence, University of Pennsylvania, Philadelphia, PA, USA. 2 Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 3 Department of Radiology, University of Pennsylvania, Philadelphia, PA, USA. 4 Division of Cardiovascular Medicine, Department of Medicine, University of Pennsylva- nia, Philadelphia, PA, USA. 5 Ultrasound in Cardiac Electrophysiology and Bio- mechanics Key Laboratory of Sichuan Province, Cardiovascular Ultrasound and Non‑Invasive Cardiology Department, Affiliated Hospital of University of Electronic Science and Technology of China, Sichuan Academy of Medi- cal Sciences, Sichuan Provincial People’s Hospital, Chengdu, Sichuan, China. 6 Oxford Center for Clinical Magnetic Resonance Research, Oxford BRC NIHR, Division of Cardiovascular Medicine, Radcliffe Department of Medicine, Uni- versity of Oxford, Oxford, UK. 7 Circle Cardiovascular Imaging Inc., Calgary, AB, Canada. 8 Department of Biophysics, University of Pennsylvania, Philadelphia, PA, USA. 9 Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 10 Perelman School of Medicine, University of Pennsylvania, 11‑135, South Pavilion, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. T1 and T1ρ relaxation time moderately correlate with LGE percent enhancement area using FWHM threshold- ing. Additionally, T1, T1ρ, and ECV distinguish HCM patients from healthy controls, irrespective of whether the patient’s myocardium demonstrated positive LGE, showing potential value as a noninvasive biomarker. Fur- ther study is needed to elucidate the role of T1ρ in risk prediction for HCM patients. 14. Yin Q, Abendschein D, Muccigrosso D, O’Connor R, Goldstein T, Chen R, et al. A non-contrast CMR index for assessing myocardial fibrosis. Magn Reson Imaging. 2017;42:69–73. Ethics approval and consent to participate 12. Witschey WR, Zsido GA, Koomalsingh K, Kondo N, Minakawa M, Shuto T, et al. In vivo chronic myocardial infarction characterization by spin locked cardiovascular magnetic resonance. J Cardiovasc Magn Reson. 2012;14:37. The study protocol was approved by the Institutional Review Board of the University of Pennsylvania and all subjects gave written informed consent prior to enrollment. 13. Stoffers RH, Madden M, Shahid M, Contijoch F, Solomon J, Pilla JJ, et al. Assessment of myocardial injury after reperfused infarction by T1rho car- diovascular magnetic resonance. J Cardiovasc Magn Reson. 2017;19(1):17. Competing interests Witschey WR, Pilla JJ, Ferrari G, Koomalsingh K, Haris M, Hinmon R, et al. Rotating frame spin lattice relaxation in a swine model of chronic, left ventricular myocardial infarction. Magn Reson Med. 2010;64(5):1453–60. y 31. Eijgenraam TR, Sillje HHW, de Boer RA. Current understanding of fibrosis in genetic cardiomyopathies. Trends Cardiovasc Med. 2020;30(6):353–61. g 21. Messroghli DR, Radjenovic A, Kozerke S, Higgins DM, Sivananthan MU, Ridgway JP. Modified Look-Locker inversion recovery (MOLLI) for high- resolution T1 mapping of the heart. Magn Reson Med. 2004;52(1):141–6. 32. 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Khan MA, Laakso H, Laidinen S, Kettunen S, Heikura T, Yla-Herttuala S, et al. The follow-up of progressive hypertrophic cardiomyopathy using magnetic resonance rotating frame relaxation times. NMR Biomed. 2018;31(2). 24. Flett AS, Hayward MP, Ashworth MT, Hansen MS, Taylor AM, Elliott PM, et al. Equilibrium contrast cardiovascular magnetic resonance for the measurement of diffuse myocardial fibrosis: preliminary validation in humans. Circulation. 2010;122(2):138–44. Competing interests KW is an employee of Circle Cardiovascular Imaging Inc., Calgary, AB, Canada. The remaining authors declare that they have no competing interests. Page 9 of 9 Thompson et al. J Cardiovasc Magn Reson (2021) 23:120 Thompson et al. J Cardiovasc Magn Reson (2021) 23:120 15. van Oorschot JW, Guclu F, de Jong S, Chamuleau SA, Luijten PR, Leiner T, et al. Endogenous assessment of diffuse myocardial fibrosis in patients with T1rho -mapping. J Magn Reson Imaging. 2017;45(1):132–8. 26. Huang L, Ran L, Zhao P, Tang D, Han R, Ai T, et al. MRI native T1 and T2 mapping of myocardial segments in hypertrophic cardiomyopathy: tissue remodeling manifested prior to structure changes. Br J Radiol. 2019;92(1104):20190634. 16. Kramer CM, Appelbaum E, Desai MY, Desvigne-Nickens P, DiMarco JP, Friedrich MG, et al. Hypertrophic Cardiomyopathy Registry: the rationale and design of an international, observational study of hypertrophic cardiomyopathy. Am Heart J. 2015;170(2):223–30. 27. Hen Y, Takara A, Iguchi N, Utanohara Y, Teraoka K, Takada K, et al. High sig- nal intensity on T2-weighted cardiovascular magnetic resonance imaging predicts life-threatening arrhythmic events in hypertrophic cardiomyopa- thy patients. Circ J. 2018;82(4):1062–9. cardiomyopathy. Am Heart J. 2015;170(2):223–30. y y 17. Berisha S, Han J, Shahid M, Han Y, Witschey WR. Measurement of myo- cardial T1rho with a motion corrected, parametric mapping sequence in humans. PLoS ONE. 2016;11(3):e0151144. 28. Gommans DHF, Cramer GE, Bakker J, Dieker HJ, Michels M, Fouraux MA, et al. High T2-weighted signal intensity for risk prediction of sudden cardiac death in hypertrophic cardiomyopathy. Int J Cardiovasc Imaging. 2018;34(1):113–20. 18. Witschey WR, Borthakur A, Elliott MA, Fenty M, Sochor MA, Wang C, et al. T1rho-prepared balanced gradient echo for rapid 3D T1rho MRI. J Magn Reson Imaging. 2008;28(3):744–54. 29. Amano Y, Aita K, Yamada F, Kitamura M, Kumita S. Distribution and clinical significance of high signal intensity of the myocardium on T2-weighted images in 2 phenotypes of hypertrophic cardiomyopathy. J Comput Assist Tomogr. 2015;39(6):951–5. 19. Witschey WR 2nd, Borthakur A, Elliott MA, Mellon E, Niyogi S, Wallman DJ, et al. Artifacts in T1 rho-weighted imaging: compensation for B(1) and B(0) field imperfections. J Magn Reson. 2007;186(1):75–85. 20. Piechnik SK, Ferreira VM, Dall’Armellina E, Cochlin LE, Greiser A, Neubauer S, et al. Shortened Modified Look-Locker Inversion recovery (ShMOLLI) for clinical myocardial T1-mapping at 1.5 and 3 T within a 9 heartbeat breathhold. J Cardiovasc Magn Reson. 2010;12:69. 30. Thompson et al. J Cardiovasc Magn Reson (2021) 23:120 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 25. Dass S, Suttie JJ, Piechnik SK, Ferreira VM, Holloway CJ, Banerjee R, et al. Myocardial tissue characterization using magnetic resonance non- contrast t1 mapping in hypertrophic and dilated cardiomyopathy. 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https://clok.uclan.ac.uk/46182/1/Journal%20of%20Neuropsychology%20-%202023%20-%20Souter%20-%20How%20do%20valence%20and%20meaning%20interact%20%20The%20contribution%20of%20semantic%20control.pdf
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How do valence and meaning interact? The contribution of semantic control
Journal of neuropsychology
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R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Nicholas E. Souter1    |  Ariyana Reddy1,2  |  Jake Walker1,3  |  Julián Marino Dávolos1  |  Elizabeth Jefferies1 Nicholas E. Souter1    |  Ariyana Reddy1,2  |  Jake Walker1,3  |  Julián Marino Dávolos1  |  Elizabeth Jefferies1 1Department of Psychology, University of York, York, UK 2Faculty of Health Sciences, University of Hull, Hull, UK 3School of Psychology and Computer Science, University of Central Lancashire, Preston, UK Correspondence Nicholas E. Souter, Department of Psychology, University of York, York YO10 5DD, UK. Email: nes522@york.ac.uk Funding information ERC Consolidator grant, Grant/Award Number: FLEXSEM – 771863 1Department of Psychology, University of York, York, UK Central Lancashire Online Knowledge (CLoK) Title How do valence and meaning interact? The contribution of semantic control Type Article URL https://clok.uclan.ac.uk/46182/ DOI https://doi.org/10.1111/jnp.12312 Date 2023 Citation Souter, Nicholas E., Reddy, Ariyana, Walker, Jake, Marino Dávolos, Julián and Jefferies, Elizabeth (2023) How do valence and meaning interact? The contribution of semantic control. Journal of Neuropsychology. ISSN 1748- 6645 Creators Souter, Nicholas E., Reddy, Ariyana, Walker, Jake, Marino Dávolos, Julián and Jefferies, Elizabeth Central Lancashire Online Knowledge (CLoK) Title How do valence and meaning interact? The contribution of semantic control Type Article URL https://clok.uclan.ac.uk/46182/ DOI https://doi.org/10.1111/jnp.12312 Date 2023 Citation Souter, Nicholas E., Reddy, Ariyana, Walker, Jake, Marino Dávolos, Julián and Jefferies, Elizabeth (2023) How do valence and meaning interact? The contribution of semantic control. Journal of Neuropsychology. ISSN 1748- 6645 Creators Souter, Nicholas E., Reddy, Ariyana, Walker, Jake, Marino Dávolos, Julián and Jefferies, Elizabeth Central Lancashire Online Knowledge (CLoK) Title How do valence and meaning interact? The contribution of semantic control Type Article URL https://clok.uclan.ac.uk/46182/ DOI https://doi.org/10.1111/jnp.12312 Date 2023 Citation Souter, Nicholas E., Reddy, Ariyana, Walker, Jake, Marino Dávolos, Julián and Jefferies, Elizabeth (2023) How do valence and meaning interact? The contribution of semantic control. Journal of Neuropsychology. ISSN 1748- 6645 Creators Souter, Nicholas E., Reddy, Ariyana, Walker, Jake, Marino Dávolos, Julián and Jefferies, Elizabeth It is advisable to refer to the publisher’s version if you intend to cite from the work. https://doi.org/10.1111/jnp.12312 or information about Research at UCLan please go to http://www.uclan.ac.uk/researc All outputs in CLoK are protected by Intellectual Property Rights law, including Copyright law. Copyright, IPR and Moral Rights for the works on this site are retained by the individual authors and/or other copyright owners. Terms and conditions for use of this material are defined in the http://clok.uclan.ac.uk/policies/ R E S E A R C H A R T I C L E DOI: 10.1111/jnp.12312 Received: 19 May 2022        Accepted: 6 March 2023 DOI: 10.1111/jnp.12312 Received: 19 May 2022        Accepted: 6 March 2023 Abstract The hub-and-spoke model of semantic cognition proposes that conceptual representations in a heteromodal ‘hub’ interact with and emerge from modality-specific features or ‘spokes’, including valence (whether a concept is posi- tive or negative), along with visual and auditory features. As a result, valence congruency might facilitate our abil- ity to link words conceptually. Semantic relatedness may similarly affect explicit judgements about valence. More- over, conflict between meaning and valence may recruit semantic control processes. Here we tested these predic- tions using two-alternative forced-choice tasks, in which participants matched a probe word to one of  two possible target words, based on either global meaning or valence. Experiment 1 examined timed responses in healthy young adults, while Experiment 2 examined decision accuracy in semantic aphasia patients with impaired controlled seman- tic retrieval following left hemisphere stroke. Across both experiments, semantically related targets facilitated valence matching, while related distractors impaired performance. Valence congruency was also found to facilitate seman- tic decision-making. People with semantic aphasia showed impaired valence matching and had particular difficulty when semantically related distractors were presented, suggesting that the selective retrieval of valence information relies on semantic control processes. Taken together, the results are consistent with the hypothesis that automatic access to the global meaning of written words affects the processing of valence, and that the valence of words is also retrieved even 2Faculty of Health Sciences, University of Hull, Hull, UK 3School of Psychology and Computer Science, University of Central Lancashire, Preston, UK How do valence and meaning interact? The contribution of semantic control Nicholas E. Souter1    |  Ariyana Reddy1,2  |  Jake Walker1,3  |  Julián Marino Dávolos1  |  Elizabeth Jefferies1 J Neuropsychol. 2023;00:1–19. wileyonlinelibrary.com/journal/jnp 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2023 The Authors. Journal of Neuropsychology published by John Wiley & Sons Ltd on behalf of The British Psychological Society. wileyonlinelibrary.com/journal/jnp J Neuropsychol. 2023;00:1–19. INTRODUCTION A representation of puppy may rely on knowledge concerning typical visual features and characteristic yapping noises, and that puppies are positive entities who evoke joy. It can be argued that valence (whether items are pleasant or unpleasant) is a core feature of heteromodal concepts. The ‘hub-and-spoke’ frame- work suggests that semantic representation relies on interactions between a transmodal hub in the ante- rior temporal lobes (ATL) and modality-specific spokes; including perceptual and motor features along with valence (Lambon Ralph et al., 2017). Amygdala and orbitofrontal cortex may support the integration of emotion-based features through connections with ATL (Riberto et al., 2019). Patients with semantic dementia (SD), following ATL atrophy, show degradation of conceptual knowledge across tasks that probe the same concepts (Jefferies & Lambon Ralph, 2006), and experience difficulty categorising facial emotions (Lindquist et al., 2014). The ability to make sense of discrete emotions may rely on conceptual representations (Lindquist et al., 2015). Concepts are grounded in valence as well as action and percep- tion (Martin, 2016). Indeed, valence benefits abstract word learning (Ponari et al., 2018) and modulates activation in the anterior cingulate cortex, important for abstract word processing (Vigliocco et al., 2014). Valence can therefore be considered a semantic feature. Conceptual information may modulate the acces- sibility of valence features and vice versa. Semantic cognition relies not only on heteromodal representations, but also on the ability to flexibly retrieve them; ‘semantic control’ (Lambon Ralph et al., 2017). Semantic control demands are maxim- ised when meaning is ambiguous and/or there is competition from task-irrelevant information (Jefferies et  al.,  2019). Neuropsychological studies reveal a double dissociation between degraded conceptual knowledge in SD, and disordered multi-modal semantic control in semantic aphasia (SA) following left frontal-temporal–parietal stroke (Jefferies & Lambon Ralph, 2006). SA patients are sensitive to executive demands of semantic tasks (Jefferies, 2013): They have difficulty retrieving non-dominant conceptual information and are susceptible to semantic distractors (Noonan et al., 2010). This frequently co-occurs with domain-general executive dysfunction (Thompson et al., 2018). SA patients are sensitive to cues that reduce the need to internally constrain retrieval (Noonan et al., 2010). Facial emotions can disambiguate interpretation of words that have both positive and negative meanings in SA (Lanzoni et al., 2019), possi- bly by modulating semantic control demands and constraining retrieval. Correspondence Nicholas E. Souter, Department of Psychology, University of York, York YO10 5DD, UK. Email: nes522@york.ac.uk Funding information ERC Consolidator grant, Grant/Award Number: FLEXSEM – 771863 Funding information ERC Consolidator grant, Grant/Award Number: FLEXSEM – 771863 J Neuropsychol. 2023;00:1–19. wileyonlinelibrary.com/journal/jnp 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2023 The Authors. Journal of Neuropsychology published by John Wiley & Sons Ltd on behalf of The British Psychological Society. J Neuropsychol. 2023;00:1–19. wileyonlinelibrary.com/journal/jnp 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2023 The Authors. Journal of Neuropsychology published by John Wiley & Sons Ltd on behalf of The British Psychological Society. 2 2 2 2 SOUTER et al. when this feature is task-irrelevant, affecting the efficiency of global semantic judgements. K E Y W O R D S aphasia, congruency, semantic, stroke, valence INTRODUCTION SA patients also show deficits in accessing emotions from facial portrayals; common processes may constrain the retrieval of meaning and emotion (Souter et al., 2021). Neuroimaging research implicates a distributed but largely left-lateralised ‘semantic control network’ (SCN) in semantic retrieval, which includes anterior left inferior frontal gyrus (IFG) and posterior middle temporal gyrus (pMTG; Jackson, 2021). These regions are adjacent to domain-general control regions (Gao et al., 2021). Lesion to and structural disconnection between left-hemisphere SCN regions predicts semantic control deficits in SA (Souter, Wang, et al., 2022). Regions of SCN are also implicated in tasks involving valence—including comparisons of lexical decision for valenced versus neutral words (Pauligk et al., 2019) and the resolution of conflict from valence incongruency (Gao et al., 2020). SCN may play a role in controlling the retrieval of emotion along with other aspects of meaning. Semantic control may be required to match words by valence when they do not share other features (puppy and cake both have positive valence but no semantic link), since a single task-relevant feature must compete with many irrelevant features. This has been reported for colour (Thompson-Schill et al., 1997). 3 VALENCE AND MEANING Global semantic similarity facilitates feature matching, reducing SCN activation (Wang et  al.,  2020). Global similarity refers to the similarity of contexts in which words are used, and should be sensitive to shared features and strength of thematic association. If access to valence irrespective of global similar- ity requires semantic control, patients with SA should be impaired at valence matching. Furthermore, a mismatch in valence may make it harder to identify global links between words. This effect, based on a single task-irrelevant feature, should be smaller than the effect of global semantic similarity on valence matching. Valence congruency between words facilitates healthy adults' detection of global semantic rela- tionships, particularly for weak associations (Marino Dávolos et al., 2020). This may be magnified in SA due to difficulty resolving competition from valence. Here, we investigated effects of (i) semantic related- ness on the ability to match words by valence and (ii) valence congruency on the ability to match words by semantic relatedness. In Experiment 1, we studied healthy young adults, asked to respond as fast and accurately as possible. In Experiment 2, we observed SA patients and age-matched controls to establish if these effects are magnified by semantic control deficits. Stimuli Stimuli were nouns taken from a database (Warriner et al., 2013) which reports mean valence and arousal of words on a scale from 1 to 9, using participant ratings. We classified words above 6 as positively valenced, between 4 and 6 as neutral, and below 4 as negative.1 We excluded words with a standard devi- ation of valence ratings above 2, which may have ambiguous meaning (e.g., ‘jam’) or diverse emotional reactions (e.g., ‘religion’). We assessed the strength of association between each probe-target and probe- foil pair using word2vec (Mikolov et al., 2013), a measure of semantic distance between words based on co-occurrence in text and an effective proxy for semantic relatedness (Pereira et al., 2016). Other approaches are available, such as asking participants to self-generate associations. Co-occurrence was the most practical way of measuring semantic relatedness while balancing psycholinguistic properties. An association was considered ‘strong’ if word2vec was above .2, ‘weak’ if between .1 and .2, and negligible if below .1. Stimuli were controlled for valence, strength of association, word frequency, and psycholinguis- tic factors (see Supporting Information section ‘Stimulus Properties’). Negative words were significantly higher in arousal than positive words. To observe for potential effects of this confound, each mixed effects model used in this study was re-run with arousal congruency between probe and target word as a predictor (see Table S2). No effect of arousal congruency was found in any model, nor did its inclusion attenuate any other effects. It is therefore likely that observed effects of valence congruency can be attrib- uted to valence itself, rather than arousal. 1 Valenced words included largely emotion-laden terms with acquired affective connotation (e.g., war, rainbow). 9.5% of stimuli could be considered emotion-label, representing affective states (e.g., hope, terror). Semantic matching task The semantic matching task required participants to match one of two words to a probe by semantic relatedness. Participants were told: “your task is to indicate which of the two words on the bottom has the strongest connection to the word on top”. Two conditions manipulated valence congruency, a third manipulated association strength. In the congruent target condition, the target had a strong association to the probe and was congruent in valence, while the foil had no association and was incongruent. In the congru- ent distractor condition, the target had a strong association to the probe but was incongruent in valence, while the foil had no association but was congruent. In the weak association condition, the target had a weak association to the probe, while the foil had no association. Valence congruency was not manipulated here due to challenges sourcing weakly associated targets while manipulating valence. The valence of the foil was congruent with the probe in half of the trials, and incongruent in the remainder. Example trials can be seen in Figure 1b. Valence matching task The valence matching task required participants to match one of two words to a probe word by valence. The target was always the same valence as the probe, while the foil was the opposite valence. Partici- pants were told: “your task is to indicate which of the two words on the bottom has the same emotional valence (positive or negative) as the word on the top”. We manipulated strength of semantic association. In the associated target condition, the probe had a strong association with the target and no association with the foil. In the no association condition, the probe had no association with either response option. In the associated distractor condition, the probe had no association with the target but a strong association with the foil. It was predicted that the associated target condition would facilitate valence matching through 1 Valenced words included largely emotion-laden terms with acquired affective connotation (e.g., war, rainbow). 9.5% of stimuli could be considered emotion-label, representing affective states (e.g., hope, terror). 17486653, 0, Downloaded from https://bpspsychub.onlinelibrary.wiley.com/doi/10.1111/jnp.12312 by Test, Wiley Online Library on [03/04/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA 4 4 SOUTER et al. F I G U R E 1   Examples of trials in each condition in the (a) valence matching and (b) semantic matching tasks. The relationship to the probe word for both the target and foil is explained for each example. Target words are underlined and in bold. F I G U R E 1   Examples of trials in each condition in the (a) valence matching and (b) semantic matching tasks. The relationship to the probe word for both the target and foil is explained for each example. Target words are underlined and in bol semantic cueing, while the associated distractor condition would impair matching by requiring inhibition of the distractor. Example trials can be seen in Figure 1a. Design A within-subjects design was used; all participants completed both the valence matching and semantic matching tasks. Procedure Block order (valence/semantic) was randomised within each session. At the start of each block partic- ipants saw instructions explaining the matching strategy and an example trial with explanation of the correct answer. Valence matching instructions did not disclose that association strength would be manipu- lated, and semantic matching instructions did not disclose that valence congruency or association strength would be manipulated. Participants were instructed to press the ‘1’ key on their keyboard to select the left response option, and ‘2’ to select the right option. Before each block, participants completed six practice trials including feedback. Between blocks, participants saw a warning of the change in task instructions. No time limit was applied. Participants Participants were neurologically healthy adults tested on the online platform Gorilla (www.gorilla.sc; Anwyl-Irvine et al., 2020). Eighty-six participants were recruited opportunistically. Participants automat- ically received an email one week after the first session, prompting them to complete the second. Partic- ipants were excluded if they did not complete the second session (N = 11), if they scored below chance (50% accuracy) on any condition (N = 14), or if their median response time for any condition was an outlier (N = 3), as determined in SPSS (version 27.0; IBM Corp., 2020). The sample consisted of 60 adults (38 female) between the ages of 19 and 41 [Mean (SD) = 25.1 (5.6)]. Trial structure The experiment was split across two sessions separated by at least a week, each containing a block of valence matching and of semantic matching. Trial order was randomised within blocks. The same response triads were used across (i) ‘valence – associated target’ and ‘semantic – congruent target’ and (ii) VALENCE AND MEANING 5 ‘valence – associated distractor’ and ‘semantic – congruent distractor’ (target response switched). Triads in the ‘valence – no association’ condition were re-used in the ‘semantic – weak association’ condition, with one response option replaced with a weakly associated target. Presentation order was counterbalanced, such that if a given triad appeared in valence matching in session 1, it appeared in semantic matching in session 2. Target responses appeared on the left in half of the trials, and on the right in the remainder. Each condition contained 27 trials, providing 81 trials per task, and 162 trials overall. Principal components analysis PCA revealed two components for accuracy and one for RT (see Table 1). The first accuracy component appears to reflect conditions which should be automatic; valence matching without semantically associated distractors and semantic matching with valence-congruent targets. The second component appears to reflect conditions which should require controlled processing; valence matching with associated distractors, semantic matching with valence-incongruent targets, and weak associations. The RT factor suggests that faster responses on a given condition are associated with faster responses on all other conditions. Alternative interpretations are possible. Results Participants' mean accuracy and RT in each condition are in Figure 2. Downloaded from https://bpspsychub.onlinelibrary.wiley.com/doi/10.1111/jnp.12312 by Test, Wiley Online Library on [03/04/2023]. See the Terms and Cond Effects of valence congruency on semantic matching were assessed by comparing performance across the congruent target and congruent distractor conditions with Wilcoxon signed-rank tests since the normality assumption was violated. We then assessed the effect of association strength by averaging across the congruent target and congruent distractor conditions to produce a strong association score, which was compared to the weak association condition using Wilcoxon signed-rank tests. This strong association score should control for valence, as trials are equally split across congruent targets and foils. While target valence congruency was not manipulated for weak association trials, the foil was congruent in half trials. Given evidence that valence congruency effects depend on association strength (Marino Dávolos et al., 2020), we examined the parametric effect of probe-target association strength (using word2vec) across the congruent target and congruent distractor conditions. For accuracy, we used a mixed effects logistic regression, predicting the probability of a correct response. For RT, a mixed effects linear regression was used. Outliers were addressed by removing RTs larger than either 10 s or 3 standard deviations above a given participant's mean RT in each condition. RTs were log transformed such that residuals were approximately normally distributed. Condition and association strength were used as fixed factors, and participant identity and item (trial) as crossed random factors. Likelihood ratio tests were used to deter- mine significance by statistically comparing the full model to nested versions with effects or interactions removed, using the chi-square distribution. We used the same method to assess effects of association strength on valence matching – restricted to the no association and associated distractor conditions, given that target strength was matched across them. This observes effects of semantically associated distractors on valence matching as a function of association strength but does not provide insight into the relationship between valence congruency and processing of meaning. This is reported in the Supporting Information section ‘Valence Congruency Mixed Effects Models – Experiment 1’. Finally, we performed two-way repeated measures ANOVA with variables of task (valence matching vs. semantic matching) and difficulty (easy [‘valence – associated target’ and ‘semantic – congruent target’] vs. hard [‘valence – associated distractor’ and ‘semantic – congruent distractor’]). This allowed us to compare performance across tasks and assess whether either difficulty manipulation was more influential. Data analysis For each condition we extracted each participant's accuracy (percent correct) and response time (RT; seconds) for correct responses. Median RT, rather than mean RT, was extracted for each condition at the individual-level to reduce effects of outliers. At the group-level, the mean of median RTs for each condition was assessed. We entered accuracy and RT on all conditions into separate principal compo- nents analyses (PCA) with varimax rotation, to assess whether performance across conditions loads onto common components. To assess the effect of semantic association on valence matching, we conducted one-way repeated measures ANOVAs, comparing accuracy and RT across the three conditions. SOUTER et al. Semantic matching Next, we contrasted performance on the semantic matching conditions that involved targets and distrac- tors of the same valence, and that involved strong and weak associations. The two comparisons for both accuracy and RT were Bonferroni-corrected. For both measures, there was a significant difference between the congruent target and congruent distractor conditions [accuracy: Z = −5.5, p < .001,3 RT: Z = −4.5, p < .001], and between strong association and weak association trials [accuracy: Z = −6.4, p < .001, RT: Z = −6.7, p < .001]. This suggests that valence-congruent targets facilitated semantic decisions relative to trials with valence-congruent distractors, and that weak associations conferred greater semantic control demands than strong associations (see Figure 2). The semantic task involved separate manipulations of valence congruency and association strength. To establish if these factors interact, we used mixed effects models. Valence congruency was included as a binary predictor (congruent target vs. congruent distractor), while association strength was continuous (word- 2vec score between target and probe word). Participant identity and item (trial) were used as crossed random factors. Results can be seen in Table 2. Stronger probe-target association predicted more accurate and faster responses, while valence congru- ency predicted faster responses. Valence congruency did not affect accuracy, contrary to the Wilcoxon test reported above. This effect may be attenuated when factoring in random variation attributable to test item. For both measures, a significant interaction was found. These interactions were parsed using the emtrends function of the emmeans package (Lenth, 2020), and visualised using the ggpredict function of the ggeffects package (Lüdecke, 2018); see Figure 3.4 For the congruent target condition, greater association strength was associated with a higher probability of a correct response and faster responses [accuracy: association = 6.07, LCL = 4.16, UCL = 7.99, RT: association = −.77, LCL = −.93, UCL = −.61]. For the TA B L E 2   Output of Experiment 1 semantic matching mixed effects regressions. Valence matching [accuracy: t(59) = 2.4, p = .039, RT: t(59) = −5.9, p < .001] and between the no association and associated distractor conditions [accuracy: t(59) = 7.8, p < .001, RT: t(59) = −2.9, p = .009].2 This suggests that seman- tically related targets facilitated valence matching, while distractors impaired performance. [accuracy: t(59) = 2.4, p = .039, RT: t(59) = −5.9, p < .001] and between the no association and associated distractor conditions [accuracy: t(59) = 7.8, p < .001, RT: t(59) = −2.9, p = .009].2 This suggests that seman- tically related targets facilitated valence matching, while distractors impaired performance. 2 The assumption of normality was not always met but non-parametric tests elicited the same outcomes. Accuracy: associated target – no association [Z = −3.0, p = .005], no association – associated distractor [Z = −5.9, p < .001]. RT: associated target – no association [Z = −4.9, p < .001], no association – associated distractor [Z = −3.6, p < .001]. 3 Note that this effect is not significant in the mixed effects model below. 4 Although RT was estimated using a linear mixed effects model, trends visualised are curved as RT was log-transformed. Similarly, accuracy was estimated using log transformation of odds ratios. ificant result. Significant results are also presented in bold. The Accuracy model was run in R using lme4 package (version 1.1-25; Bates et al., 2015). As this is a logistic model, estimate coefficients reflect log transformation of odds ratios (Larsen et al., 2000). The Response Time model was run in R using lmerTest package (version 3.1-3; Kuznetsova et al., 2017), response time values are log transformed. Abbreviation: CI, confidence interval. as estimated using a linear mixed effects model, trends visualised are curved as RT was log-transformed. Similarly, accuracy was og transformation of odds ratios. Valence matching Repeated measures ANOVAs were used to examine performance across the valence matching conditions. We found significant effects of condition for accuracy [F(1.3, 73.9) = 55.0, p < .001, 𝐴𝐴 𝐴𝐴2 𝑝𝑝  = .48] and RT [F(2, 118) = 38.2, p < .001, 𝐴𝐴 𝐴𝐴2 𝑝𝑝  = .39]. Post-hoc contrasts (Bonferroni-corrected for two comparisons for each ANOVA) revealed significant differences between the associated target and no association conditions VALENCE AND MEANING 7 F I G U R E 2   Participants' (a) mean accuracy (percentage correct) and (b) mean response time (seconds) for each task and di i i E i 1 E b fl d d f h Th ‘S A i i ’ b fl h f VALENCE AND MEANING F I G U R E 2   Participants' (a) mean accuracy (percentage correct) and (b) mean response time (seconds) for each task and ondition in Experiment 1. Error bars reflect one standard error of the mean. The ‘Strong Association’ bars reflect the average of performance on the ‘Congruent Target’ and ‘Congruent Distractor’ conditions. F I G U R E 2   Participants' (a) mean accuracy (percentage correct) and (b) mean response time (seconds) for each task and condition in Experiment 1. Error bars reflect one standard error of the mean. The ‘Strong Association’ bars reflect the average of performance on the ‘Congruent Target’ and ‘Congruent Distractor’ conditions. TA B L E 1   Rotated component matrices for principal components analysis of Experiment 1 with varimax rotation, examining accuracy and response time across conditions. TA B L E 1   Rotated component matrices for principal components analysis of Experiment 1 with varimax rotation, examining accuracy and response time across conditions. Matching task Condition Accuracy Response time Component 1 (Eigenvalue = 1.83) Component 2 (Eigenvalue = 1.73) Component 1 (Eigenvalue = 4.65) Valence Associated target .772 −.206 .845 No association .806 .099 .871 Associated distractor .149 .701 .836 Semantic Congruent target .705 .122 .903 Congruent distractor −.129 .716 .913 Weak association .032 .841 .908 Note: Strong loadings for each component in bold SOUTER et al. [accuracy: t(59) = 2.4, p = .039, RT: t(59) = −5.9, p < .001] and between the no association and associated distractor conditions [accuracy: t(59) = 7.8, p < .001, RT: t(59) = −2.9, p = .009].2 This suggests that seman- tically related targets facilitated valence matching, while distractors impaired performance. n of normality was not always met but non-parametric tests elicited the same outcomes. Accuracy: associated target – no association 005], no association – associated distractor [Z = −5.9, p < .001]. RT: associated target – no association [Z = −4.9, p < .001], no association – [Z = −3.6, p < .001]. Note: *Reflects a significant result. Significant results are also presented in bold. The Accuracy model was run in R using lme4 package (version 1.1-25; Bates et al., 2015). As this is a logistic model, estimate coefficients reflect log transformation of odds ratios (Larsen et al., 2000). The Response Time model was run in R using lmerTest package (version 3.1-3; Kuznetsova et al., 2017), response time values are log transformed. Abbreviation: CI, confidence interval. Task comparison We compared the effect of congruency/relatedness across tasks using repeated-measures ANOVA. Results are in Table 3. There were significant effects of task and difficulty and a significant task by diffi- culty interaction for both accuracy and RT. This reflects more accurate and faster responses for semantic matching than valence matching, and for congruent/related than incongruent/distractor trials. The inter- actions reflect larger effects of semantic relatedness on valence matching than of valence congruency on semantic matching (see Figure 2), as expected as valence is only one of many semantic features. Semantic matching Measure Effect Result Accuracy Task F (1, 59) = 45.1, p < .001, 𝐴𝐴 𝐴𝐴2 𝑝𝑝  = .43* Difficulty F (1, 59) = 79.7, p < .001, 𝐴𝐴 𝐴𝐴2 𝑝𝑝  = .58* Task by difficulty F (1, 59) = 20.4, p < .001, 𝐴𝐴 𝐴𝐴2 𝑝𝑝  = .26* Response Time Task F (1, 59) = 201.1, p < .001, 𝐴𝐴 𝐴𝐴2 𝑝𝑝  = .77* Difficulty F (1, 59) = 72.7, p < .001, 𝐴𝐴 𝐴𝐴2 𝑝𝑝  = .55* Task by difficulty F (1, 59) = 36.6, p < .001, 𝐴𝐴 𝐴𝐴2 𝑝𝑝  = .38* Note: *Reflects a significant effect. TA B L E 3   Experiment 1 task comparison ANOVA results. Measure Effect Result Accuracy Task F (1, 59) = 45.1, p < .001, 𝐴𝐴 𝐴𝐴2 𝑝𝑝  = .43* Difficulty F (1, 59) = 79.7, p < .001, 𝐴𝐴 𝐴𝐴2 𝑝𝑝  = .58* Task by difficulty F (1, 59) = 20.4, p < .001, 𝐴𝐴 𝐴𝐴2 𝑝𝑝  = .26* Response Time Task F (1, 59) = 201.1, p < .001, 𝐴𝐴 𝐴𝐴2 𝑝𝑝  = .77* Difficulty F (1, 59) = 72.7, p < .001, 𝐴𝐴 𝐴𝐴2 𝑝𝑝  = .55* Task by difficulty F (1, 59) = 36.6, p < .001, 𝐴𝐴 𝐴𝐴2 𝑝𝑝  = .38* Note: *Reflects a significant effect. TA B L E 3   Experiment 1 task comparison ANOVA results. TA B L E 3   Experiment 1 task comparison ANOVA results. Note: *Reflects a significant effect. congruent distractor condition, no effect of association strength was observed [accuracy: association = −.47, LCL = −5.20, UCL = 4.27, RT: association = .11, LCL = −.34, UCL = .56]. This suggests that benefits of association strength may not occur when participants must resolve inconsistency between valence and meaning. Stronger associations appear more advantageous when incongruency is not present. Semantic matching Measure Variable Estimate Lower 95% CI Upper 95% CI Likelihood ratio test Accuracy Intercept 2.49 2.04 2.94 – Valence congruency 1.28 −.51 3.08 χ(1) = 1.94, p = .163 Association strength 6.07 4.16 7.99 χ(1) = 37.69, p < .001* Valence by strength −6.54 −11.6 −1.43 χ(1) = 6.10, p = .014* Response time Intercept .97 .91 1.04 – Valence congruency −.37 −.54 −.20 χ(1) = 17.39, p < .001* Association strength −.77 −.93 −.61 χ(1) = 71.23, p < .001* Valence by strength .88 .40 1.36 χ(1) = 12.62, p < .001* TA B L E 2   Output of Experiment 1 semantic matching mixed effects regressions. Note: *Reflects a significant result. Significant results are also presented in bold. The Accuracy model was run in R using lme4 package (version 1.1-25; Bates et al., 2015). As this is a logistic model, estimate coefficients reflect log transformation of odds ratios (Larsen et al., 2000). The Response Time model was run in R using lmerTest package (version 3.1-3; Kuznetsova et al., 2017), response time values are log transformed. Abbreviation: CI, confidence interval. Note: *Reflects a significant result. Significant results are also presented in bold. The Accuracy model was run in R using lme4 package (version 1.1-25; Bates et al., 2015). As this is a logistic model, estimate coefficients reflect log transformation of odds ratios (Larsen et al., 2000). The Response Time model was run in R using lmerTest package (version 3.1-3; Kuznetsova et al., 2017), response time values are log transformed. Abbreviation: CI, confidence interval. 9 VALENCE AND MEANING F I G U R E 3   Associations between probe-target association strength and both the likelihood of a correct response (left) and response time (right) for the Experiment 1 semantic matching task. Grey shaded areas reflect confidence intervals based on the standard errors. F I G U R E 3   Associations between probe-target association strength and both the likelihood of a correct response (left) and response time (right) for the Experiment 1 semantic matching task. Grey shaded areas reflect confidence intervals based on the standard errors. TA B L E 3   Experiment 1 task comparison ANOVA results. Participants Participants included five patients and 15 neurologically healthy controls. All patients had left hemisphere stroke. They had an average age of 61.5 (SD = 6.3), average age of leaving education of 19.8 (SD = 3.6), and an average of 13.6 years (SD = 5.0) since stroke. Controls had an average age of 65.0 (SD = 6.7) and average age of leaving education of 21.2 (SD = 3.0). Patients were selected from a database of SA patients who were recruited from communication support groups across Yorkshire. Patients in the current sample were those able to engage with remote testing due to restrictions during the COVID-19 pandemic. Infor- mation on lesion location, when available, is reported in the Supporting Information section ‘Lesion Analysis’ and displayed in Figure S3. EXPERIMENT 2: SEMANTIC APHASIA PATIENTS Experiment 2 employed the same tasks, with SA patients and age-matched controls. 10 10 SOUTER et al. Background neuropsychological testing Patients were tested on language, memory, visuospatial processing, executive function, and semantic cognition. Description of patients' performance on specific assessments can be seen in the Supporting Information section ‘Background Neuropsychology’. Individual patients' performance on non-semantic and semantic assessments is in Tables S5 and S6, respectively. Patients showed minimal impairment in word repetition, but all showed impaired verbal fluency. Four had impaired verbal working memory. All had preserved visuospatial processing. Two were impaired on at least one test of executive function. p p p g p On the Cambridge Semantic Battery (Bozeat et al., 2000), patients showed variable performance on picture naming, but invariably improved following phonemic cueing. Patients performed near ceiling on word-picture matching and showed at least some impairment on picture and word versions of the associative Camel and Cactus Test. All showed impairment on assessments which manipulated semantic control: including difficulty retrieving subordinate thematic associations, deleterious effects of semantic distractors, and benefits of contextual cueing. Given relatively preserved performance on aspects of the Cambridge Semantic Battery, patients should be conceptualised as presenting with impairments in seman- tic control, rather than deficits in semantic representation as in SD (Jefferies & Lambon Ralph, 2006). All patients were impaired on at least one verbal and non-verbal measure of semantic control, consistent with Jefferies and Lambon Ralph (2006), although the current sample may have relatively mild impairment due to the use of demanding semantic tasks. Our sample is also consistent with the original definition of SA as impairment in the flexible manipulation of information for abstract and symbolic processing (Head, 1926). Patients' deficits extend beyond those reported by Head (1926), with added evidence of impaired language, working memory, and executive function. Patients were not excluded based on impair- ments beyond the semantic domain. Patients were grouped based on the presence of shared semantic control impairments, as in prior studies (Stampacchia et al., 2018). Using this group, we can ask whether semantic control impairments in SA extend to valence matching, but cannot rule out the contribution of non-semantic impairments. Patients' degree of semantic control impairment was quantified using the results of PCA previously conducted on a larger sample (N = 17, including the current five; Souter, Stampacchia, et al., 2022). Regression scores were taken as patients' semantic control composite scores. These can be seen in Table S6. Loadings for this component are in Table S7. Design We used a mixed design, with patients and controls completing both the valence matching and semantic matching tasks. VALENCE AND MEANING 11 Procedure The paradigm was coded in PsychoPy3 (Peirce et al., 2019) and run remotely over Zoom (Zoom Video Communications Inc., 2016). The researcher shared their screen such that the participant could see the exper- iment, and gave them remote control of the cursor. At the start of each session, participants were shown instructions and practice trials as in Experiment 1 (see Procedure Section). To respond, participants moved the cursor over the response they wished to select. The researcher then recorded their choice by pressing a button – an analogue to pointing at the screen, the method typically employed during our in-person testing. Data analysis Accuracy (percent correct) was the dependent measure. Each patient was classified as either impaired or not impaired on each condition using Singlims (Crawford et al., 2010), which compares an individual score to the respective control mean and standard deviation. One-tailed p-values below .05 were taken as reflecting impairment. As sample size was insufficient to run ANOVAs as in Experiment 1, we used mixed effects logistic regressions in R (R Core Team, 2020). All models were fit by maximum likelihood, based on Gaussian Hermite approximation, and run using the lme4 package (version 1.1-25; Bates et al., 2015). Models predicted the likelihood of a correct response for a given trial under varying conditions and included participant identity and item as random factors. Likelihood ratio tests determined the contribution of specific effects and interactions, by statistically comparing full models to nested versions with the respec- tive effect removed, using the chi-square distribution. Four models were created. (1) A ‘valence matching’ model restricted to the valence matching task used group (patients vs. controls), condition (associated target vs. no association vs. associated distractor), and their interaction as fixed effects. (2) A ‘semantic matching (binary)’ model restricted to the semantic matching task used group (patients vs. controls), binary association strength (strong association vs. weak association), and their interaction as fixed effects. As in Experiment 1, strong association trials were comprised of both congruent target and congruent distractor trials. (3) This was followed by a ‘semantic matching (parametric)’ model, which allowed us to consider the interaction between valence congruency (congruent target vs. congru- ent distractor) and parametric probe-target association strength (using word2vec scores), across groups (patients vs. controls).5 (4) Finally, a ‘task comparison’ model included group (patients vs. controls), task (valence vs. semantic), and difficulty (easy [‘valence – associated target’ and ‘semantic – congruent target’] vs. hard [‘valence – associated distractor’ and ‘semantic – congruent distractor’]) as fixed effects. Each possible interaction was included. When necessary, interactions were followed by post-hoc contrasts in emmeans (Lenth, 2020), which quantify differences based on odds ratios (OR), with Bonferroni correc- tion applied. Results Impairment of individual patients assessed with Singlims Impairment of individual patients assessed with Singlims Each patient's percentage accuracy for each condition, and average accuracy for patients and controls, can be seen in Figure 4. Conditions on which patients were impaired, determined in Singlims, are reflected by asterisks. In the valence matching task, patients performed near ceiling on the associated target condition, with none impaired. Two patients were impaired on the no association condition. Three were impaired on 5 As in Experiment 1, we used the same method to assess the effect of association strength on valence matching – restricted to the no association and associated distractor conditions. This analysis is reported in the Supporting Information section ‘Valence Congruency Mixed Effects Models – Experiment 2’. Experiment 2’. 12 12 12 SOUTER et al. F I G U R E 4   Percentage correct for each condition in the valence and semantic matching tasks for each patient and for the average of the patient and controls groups. *Reflects impairment relative to controls based on Singlims analysis. The dotted line reflects chance level performance (50%). Error bars reflect one standard error of the mean. Patients are ordered left to right in descending order of semantic control impairment, on the basis of their semantic control composite score. F I G U R E 4   Percentage correct for each condition in the valence and semantic matching tasks for each patient and for the average of the patient and controls groups. *Reflects impairment relative to controls based on Singlims analysis. The dotted line reflects chance level performance (50%). Error bars reflect one standard error of the mean. Patients are ordered left to right in descending order of semantic control impairment, on the basis of their semantic control composite score. the associated distractor condition, performing at or below chance-level. In the semantic matching task, patients generally performed near ceiling on the congruent target condition, with only one impaired. None were impaired on the congruent distractor condition. Only one was impaired on strong association trials (the confluence of congruent target and congruent distractor). Three patients were impaired on the weak association condition, with one performing close to chance. Experiment 2 mixed effects logistic regressions are in Table 4. Valence matching g The valence matching model revealed significant effects of group and condition, and a group by condi- tion interaction. The effect of group reflected higher accuracy in controls than patients. To parse the interaction, contrasts in emmeans compared performance on each condition between groups. While no difference was found for the associated target condition (OR = .48, p > 1), controls were more likely than patients to produce a correct response in the no association (OR = .13, p = .002) and associated distractor (OR = .08, p < .001) conditions. This suggests impaired valence matching in patients, most notable in the presence of related distractors, that is ameliorated by related targets. When running within-group contrasts (Table S8), both groups show reduced accuracy following associated distractors, relative to base- line. Neither sees a significant improvement from associated targets. While patients do not benefit from related targets in absolute terms, this is the only condition on which they do not present with impairment relative to controls. Semantic matching (binary) 17486653, 0, Downloaded from https://bpspsychub.onlinelibrary.wiley.com/doi/10.1111/jnp.12312 by Test, Wiley Online Library on [03/04/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Libra 13 VALENCE AND MEANING TA B L E 4   Output of Experiment 2 mixed effects logistic regressions. p p g g Model Variable Estimate Lower 95% CI Upper 95% CI Likelihood ratio test Valence matching Intercept 3.08 2.27 3.88 – Group 1.64 .80 2.49 χ(1) = 11.9, p = .001* Condition – – – χ(2) = 57.0, p < .001* Group by condition – – – χ(2) = 9.60, p = .008* Semantic matching (binary) Intercept 3.91 3.04 4.78 – Group 1.32 .41 2.24 χ(1) = 7.44, p = .006* Association strength −3.07 −3.84 −2.31 χ(1) = 69.5, p < .001* Group by association strength .90 .21 1.58 χ(1) = 6.53, p = .011* Semantic matching (parametric) Intercept .80 −.05 1.66 – Group 2.38 1.49 3.26 χ(1) = 19.2, p < .001* Valence congruency 4.15 1.07 7.23 χ(1) = 7.09, p = .008* Probe-Target association 10.12 6.70 13.54 χ(1) = 42.9, p < .001* Group by congruency −1.27 −4.05 1.50 χ(1) = .79, p = .374 Group by association −2.93 −6.36 .51 χ(1) = 2.79, p = .095 Valence congruency by association −13.22 −21.73 −4.71 χ(1) = 8.33, p = .004* Group by association by congruency 2.18 −5.50 9.86 χ(1) = .30, p = .584 Task comparison Intercept 2.81 1.91 3.72 – Group 1.80 .87 2.73 χ(1) = 12.4, p < .001* Task .80 −.14 1.74 χ(1) = 2.82, p = .093 Difficulty .98 −.22 2.18 χ(1) = 2.65, p = .104 Group by task −.20 −1.15 .75 χ(1) = .17, p = .682 Group by difficulty −.98 −2.10 .14 χ(1) = 3.00, p = .083 Task by difficulty −4.44 −6.03 −2.85 χ(1) = 32.1, p < .001* Group by task by difficulty 1.89 .44 3.34 χ(1) = 6.58, p = .010* Note: *Reflects significance at the 05 threshold Significant results are also presented in bold Models were run in R using lme4 package (version Note: *Reflects significance at the .05 threshold. Significant results are also presented in bold. Models were run in R using lme4 package (version 1.1-25; Bates et al., 2015). As these are logistic models, estimate coefficients reflect log transformation of odds ratios (Larsen et al., 2000). The valence matching condition effect and group by condition interaction do not include an estimate value, as these effects are not provided by the overall model. Semantic matching (binary) The first semantic matching model observed for binary effects of association strength. Again, controls were more likely to produce a correct response than patients. There was also a significant effect of associ- ation strength, reflecting higher accuracy on strong association than weak association trials. Finally, a significant group by association strength interaction reflects that patients were disproportionately impaired by weak associations (see Figure 4). 17486653, 0, Downloaded from https://bpspsychub.onlinelibrary.wiley.com/doi/10.1111/jnp.12312 by Test, Wiley Online Library on [03/04/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Libra The respective likelihood ratio test results were obtained by comparing the full model to nested versions in which all condition main effects or interactions were removed. Note: *Reflects significance at the .05 threshold. Significant results are also presented in bold. Models were run in R using lme4 package (version 1.1-25; Bates et al., 2015). As these are logistic models, estimate coefficients reflect log transformation of odds ratios (Larsen et al., 2000). The valence matching condition effect and group by condition interaction do not include an estimate value, as these effects are not provided by the overall model. The respective likelihood ratio test results were obtained by comparing the full model to nested versions in which all condition main effects or interactions were removed. Abbreviation: CI, confidence interval. Semantic matching (parametric) g (p ) The second semantic matching model looked for parametric effects of association strength, and interac- tions with group and valence congruency. Controls were more likely to produce a correct response than patients. We observed an effect of valence congruency, reflecting higher accuracy in the congruent target than congruent distractor condition (see Figure 4). We observed a significant effect of probe-target association strength, and an interaction between strength and valence congruency. This interaction was parsed using the emtrends function of the emmeans package (Lenth, 2020), and visualised using the ggpredict function of the ggeffects package (Lüdecke, 2018; Figure 5). Across groups, a positive effect of association strength on accuracy was found for the congruent target condition (association = 8.66, LCL = 5.79, UCL = 11.52). In the congruent distractor condition, no effect was observed (association = −3.48, LCL = −9.89, UCL = 2.94). 14 14 SOUTER et al. F I G U R E 5   Associations between probe-target association strength and the likelihood of a correct response for the Experiment 2 semantic matching task in (a) semantic aphasia patients and (b) control participants. Grey shaded areas reflect confidence intervals based on the standard errors. F I G U R E 5   Associations between probe-target association strength and the likelihood of a correct response for the Experiment 2 semantic matching task in (a) semantic aphasia patients and (b) control participants. Grey shaded areas reflect confidence intervals based on the standard errors. Task comparison A significant effect of group reflected that controls were more likely to provide correct responses than patients. 17486653, 0, Downloaded from https://bpspsychub.onlinelibrary.wiley.com/doi/10.1111/jnp.12312 by Test, Wiley Online Library on [03/04/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Libra There was a task by difficulty interaction, and a group by task by difficulty interaction. As reported in the Valence matching Section, patients were less likely to produce a correct response than controls for valence – associated distractor trials but not for valence – associated target trials. No group differ- ences were observed for semantic – congruent target (OR = .13, p = .469), or semantic – congruent distractor trials (OR = .45, p = .746). Effects of semantic relatedness on valence matching were larger than effects of valence congruency on semantic matching, particularly for patients (see Figure 4). This might reflect difficulty selecting goal-relevant features when semantic control demands are high. DISCUSSION This may account for why valence matching was impaired by related distractors; this requires inhibition of task-irrelevant features. Accordingly, SA patients were dispropor- tionately affected by this manipulation. SA patients were frequently impaired on valence matching even in the absence of distractors. Patients were not impaired relative to controls in the context of semantically related targets, suggesting facilitatory effects of global relatedness in accessing concept valence. The observed effects of cueing and miscueing in SA are consistent with prior evidence (Noonan et al., 2010). The current findings suggest an important role of valence in the lexicon, such that it may facilitate access to other featural and contextual aspects of concepts. Due to dominance of global relatedness over specific features (Thompson-Schill et al., 1997), effects of valence on semantic judgements were predicted to be modest. Nevertheless, we saw improved seman- tic matching in the context of valence-congruency. This is consistent with prior evidence of facilitatory effects of valence congruency on semantic matching (Marino Dávolos et al., 2020). Due to the design employed, we could not replicate previous analysis from Marino Dávolos et al. (2020), demonstrating that valence congruency is particularly helpful for retrieving weak associations. Valence congruency was only manipulated for strongly associated word pairs. We instead looked for effects of parametric probe-target association strength under conditions of valence-congruency and incongruency. Greater association strength facilitated semantic matching when the probe and target were congruent in valence, but not when they were incongruent. Benefits of stronger associations were reduced when participants needed to resolve valence incongruency between the probe and target, while disregarding valence-congruent distractors. Given the results of Marino Dávolos et al. (2020), we might expect this interaction to take a different form when weaker associations are presented, reflecting the changing contribution of decisional uncertainty and controlled retrieval demands as task parameters vary. Current findings suggest that access to valence is susceptible to control demands. Indeed, PCA in Experiment 1 suggests that accuracy on conditions that were more automatic or control-demanding loaded onto separate factors, regardless of task (semantic vs. valence). The involvement of control in valence processing is highlighted by evidence that divided attention can disrupt emotion-enhanced memory effects of valenced stimuli (Kang et al., 2014). Specific neural substrates may support controlled processing of valence. Zhuang et al. (2021) compared neural activation during tasks requiring domain-general response inhibition to those involving the manipulation of emotional context. DISCUSSION The hub-and-spoke model implicates valence as a feature of semantic concepts (Lambon Ralph et al., 2017), supported by research into abstract word processing (Ponari et al., 2018; Vigliocco et al., 2014). Accordingly, valence may influence judgements of semantic relatedness. Due to modulation of semantic control demands, global semantic similarity may influence ability to match words by valence. Such effects may be exaggerated in SA patients with impairments in constraining internal information. In young adults with a sensitive measure of RT (Experiment 1) and in five left-hemisphere stroke patients with SA and age-matched controls (Experiment 2), we found evidence that (i) accessing word valence is vulnerable to interference from overall meaning; (ii) valence congruency can facilitate access to word meaning, (iii) effects of semantic relatedness on valence matching are larger than effects of valence congruency on semantic matching, and (iv) effects of semantic distractors on valence matching are increased in SA. We further demonstrated that in the context of strong semantic associations, parametric increases in probe-target association strength facilitate responses only when words are congruent in valence. Finally, participants were more accurate and faster when retrieving strong than weak semantic associations – heightened in SA. Valence can be considered a semantic feature, as concepts are grounded in valence as they are for action and perception (Martin, 2016). A distinction can be made between ‘affective’ valence; experiencing something as negative – and ‘semantic’ valence; knowing something is negative (Itkes & Kron, 2019). One VALENCE AND MEANING 15 may understand that a flower is a positive entity without deriving joy. This distinction may be related to the separation between emotion-laden words that are imbued with affective connotations, and emotion-label words that convey affective states (Zhang et al., 2017). Only 9.5% of words across conditions were emotion-label, meaning terms were largely emotion-laden. While we had insufficient emotion-label stim- uli to explore differential effects of these categories on semantic matching, future researchers may wish to test this distinction. The intersection of valence and meaning is consistent with theory that perception of discrete emotions relies on semantic knowledge (Lindquist et al., 2015). Indeed, this ability is impaired following deficits in semantic storage (Lindquist et al., 2014) and control (Souter et al., 2021). Matching words by valence may require participants to focus on a specific feature while disregarding others that together determine global similarity. DISCUSSION Lateral frontal regions were engaged regardless, while the ventral striatum and medial orbitofrontal cortex were sensitive to emotional context. Similarly, SCN regions including bilateral IFG and left pMTG (Jackson, 2021) are reliably activated for tasks requiring reappraisal of valenced stimuli (Messina et al., 2015). Messina et al. (2015) argue for contri- butions of semantic processing and executive control to emotion reappraisal, due to the need to access alternative representations of affective stimuli. SCN has been argued to allow for the integration of long- term abstract memory representations with goal states (Wang et al., 2020). This network, damaged in SA (Souter, Wang, et al., 2022), may support the control of both meaning and emotion. Limitations Due to social distancing restrictions during the COVID-19 pandemic, Experiment 2 was conducted remotely. The demands of this method (e.g., self-directed computer use) led to the exclusion of more 16 16 SOUTER et al. impaired patients from our database, reducing our sample size. For the same reason, it was not possible to obtain neuroanatomical scans for all patients, preventing us from relating behavioural impairment with lesion profile. We saw evidence of individual-level task impairments, determined by Singlims. These impairments were not consistent across all patients. Further work with larger groups may be helpful in confirming our observations. We saw group-level differences in mixed effects models while controlling for random variation attributable to participant identity, suggesting meaningful group differences. Despite this, this small sample size limits out ability to predict whether effects would generalise to other patients with this symptom profile. Second, it should be noted that judgements of valence are subjective. It may be that ‘Gallery’, for instance, was positive for some participants but negative for others. Despite this, participants without semantic control impairment performed at ceiling even in the no association condition (Experiment 1 = 95.7%, Experiment 2 controls = 97.8%), suggesting consensus on categorical valence. Furthermore, we used valence congruency as a binary predictor (positive/negative). One could instead observe parametric effects using participant ratings, with very positive words being more congruent with other very positive words than with mildly posi- tive words. Doing so may provide a more sensitive measure. While a binary predictor was found to be sufficient in revealing behavioural effects, future researchers may wish to employ a continuous measure. Finally, it has been argued that valence is more important in the representation of abstract concepts which lack physical properties (Kousta et al., 2011). Evidence suggests interactions between valence and word concreteness in the recruitment of semantic control regions (Pauligk et al., 2019). In the current investigation, we did not manipulate concreteness; future research may benefit from considering this factor. CONCLUSION This study suggests that access to valence information during an explicit matching task is not auto- matic; task-irrelevant semantic information can impact retrieval. Such effects are particularly prominent in patients with impaired semantic control, likely due to difficulty in constraining internal information. Simi- larly, valence congruency facilitates judgements of global semantic relatedness, suggesting that valence constitutes an important feature of heteromodal concepts. These results provide novel insights into the relationship between semantic retrieval and valence processing. AUTHOR CONTRIBUTIONS Nicholas E. Souter: Conceptualization; data curation; formal analysis; investigation; methodology; project administration; supervision; visualization; writing – original draft; writing – review and editing. Ariyana Reddy: Investigation; methodology; writing – review and editing. Jake Walker: Investigation; methodology; writing – review and editing. Julián Marino Dávolos: Conceptualization; writing – review and editing. Elizabeth Jefferies: Conceptualization; funding acquisition; methodology; supervision; writ- ing – review and editing. ACKNOWLEDGEMENTS Thank you to Zhiyao Gao for his assistance in obtaining Word2Vec scores. FUNDING INFORMATION The study was funded by an ERC Consolidator grant to EJ (FLEXSEM – 771863). FUNDING INFORMATION CONFLICT OF INTEREST STATEMENT The authors have no competing interests to disclose. 17 VALENCE AND MEANING ORCID Nicholas E. Souter https://orcid.org/0000-0002-0999-1811 Elizabeth Jefferies https://orcid.org/0000-0002-3826-4330 OPEN RESEARCH BADGES This article has earned Open Data and Open Materials badges. Data and materials are available at https:// osf.io/fgjcs/. REFERENCES https://doi.org/10.1093/brain/awl153 , , p , ( ) p p comparison. Brain, 129, 2132–2147. https://doi.org/10.1093/brain/awl153 Jefferies, E., Thompson, H., Cornelissen, P., & Smallwood, J. (2019). The neurocognitive basis of knowledge about object identity and events: Dissociations reflect opposing effects of semantic coherence and control. Philosophical Transactions of the Royal Society B, 375, 20190300. https://doi.org/10.1098/rstb.2019.0300 375, 20190300. https://doi.org/10.1098/rstb.2019.0300 Kang, C., Wang, Z., Surina, A., & Lü, W. (2014). 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M., & Becker, B. (2021). Segregating domain-general from emotional context-specific inhibitory control systems – Ventral striatum and orbitofrontal cortex serve as emotion-cognition integration hubs. NeuroImage, 238, 118269. https://doi.org/10.1016/j. neuroimage.2021.118269 Zhuang, Q., Xu, L., Zhou, F., Yao, S., Zheng, X., Zhou, X., Li, J., Xu, X., Fu, M., Li, K., Vatansever, D., Kendrick, K. M., & Becker, B. (2021). 7486653, 0, Downloaded from https://bpspsychub.onlinelibrary.wiley.com/doi/10.1111/jnp.12312 by Test, Wiley Online Library on [03/04/2023]. See the Terms and Con Segregating domain-general from emotional context-specific inhibitory control systems – Ventral striatum and orbitofrontal cortex serve as emotion-cognition integration hubs. NeuroImage, 238, 118269. https://doi.org/10.1016/j. neuroimage.2021.118269 Zoom Video Communications Inc. (2016). Security guide. Zoom Video Communications Inc. https://d24cgw3uvb9a9h.cloudfront.net/ static/81625/doc/Zoom-Security-White-Paper.pdf Zoom Video Communications Inc. (2016). Security guide. Zoom Video Communications Inc. https://d24cgw3uvb9a9h.cloudfront.net/ static/81625/doc/Zoom-Security-White-Paper.pdf 17486653, 0, Downloaded from https://bpspsychub.onlinelibrary.wiley.com/doi/10.1111/jnp.12312 by Test, Wiley Online Library on [03/04/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA artic 19 VALENCE AND MEANING SUPPORTING INFORMATION Additional supporting information can be found online in the Supporting Information section at the end of this article. How to cite this article: Souter, N. E., Reddy, A., Walker, J., Marino Dávolos, J., & Jefferies, E. (2023). How do valence and meaning interact? The contribution of semantic control. Journal of Neuropsychology, 00, 1–19. https://doi.org/10.1111/jnp.12310
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Genetic aspects of autism spectrum disorders: insights from animal models
Frontiers in cellular neuroscience
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INTRODUCTION manifestations of its core symptoms, gradual changes over time, and differing degrees of response to interventions (Abrahams and Geschwind, 2008; Levitt and Campbell, 2009). 10–25% of ASD cases seem to have an underlying genetic disorder such as fragile X syndrome, tuberous sclerosis (TSC),and Rett syndrome (Betancur et al., 2009). Autism spectrum disorders (ASDs) are a complex set of het- erogeneous neurodevelopmental disorders categorized by a triad of key behavioral anomalies. Characteristic behavioral abnor- malities consist of restricted interests accompanied by repetitive behavior, deficits in language and communication, and the inabil- ity to engage in reciprocal social interactions (Abrahams and Geschwind,2008; Betancur et al.,2009; Levitt and Campbell,2009; Peca et al., 2011b; Zoghbi and Bear, 2012). Autism is not a singu- lar disease entity. The disorder encompasses a spectrum of wide ranging phenotypic manifestations which span from debilitating impairments to mild behavioral and personality traits. Therefore, autism is rightfully referred to as “autism spectrum disorders” (Persico and Bourgeron, 2006). Recent studies have highlighted numerous potential risk fac- tors that may contribute to ASD. These risk factors range from genetic, to epigenetic, to environmental factors. Detection of copy number variations (CNV), point mutations, and identification of rare variants in synaptic cell adhesion proteins and pathways are some of the ways researchers are providing insight into the patho- physiology of ASD (Sebat et al., 2007; Malhotra and Sebat, 2012; Zoghbi and Bear,2012). It is worthwhile to note that the genes and the genetic pathways implicated in ASD, and the identification of any causal rare variants are accessible to modeling in experimen- tal systems. Research findings both from studying human genetics and animal models of ASD suggest that disruption of synapse formation and stabilization processes is a key underlying feature in ASD etiology. Dysfunctions in the assembly or structure of transmembrane and scaffolding proteins needed for building and maintaining synapses, and disruption in cellular signaling path- ways controlling synaptogenesis are major contributing factors in ASD. Autism spectrum disorder appears to be involved in early brain development. Obvious signs and symptoms show early onset within the first 3 years of life and persist into adult- hood. According to the recent reports from the Center for Disease Control, an estimated 1 in 88 children has been identi- fied with ASD. Interestingly, these disorders show a gender bias where males are affected almost five times more than females (http://www.cdc.gov/Features/CountingAutism/). Swati Banerjee*, Maeveen Riordan and Manzoor A. Bhat* Autism spectrum disorders (ASDs) are a complex neurodevelopmental disorder that display a triad of core behavioral deficits including restricted interests, often accompanied by repetitive behavior, deficits in language and communication, and an inability to engage in reciprocal social interactions. ASD is among the most heritable disorders but is not a simple disorder with a singular pathology and has a rather complex etiology. It is interesting to note that perturbations in synaptic growth, development, and stability underlie a variety of neuropsychiatric disorders, including ASD, schizophrenia, epilepsy, and intellectual disability. Biological characterization of an increasing repertoire of synaptic mutants in various model organisms indicates synaptic dysfunction as causal in the pathophysiology of ASD. Our understanding of the genes and genetic pathways that contribute toward the formation, stabilization, and maintenance of functional synapses coupled with an in-depth phenotypic analysis of the cellular and behavioral characteristics is therefore essential to unraveling the pathogenesis of these disorders. In this review, we discuss the genetic aspects of ASD emphasizing on the well conserved set of genes and genetic pathways implicated in this disorder, many of which contribute to synapse assembly and maintenance across species.We also review how fundamental research using animal models is providing key insights into the various facets of human ASD. Frontiers in Cellular Neuroscience REVIEW ARTICLE published: 24 February 2014 doi: 10.3389/fncel.2014.00058 REVIEW ARTICLE published: 24 February 2014 doi: 10.3389/fncel.2014.00058 Reviewed by: Reviewed by: Eunjoon Kim, Korea Advanced Institute of Science and Technology, South Korea John Jay Gargus, University of California Irvine, USA *Correspondence: Swati Banerjee and Manzoor A. Bhat, Department of Physiology, Center for Biomedical Neuroscience, School of Medicine, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA e-mail: banerjees@uthscsa.edu; bhatm@uthscsa.edu Keywords: autism spectrum disorder, synapse, animal models, genetics, epigenetics, environment, cell adhesion molecules, scaffolding proteins Keywords: autism spectrum disorder, synapse, animal models, genetics, epigenetics, environment, cell adhesion molecules, scaffolding proteins Genetic aspects of autism spectrum disorders: insights from animal models Swati Banerjee*, Maeveen Riordan and Manzoor A. Bhat* Department of Physiology, Center for Biomedical Neuroscience, School of Medicine, University of Texas Health Science Center, San Antonio, TX, USA Edited by: Edited by: Hansen Wang, University of Toronto, Canada Hansen Wang, University of Toronto, Canada www.frontiersin.org MANY FACETS OF AUTISM SPECTRUM DISORDERS GENETICS – COPY NUMBER VARIATION While epigenetic mechanisms are implicated in the develop- ment of many disorders, they are also an intrinsic phenomenon for normal brain development. Genomic imprinting is an example of an epigenetic mechanism that occurs normally throughout life. This is when one of the two parental alleles for an imprinted gene becomes inactive due to DNA methylation resulting in monoal- lelic gene expression. This phenomenon occurs quite frequently in humans but was also discovered in fungi, plants, and other animals (Martienssen and Colot, 2001; Jiang and Kohler, 2012). Using genome-wide scans, several areas on chromosomes known as, hot spots for genomic imprinting, were located on loci 7q and 15q (Reik and Walter, 2001; Luedi et al., 2007). Interestingly, these loci are highly affected in individuals with ASD (International Molecular Genetic Study of Autism Consortium,2001; Lamb et al., 2005). Several studies have linked duplication or deletion events on the active chromosome toASD (Cook et al.,1997; Schroer et al., 1998; Koochek et al., 2006). Individuals with Angelman syndrome (Mabb et al., 2011; Huang et al., 2012) and Prader–Willi syndrome (Miyake et al., 2012) show a defect in the active allele that leads to loss of gene expression. Such correlations provide compelling evidence for the role of genetic and epigenetic mechanisms in the etiology of ASD. Copy number variation is among the most widespread of struc- tural variations in the human genome, and is increasingly being implicated as a major contributor to the pathophysiology of complex neurodevelopmental disorders (Sebat et al., 2007, 2009; Christian et al., 2008; Kumar et al., 2008; Marshall et al., 2008; Weiss et al.,2008; Bucan et al.,2009; Glessner et al.,2009; Merikan- gas et al., 2009; Luo et al., 2012; Malhotra and Sebat, 2012). CNVs largely comprise of duplications and deletions and can be de novo or familial. De novo CNVs are more prevalent in causing sporadic genomic disorders (McCarroll et al., 2008). The duplication or deletion events disrupt gene structure, expres- sion, and function and are a common cause of developmental delay. Several studies suggest important role of CNVs in disease etiology, susceptibility, and inheritance (Beckmann et al., 2007; Estivill and Armengol, 2007). Large-scale genome-wide associ- ation studies are credited for detection of CNVs in rare cases of ASD (Ma et al., 2009a). Duplications and microdeletions in many loci are associated with ASD. EPIGENETICS Epigenetic mechanisms underlie several human neurodevelop- mental disorders. Genomic imprinting, epimutations, DNA methylation, and histone modifications are all examples of epige- netic mechanisms linked to the development of certain disorders. These mechanisms involve modifications of nucleotides or chro- mosomes without altering the genetic sequence (Zoghbi, 2003; Egger et al., 2004). Thus causing modifications in gene expression that may increase the likelihood of developing a particular disease. Epigenetic mechanisms are believed to function at the interface between genetic and environmental factors (Jiang et al., 2004; Qiu, 2006). Studies linking these two factors are gaining importance for understanding the etiologies of complex disorders and could play a role in the development of ASD. HISTORICAL OVERVIEW OF AUTISM SPECTRUM DISORDERS HISTORICAL OVERVIEW OF AUTISM SPECTRUM DISORDERS Leo Kanner, a psychiatrist, initially described autism well over half a century ago (Kanner, 1943, 1968, 1971). Studies on the relationship between autism and abnormal electroencephalogram were among the first to suggest autism as a disorder of brain function (Creak and Pampiglione, 1969). Despite these ground- breaking observations on autism, early identification of autism was marred by lack of adequate diagnostic criteria. It was not until the introduction of the concept of “autism triad” that high- lighted the now well-established characteristics of impairment in social interaction, language and communication did Autism become a recognizable disorder. Since then the clinical concep- tualizations of ASD have consistently evolved together with a steady rise in the number of ASD cases. Our current under- standing of ASD is that of a complex neurological disorder that continues to challenge our ability to identify the underlying causal mechanisms. Frontiers in Cellular Neuroscience INTRODUCTION ASD is among the most heritable disorders evidenced by family and twin stud- ies with a concordance rate of 70–90% for monozygotic twins (Folstein and Rutter, 1977; Steffenburg et al., 1989; Bailey et al., 1995; Folstein and Rosen-Sheidley, 2001). Nevertheless, heri- tability in this case is more complex due to the differences in A large portion of this review will emphasize the well-conserved sets of genes and genetic pathways implicated in ASD, many of which contribute to synapse assembly and maintenance across February 2014 | Volume 8 | Article 58 | 1 www.frontiersin.org www.frontiersin.org www.frontiersin.org Genetics of autism spectrum disorders Banerjee et al. Recent findings further reiterate a correlation between synapse formation and autism (Glessner et al., 2009; Mitne-Neto et al., 2011). In addition to these genes, some of the other genes rec- ognized as risk factors in ASD include NEUREXIN 1 (NRXN1; Kim et al., 2008; Bucan et al., 2009), SHANK2 (Berkel et al., 2010), CNTN4 (Fernandez et al., 2004), CNTNAP2 (Bakkaloglu et al., 2008; Penagarikano et al., 2011), DPYD and DPP6 (Marshall et al., 2008); NLG1 (Glessner et al., 2009) and SYNGAP1, DLGAP2 (Pinto et al., 2010). A detailed list of genes, their potential func- tions and genetic pathways linked to ASD are summarized in Table 1. different species. Given the complexity and heterogeneity of this disorder,ithasprovedchallengingtounraveltheunderlyingcauses of ASD from human clinical population alone. Nevertheless, numerous animal models have been utilized that have enormously contributed toward understanding specific aspects that constitute the spectrum of these disorders. MANY FACETS OF AUTISM SPECTRUM DISORDERS GENETICS – COPY NUMBER VARIATION Several studies identified duplication CNVs within 15q13 (Christian et al., 2008; Miller et al., 2009) and microdeletions at many loci in 16p11.2 (Sebat et al., 2007; Marshall et al., 2008; Weiss et al., 2008; Levy et al., 2011; Sanders et al., 2011), Williams syndrome locus 7q11.23, DiGeorge syndrome locus 22q11.2, 1q21.1, and Prader–Willi and Angelman syndromes at 15q11-13 (Glessner et al., 2009; Sanders et al., 2011). Additionally, DNA methylation is an important basic step in epigenetic gene control. Methyl-CpG binding proteins bind to the methylated DNA regions to control gene expression. Mutations in methyl-CpG binding protein 2 (MeCP2) cause Rett syndrome which shows characteristic autistic-like behavior in addition to seizures, ataxia, and stereotypic hand movements (Amir et al., 1999). More recently, MeCP2 was shown to regulate several genes involved in synaptic plasticity, neuronal cell proliferation and neu- ronal transcription factors including: brain-derived neurotrophic Interestingly, genes associated with CNVs in ASD are involved in regulating synaptogenesis. Some of the genes include NEU- ROLIGIN 4 (NLGN4; Jamain et al.,2003; Laumonnier et al.,2004), SHANK3 (Durand et al., 2007; Moessner et al., 2007; Gauthier et al., 2009), TBX1, PCDH10, and NHE9 (Morrow et al., 2008). February 2014 | Volume 8 | Article 58 | 2 Frontiers in Cellular Neuroscience www.frontiersin.org Genetics of autism spectrum disorders Banerjee et al. Table 1 | Conserved genes implicated in ASD. Gene Protein description Nature of abnormality Reference NRXN1 Transmembrane Mutation, CNVs Feng et al. (2006) NRXN2 Transmembrane Mutation Arstikaitis et al. (2011) NRXN3 Transmembrane Mutation Vaags et al. (2012) NLGN1 Transmembrane Genetic association Glessner et al. (2009) NLGN3 Transmembrane Mutation Jamain et al. (2003) NLGN4 Transmembrane Mutation, CNVs Jamain et al. (2003) CNTN3 Ig-CAM Mutation, CNVs Morrow et al. (2008) CNTN 4 Ig-CAM Mutation Roohi et al. (2009) CNTNAP2 Transmembrane Mutation, genetic association Arking et al. (2008) NrCAM Ig-CAM Genetic association Marui et al. (2009) CDH9/10 Transmembrane Genetic association Bucan et al. (2009) CDH18 Transmembrane Chromosomal abnormality Marshall et al. (2008) PCDH9 Transmembrane Mutation Marshall et al. (2008) PCDH10 Transmembrane Mutation Morrow et al. (2008) PCDH19 Transmembrane Mutation Dibbens et al. (2008) SHANK1 Scaffolding Mutation Sato et al. (2012) SHANK2 Scaffolding Mutation Berkel et al. (2010) SHANK3 Scaffolding Mutation Durand et al. (2007) DLG4 (disk large homolog 4) Scaffolding SNPs Feyder et al. (2010) HOMER1 Scaffolding Mutation Kelleher et al. (2012) cAMP-GEF (guanine exchange factor) Cytoskeletal Mutation Bacchelli et al. MANY FACETS OF AUTISM SPECTRUM DISORDERS GENETICS – COPY NUMBER VARIATION (2003) RELN (Reelin) Secreted Genetic association Persico et al. (2001) EN2 (Engrailed 2) Transcription factor Genetic association Gharani et al. (2004) Table 1 | Conserved genes implicated in ASD. chemicals of concern to human health that can either directly or indirectly affect signaling pathways impaired in ASD. factor (BDNF), distal-less homeobox 5 (DlX5), and insulin-like growth factor binding protein 3 (IGF3; Chen et al., 2003; Mar- tinowich et al., 2003; reviewed in Miyake et al., 2012). Thus, epigenetic misregulation of synaptic genes could potentially con- tribute to ASD (Beaudet, 2007). Yet another set of studies suggest that extrinsic factors like the environment can alter epigenetic make up leading to defective neuronal functions (Jessberger et al., 2007; Ma et al., 2009b). Prenatal exposure to certain pesticides and insecticides are known to inhibit acetylcholine (ACh) and γ-aminobutyric acid (GABA; Shelton et al., 2012). Studies show these neurotransmitter systems are altered in a subset of autistic individuals. Similarly, environmentally induced alterations in calcium signaling path- ways caused by organic pollutants, impact a broad range of neurotransmitter systems like the cholinergic, GABAergic, and dopaminergic systems (Pessah et al., 2008; Corrales and Herbert, 2011). In addition to disruptions in important neuronal signaling pathways, pesticides can cause oxidative stress, neuroinflamma- tion, and mitochondrial dysfunction, all contributors to neuronal cell-death and dysfunction (Herbert, 2010; Shelton et al., 2012). Furthermore, cytokine-mediated influences and immune-related proteins are also listed as modulating factors for ASD (Ashwood et al., 2011; Onore et al., 2012). Families with ASD often show clustering of autoimmune disorders (Croen et al., 2005; Currenti, 2010). Several reports indicate the presence of serum antibody reactivity against human cortical and cerebellar regions of the brain in autistic patients (Silva et al.,2004). This process is thought to begin in utero and is associated with placental transfer of mater- nal autoantibodies to neuronal proteins potentially leading to neuronal dysfunction. Frontiers in Cellular Neuroscience RODENTS Mouse models recapitulating symptoms of ASD through selective manipulations of genes and neural circuitry is a much more amenable model system compared to NHP models. Currently, there is a sizeable number of autism mouse models available made possible due to generation of specific gene knockouts; mutations in these genes are thought to contribute to ASD together with the emergence of CNVs and high-end genome-wide sequenc- ing studies. Mouse models of human disorders have limitations in recapitulating the entire phenotypic spectrum (Arguello and Gogos, 2006). The validity of mouse models of human disorders are based on three criteria: (i) construct validity as provided by knock outs that carry a mutation in a gene that is affected in the human disorder (Peca et al., 2011a), (ii) face validity as reflected in animals that bear many of the core and ancillary physical or behavioral resemblances to the human disorder (Crawley, 2004), and (iii) predictive validity, which by far, is the most challenging to accomplish and indicates a similar response in the mouse model to an intervention that is known to be effective in human patients with that disorder. One of the animal models largely believed to help bridge the gap between humans and lower vertebrate systems is the non-human primate (NHP) model. NHP model is relevant for understanding ASD due to its high degree of correspondence to human behav- ior and their striking homology in the anatomy of neural circuits that mediate social behavior (for review, see Ongur and Price, 2000; Watson and Platt, 2012). Some of the behavioral corre- lates that NHPs have with human behavioral deficits seen in ASD include repetitive behaviors (Lutz et al.,2003; Alarcon et al., 2008), social communication (Ghazanfar and Santos, 2004), and their ability to follow other’s gazes, a tendency that is compromised in Autism. For example, ablation studies in NHPs especially of the superior temporal sulcus region reveal difficulties in respond- ing to social cues like eye gaze (Campbell et al., 1990). The lesion model involving the amygdala in NHPs is used to study alter- ations in socio-emotional behaviors (Amaral et al., 2003). Some papers speculate on the involvement of mirror neurons in the development of ASD (Oberman et al., 2005). In early childhood development, mirror neurons may play a key role in mimicking behaviors, actions, and language. ENVIRONMENT AND OTHER FACTORS Environmental contributions and other modulating factors are emerging as potential risk factors for ASD. Heavy metals, parental age, immunological proteins, environmental pesticides and insec- ticides, and food contaminants are thought to act as modulators of ASD (Durkin et al., 2008). These factors could contribute toward an increase in the prevalence of ASD but may not be sufficient to cause ASD. Nonetheless, a major challenge is to identify environ- mental factors relevant to ASD that could influence susceptibility, severity, and intervention outcomes. Heritable genetic vulnerabil- ities can magnify the adverse effects triggered by environmental factors. If both genes and environment converge, a resulting dysfunction of neurotransmitters and signaling pathways could take place at key developmental time points (Pessah et al., 2008). The toxicological literature point toward several environmental February 2014 | Volume 8 | Article 58 | 3 www.frontiersin.org www.frontiersin.org Genetics of autism spectrum disorders Banerjee et al. ANIMAL MODELS OF AUTISM SPECTRUM DISORDERS ASD. Autoantibodies present in children with ASD have prompted investigators to analyze the affects of maternal IgG antibodies on the fetal brain during gestation. Injections of IgG antibod- ies from human mothers who had multiple children with ASD to pregnant rhesus monkeys resulted in abnormal stereotyped behav- iors in offspring, and increased activity of offspring compared to controls (Martin et al., 2008). Although use of NHP model has the capability of contributing to some of the more behav- ioral aspects of ASD research, the absence of genetic knockouts in NHPs modeling ASD together with the careful considerations of ethical implications of NHP research tend to pose limitations that can be better addressed using rodents and invertebrate model systems. ASD. Autoantibodies present in children with ASD have prompted investigators to analyze the affects of maternal IgG antibodies on the fetal brain during gestation. Injections of IgG antibod- ies from human mothers who had multiple children with ASD to pregnant rhesus monkeys resulted in abnormal stereotyped behav- iors in offspring, and increased activity of offspring compared to controls (Martin et al., 2008). Although use of NHP model has the capability of contributing to some of the more behav- ioral aspects of ASD research, the absence of genetic knockouts in NHPs modeling ASD together with the careful considerations of ethical implications of NHP research tend to pose limitations that can be better addressed using rodents and invertebrate model systems. Autism spectrum disorder is a complex disorder with no singu- lar pathology and because of this a collective and collaborative approach is the key to understanding its etiology and design of rational interventions. Studies in animals are aimed at modeling the core phenotypes associated with ASD, including communi- cation and social impairments, restricted interests, and repetitive behaviors in an attempt to uncover the mechanisms that under- score the entire spectrum of the disorder. In this section, we will uncover the wide range of both invertebrate and vertebrate model systems utilized by researchers that have collectively made signif- icant contributions toward understanding the mechanisms that underlie ASD (summarized in Table 3). February 2014 | Volume 8 | Article 58 | 4 RODENTS Other examples of mouse models tostudycharacteristicsof ASD includePurkinje-specificknockout of TSC1 (Tsai et al., 2012), chromosome-engineered mouse model for human 15q11-13 (Nakatani et al., 2009), model for 16p11.2 lesion found in autism (Horev et al., 2011), 22q11.2 mice lacking PTEN (Zhou et al., 2009), CNTNAP2 (Penagarikano et al., 2011), SHANK2 (Won et al., 2012), and SCN1A (Han et al., 2012). The impressive array of mouse models displaying behaviors that are reflective of the human behavioral and cognitive ASD symptoms is highly informative. On the other hand, the behavioral phenotypes between mouse models with ablation of the same gene show vari- ations based on either their genetic backgrounds (Crabbe et al., 1999), or how individual laboratories conduct their behavioral assays further underscoring the impact of the genetic background or the local environment on the displayed phenotypes. In any case, a complete understanding of the similarities and differences in the behavioral phenotypes across the ASD mouse models will pro- vide key insights into the underlying neural circuitry behind these behaviors. Other emerging rodent models of ASD include rat and prairie vole (McGraw and Young, 2010). Rats that are injected with val- proic acid (VPA) serve as an environmentally triggered model of autism and this method has emerged as a new way to study ASD in rats (Rodier et al., 1997). VPA injected to gestational mothers before neural tube closure causes autistic-like phenotypes in off- spring such as a reduction in the number of cerebellar Purkinje cells and disruption in inhibitory circuits (Gogolla et al., 2009). Furthermore, these animals show similar behavioral phenotypes Table 4 | Receptors, transporters, and channel proteins in ASD. Gene Gene name Genetic abnormality Reference AGTR2 Angiotensin II receptor, type 2 Mutation Vervoort et al. (2002) ADRB2 Adrenergic β-2 receptor Genetic association Cheslack-Postava et al. (2007) AVPR1A Arginine vasopressin receptor 1A Mutation, genetic association Yirmiya et al. (2006) DRD3 Dopamine receptor D3 Genetic association de Krom et al. (2009) ESRRB Estrogen-related receptor β Genetic association Wang et al. (2009) GABRB3 GABA A receptor, β3 Genetic association Cook et al. (1998) GABR4/GABRB1 GABA A receptor, α4/β1 Genetic association Ma et al. (2005) GRIP1 Glutamate receptor interacting protein Mutation, genetic association Mejias et al. (2011) GRIK2 Glutamate receptor, kainate 2 Genetic association Jamain et al. (2002) GRIN2A Glutamate receptor, ionotropic, N-methyl D-aspartate 2A Mutation Barnby et al. (2005) GRIN2B Glutamate recepto14r, ionotropic, N-methyl D-aspartate 2B Mutation O’Roak et al. RODENTS A failure in the development or proper organization of mirror neurons might be linked to some of the behavioral phenotypes associated with ASD (Williams et al., 2001). NHP, like humans, possess mirror neurons and their use as a model system could provide some insight into the involvement of mirror neurons in ASD. NHP models are also used to investigate immunological factors in the etiology of Some of the mouse models representing syndromic forms of ASD include mice modeling Phelan–McDermid syndrome (SHANK3; Bangash et al., 2011; Peca et al., 2011a), Rett syndrome (MeCP2; Shahbazian et al., 2002; Moretti et al., 2006), fragile X syndrome (FMR1; Ronesi et al., 2012), Timothy syndrome (TS; CACNA1C; Bader et al., 2011), and others (see also Table 2). Neu- roligin 3 knock out mice serve as a model for non-syndromic Table 2 | Genetic syndromes with ASD-related phenotypes. Syndrome Chromosome Genes Reference Angelman 15q11 Ube3A Nurmi et al. (2001) Phelan–McDermid 22q13 Shank3 Durand et al. (2007) Rett Xq28 MeCP2 Amir et al. (1999) Tuberous sclerosis 9q34 TSC1 Baker et al. (1998), Smalley (1998) 16p13 TSC2 Timothy 16p13 CACNA1C Splawski et al. (2004) Fragile X Xq27 FMR1 Rogers et al. (2001) Cortical dysplasia-focal epilepsy syndrome 7q35 CNTNAP2 Arking et al. (2008) Smith–Lemli–Opitz 11q13 DHCR7 Tierney et al. (2001) Frontiers in Cellular Neuroscience www.frontiersin.org February 2014 | Volume 8 | Article 58 | 4 Table 2 | Genetic syndromes with ASD-related phenotypes. Genetics of autism spectrum disorders Banerjee et al. Table 3 | Phenotypic analyses and relevant animal models of ASD. Table 3 | Phenotypic analyses and relevant animal models of ASD. Table 3 | Phenotypic analyses and relevant animal models of ASD. Phenotype Animal models Non-human primate Mouse Rat Prairie vole Songbird Zebrafish Drosophila Aplysia C. elegans Genetic analyses + + + + + + Molecular analyses + + + + + + + Hyperactivity and repetitive behavior + + + Social communication + + + + + Cognition + + Impaired vocalization + + Animal models Non-human primate Mouse Rat Prairie vole Songbird Zebrafish Drosophila Aplysia C. elegans autism (Baudouin et al., 2012). Frontiers in Cellular Neuroscience RODENTS (2011) GRID1 Glutamate receptor, δ1 Mutation Glessner et al. (2009) GRID2 Glutamate receptor, δ2 Mutation Schaaf et al. (2011) GRM5 Glutamate receptor metabotropic 5 Mutation Iossifov et al. (2012) OXTR Oxytocin receptor Genetic association Wu et al. (2005) SL6A4 Solute carrier family 6 (serotonin transporter) Genetic association Sutcliffe et al. (2005) SLC25A13 Solute carrier family 25 (aspartate-glutamate carrier) Genetic association Ramoz et al. (2004) CACNA1C Calcium channel, α1B subunit Mutation Splawski et al. (2004) SCN1A/SCN2A Sodium channel, α subunit Mutation Weiss et al. (2003) KCNJ10 Potassium channel subfamily Genetic association Sicca et al. (2011) Frontiers in Cellular Neuroscience www.frontiersin.org February 2014 | Volume 8 | Article 58 | 5 Table 4 | Receptors, transporters, and channel proteins in ASD. February 2014 | Volume 8 | Article 58 | 5 Frontiers in Cellular Neuroscience www.frontiersin.org Genetics of autism spectrum disorders Banerjee et al. learning, a homologous underlying neural circuitry involving a loop between the cerebral cortex, basal ganglia, and thalamus, and a role for social influences in the learning of vocalizations (Panaitof, 2012). Studies from songbird indicate that CNTNAP2, which is implicated in human ASD and is enriched in human lan- guage related neural circuits (Alarcon et al., 2008), might play a role in vocal communication in songbirds as well (Panaitof et al., 2010). SimilartodevelopinghumanbrainwhereCNTNAP2shows a gradient distribution in frontal cortical areas, Cntnap2 expres- sion is either enhanced or reduced in key song control nuclei in songbird brain. In the absence of an animal model that can address language deficits, songbird model may prove useful for further exploration of the cellular and molecular mechanisms underlying the homologous neural circuitry that underscore lan- guage development in humans. Recently, using microarray and in situ hybridization analyses, large databases have been compiled that reveal expression patterns of certain genes in specific regions of the brain (Warren et al., 2010). Expansion on the molecular aspects of these observations has further increased the validity of behavioral songbird research. While linking behavior and genet- ics in songbirds is a tall order, it might still provide important clues about neuronal circuitry and language acquisition in the complex and heterogeneous nature of ASD and its behavioral manifestations. associated with autism including lower sensitivity to pain and higher sensitivity to non-painful stimuli, repetitive behaviors, hyperactivity, and decreased number of social behaviors. They also show delayed mental impairments and lower body weight (Schneider and Przewlocki, 2005; Favre et al., 2013). ZEBRAFISH Zebrafish are widely used as a model for studying vertebrate development and although not as popular as the mouse model for studying ASD, zebrafish are being used to dissect the genetic basis of autism due to a multitude of genetic techniques avail- able. These include lineage tracing using fluorescent tracers or labeling cells with lipophilic dyes, loss of function analyses using chemicals, transposable elements, and gain of function assays such as those involving microinjection of synthetic mRNA. In addition, morpholino technology is widely used as an efficient reverse genetic approach to understand gene function (Tropepe and Sive, 2003). Furthermore, fast oogenesis and embryogene- sis, and high fecundity allows for rapid experimental assays in this model. Transparency and external development of embryos allows for the study of growth and development of cells and tissues in live embryos (Tropepe and Sive, 2003). Additionally, zebrafish are an excellent model for use in carrying out genetic screens to identify new genes of interest and are useful in designing genetic screens to uncover specific enhancers or suppressors of particular phenotype (Dooley and Zon, 2000). These screens identified several candi- date genes, such as Reelin and MET that confers susceptibility to human ASD in zebrafish (Rice and Curran, 2001). The presence of structurally and functionally homologous regions in zebrafish brain, which are perturbed in human autistic patients, is another avenue that zebrafish researchers are taking advantage of to study brain development and neuronal connections. Although zebrafish is an excellent model to study some of the genetic aspects of ASD, the behavioral phenotypes associated with ASD are difficult to recapitulate (Tropepe and Sive, 2003). Thus, other model systems might be useful in studying some of the behavioral phenotypes associated with ASD. Frontiers in Cellular Neuroscience INVERTEBRATE MODELS Despite being millions of years apart on the evolutionary scale there is a surprising degree of genetic conservation between inver- tebrates and humans. Invertebrate models have made seminal contributions toward a basic understanding of human neurologi- cal disorders that are hard to ignore. One such classic invertebrate model for studying human neurodevelopmental disorders is the fruit fly, Drosophila. The fruit fly has proven to be an impor- tant model time and again to study various disorders where a single, causative genetic defect has been identified in Rett syn- drome (Cukier et al., 2008), fragile X syndrome (Morales et al., 2002), and Angelman syndrome (Wu et al., 2008; see also Table 2). Drosophila studies have advanced our fundamental understanding of some of these human disease gene functions, which show fea- tures of ASD. Recent studies in Drosophila have started to unravel some of the key genes, such as Neurexin 1 (Li et al., 2007; Zeng et al., 2007), Neuroligin 1 (Banovic et al., 2010), and Neuroli- gin 2 (Chen et al., 2012), which are the fly homologs of human NRXNs and NLGNs, respectively, that are implicated in ASD (De Jaco et al., 2005; Sudhof, 2008). With the unmatched power of Drosophila genetics and the potential of carrying out large scale screens using the sophisticated genetic tools available, Drosophila will undoubtedly continue to provide key mechanistic insights to dissect the genetic basis of ASD and likely facilitate the design of therapeutics. Apart from Drosophila, other invertebrate mod- els that are being used to study aspects of ASD are C. elegans (Calahorro and Ruiz-Rubio, 2011, 2012) and Aplysia (Choi et al., 2011; Ye and Carew, 2011). RODENTS Prairie voles also generated interest in the area of ASD because of their ability to form lasting social bonds and their nurturing behavior. Impaired social behaviors and deficits in various aspects of social cognition are some of the signature of ASD in humans. Thus, development of genetic, molecular, and genomic tools in prairie vole will likely be useful in basic and translational research that may be relevant to ASD (McGraw and Young, 2010). www.frontiersin.org SONGBIRDS This study showed that long-term facilita- tion and associated pre-synaptic growth were compromised when neurexin or neuroligin was depleted from pre- and post-synaptic machineries. In addition, an introduction of R451C mutation of NLGN3 associated with human ASD blocked both intermediate- and long-term facilitation in Aplysia (Choi et al., 2011). Another genetically tractable animal model is the nematode, C. elegans, which are utilized to understand the underlying mecha- nisms and abnormalities in neuronal synaptic communications in complex human neurological disorders like Alzheimer’s and ASD (Calahorro and Ruiz-Rubio, 2012). C. elegans have orthologs for ASD-related genes such as NLGNs, NRXNs, and SHANK. Recent reports highlight behavioral phenotypes in C. elegans reminiscent of ASD following removal of neuroligin homolog, nlg-1 (Hunter et al., 2010; Calahorro and Ruiz-Rubio, 2012) and subsequent functional phenotypic rescue by human NLGN1 (Calahorro and Ruiz-Rubio, 2012). Additionally, trans-synaptic NRX-1 and NLG- 1 in C. elegans are found to mediate retrograde synaptic inhibition of neurotransmitter release at the neuromuscular junction (Hu et al., 2012) further underscoring the function of these molecules in synaptic modulation. Thus determining the biological underpinnings of ASD will require a concerted effort involving studies from human clinical populations and several different animal models. This effort will provide complementary and critical insights toward understand- ing the complex and unknown ASD etiology. Since testing the causality and exploring the molecular and cellular mechanisms of ASD are either severely limited or off limits in human popu- lations, research using various animal models will provide clues to the range of functional deficits that cause the disorder and may even hint at the underlying neural circuits that drive the behavioral deficits. Recent studies on null mutants of Drosophila neuroligin 2 (dnlg2), which is the fly homolog of human NlgN3 showed reduced synaptic bouton numbers and synaptic transmission (Chen et al., 2012). Interestingly, dnlg2 is required both pre- and post-synaptically for proper synapse structure and func- tion. Another study in C. elegans reported presence of Neuroligin at both pre- and post-synaptic regions (Feinberg et al., 2008). These studies highlight the exceptions to the traditional role and localization of NLGNs at the post-synaptic terminals as seen at most vertebrate synapses. It also raises interesting questions about how synaptic organization might be fine tuned, and how signaling pathways might regulate the expression of pre- and post-synaptic proteins during synaptic development and function. Neurexins Neurexins (NRXNs) are predominantly presynaptic CAMs (Ichtchenko et al., 1995), although they were also reported to be expressed post-synaptically (Taniguchi et al., 2007). There are three Nrxn genes, Nrxn1, Nrxn2, and Nrxn3, each of which encode α- and β-isoforms. The α-Neurexin extracellular domain con- sists of six LNS domains interspersed by three EGF-like repeats and interacts with various proteins in the synaptic cleft. Mouse knock out mutants of individual Nrxns do not show gross abnor- malities in synaptic ultrastructure or in synapse number while triple α-Nrxn knock out mice die prenatally due to respiratory complications. These mice show impaired synaptic transmission, but not synapse formation suggesting that like their Nlgn lig- ands, α-NRXNs are required for proper synaptic maintenance and function, and not initial synapse formation (Missler et al., 2003). SONGBIRDS Songbirds can be used both as a molecular and a behavioral model for understanding the etiology of ASD. Songbirds are socially sophisticated and display characteristically human traits like monogamy and cultural inheritance while demonstrating the ability to learn vocalizations (Clayton et al., 2009). Vocal learning is an important element of language. Impairments in vocal and language learning are some of the core deficits in autism. Thus, understanding vocal learning through songbirds has emerged as an important model to study some aspects of ASD. A recent study in Aplysia showed that trans-synaptic Neurexin– Neuroligin interactions govern synaptic remodeling and regulates signaling required for the storage of long-term memory, includ- ing emotional memory, an ability that is affected in ASD patients Speech in humans and bird songs display striking parallels in that both seem to have a critical developmental time window for February 2014 | Volume 8 | Article 58 | 6 www.frontiersin.org www.frontiersin.org www.frontiersin.org Genetics of autism spectrum disorders Banerjee et al. of NlgN3 and NlgN4X showed retention of the mutant pro- teins in endoplasmic reticulum resulting in reduced cell surface binding to Neurexin (NRXN; Chih et al., 2004; Comoletti et al., 2004; Boucard et al., 2005). Some of the mutations of NLGNs seen in ASD patients were generated in mice and other model systems. For example, NlgN3 R451C knock-in mice showed challenged social interactions, enhanced inhibitory synaptic trans- mission, and altered spatial learning abilities (Jamain et al., 2003; Tabuchi et al., 2007; Chadman et al., 2008). This arginine to cys- teine point mutation at the analogous position as the human NLGN3 R451C was recently made in Aplysia revealing abnor- mal synaptic facilitation (Choi et al., 2011). NLGN3 knock out mice showed reduced ultrasound vocalization together and a lack of social novelty preference (Radyushkin et al., 2009). NlgN4 knock out mice displayed reduced reciprocal social interac- tions and vocalizations consistent with observations in human ASD patients (Jamain et al., 2008). Studies on NlgN1 knock out mice showed impaired NMDA receptor signaling, while NlgN2 knock out mutants revealed reduced inhibitory synap- tic transmission (Chubykin et al., 2007). Studies in vertebrate and invertebrates alike have now established that NLGNs are essential for proper synapse maturation, maintenance, and func- tion as opposed to initial synapse formation (Chih et al., 2005; Varoqueaux et al., 2006; Sudhof, 2008; Banovic et al., 2010; Chen et al., 2012). (Choi et al., 2011). SONGBIRDS Thus, studies on NLGNs using various model systems will pro- vide key insights into how these synaptic CAMs are involved in human ASD. GENES IMPLICATED IN ASD With the growing repertoire of synaptic genes implicated in ASD, it is becoming increasingly clear that synaptic dysfunction at mul- tiple levels may underlie ASD. Synapses comprise of pre- and post-synaptic elements (Figure 1; see also review by Delorme et al., 2013) like synaptic cell adhesion molecules (CAMs), ion channels, neurotransmitter receptors, scaffolding and cytoskeletal proteins that work harmoniously to provide synaptic structural integrity and functionality (refer Tables 1 and 4). Perturbations in synaptic assembly or function are commonly reported in many neuropsychiatric disorders (Blanpied and Ehlers, 2004). Frontiers in Cellular Neuroscience CELL ADHESION MOLECULES Neuroligins The cellular machinery of synapses is comprised of transmembrane heterophilic (such as Neurexin and Neuroligin) and homophilic cell-adhesion molecules (such as Cadherins and NCAM), cytoplasmic scaffolding proteins (such as PSD-95, Cask, and Shank) and cytoskeletal proteins (such as Homer and Cortactin) that link transmembrane and membrane-associated protein complexes with the underlying actin cytoskeleton. One of the emerging models in ASD is based on synaptic dysfunction in a molecular pathway that is orchestrated by trans-synaptic Neurexin–Neuroligin-dependent proteins complexes. This molecular assembly aligns the pre- and post-synaptic neuropsychiatric disorders. A schematic illustration of an ensemble of pre- and post-synaptic proteins. The majority of these proteins are highly conserved across species, and thought to confer susceptibility to a host of neurodevelopmental and neuropsychiatric disorders including ASD. The cellular machinery of synapses is comprised of transmembrane heterophilic (such as Neurexin and Neuroligin) and homophilic cell-adhesion molecules (such as Cadherins and NCAM), cytoplasmic scaffolding proteins (such as PSD-95, Cask, and Shank) and cytoskeletal proteins (such as Homer and Cortactin) that link transmembrane and membrane-associated protein complexes with the underlying actin cytoskeleton. One of the emerging models in ASD is based on synaptic dysfunction in a molecular pathway that is orchestrated by trans-synaptic Neurexin–Neuroligin-dependent proteins complexes. This molecular assembly aligns the pre- and post-synaptic overlap between the two neurodevelopmental disorders (Betancur et al., 2009; Rujescu et al., 2009). Pre-synaptic calcium channel function is also disrupted in α-Nrxn knock out mice. Interestingly, compared to three NRXNs in mam- mals, Drosophila has a single neurexin-1 (dnrx) gene which like its vertebrate counterparts is pre-synaptic and is required for proper synaptic growth and neurotransmission (Li et al., 2007). Frontiers in Cellular Neuroscience CELL ADHESION MOLECULES Neuroligins Synaptic proteins implicated in neurodevelopmental and FIGURE 1 | Synaptic proteins implicated in neurodevelopmental and neuropsychiatric disorders A schematic illustration of an ensemble of pre apparatus facilitating functional activation and modulation of ion channels that are in proximity to the neurotransmitter containing synaptic vesicles on the pre-synaptic side. These protein complexes recruit other proteins both pre- and post-synaptically and help organize functional neural networks. The cytoskeletal scaffolding protein Cask is one such notable protein which binds to the C-terminus of Neurexin. At the post-synaptic density, Neuroligin binds to PSD-95, other molecules such as PSD-93, SAP97, and the SAPAP family of proteins as well as the Shank family of scaffolding proteins help orchestrate the post-synaptic area. Homer and Shank function is thought to stabilize the post-synaptic density and serve as a platform to incorporate the post-synaptic receptors (such as NMDAR, AMPAR, and mGluR) into the machinery. The synaptic dysfunction in ASD may occur at multiple levels whereby failure to organize proper protein–protein interactions at the synapse may compromise neuronal functions (refer text for more details). apparatus facilitating functional activation and modulation of ion channels that are in proximity to the neurotransmitter containing synaptic vesicles on the pre-synaptic side. These protein complexes recruit other proteins both pre- and post-synaptically and help organize functional neural networks. The cytoskeletal scaffolding protein Cask is one such notable protein which binds to the C-terminus of Neurexin. At the post-synaptic density, Neuroligin binds to PSD-95, other molecules such as PSD-93, SAP97, and the SAPAP family of proteins as well as the Shank family of scaffolding proteins help orchestrate the post-synaptic area. Homer and Shank function is thought to stabilize the post-synaptic density and serve as a platform to incorporate the post-synaptic receptors (such as NMDAR, AMPAR, and mGluR) into the machinery. The synaptic dysfunction in ASD may occur at multiple levels whereby failure to organize proper protein–protein interactions at the synapse may compromise neuronal functions (refer text for more details). FIGURE 1 | Synaptic proteins implicated in neurodevelopmental and neuropsychiatric disorders. A schematic illustration of an ensemble of pre- FIGURE 1 | Synaptic proteins implicated in neurodevelopmental and neuropsychiatric disorders. A schematic illustration of an ensemble of pre- and post-synaptic proteins. The majority of these proteins are highly conserved across species, and thought to confer susceptibility to a host of neurodevelopmental and neuropsychiatric disorders including ASD. CELL ADHESION MOLECULES Neuroligins Neuroligins (NLGNs) are post-synaptic CAMs localized at gluta- matergic or GABAergic synapses (Song et al., 1999; Varoqueaux et al., 2004). NLGNs have a large extracellular cholinesterase- like domain, and a small intracellular domain with PDZ-binding motif. There are five NLGNs, which include two X-linked genes (NlgN3 and NlgN4X) and one Y-linked (NlgN4Y). The identi- fication of ASD-linked mutations in NLGN3 and NLGN4X was an important finding that implicated these genes in the etiol- ogy of ASD (Jamain et al., 2003) and linked ASD to molecules with synaptic function. In vitro studies using mutant forms February 2014 | Volume 8 | Article 58 | 7 Frontiers in Cellular Neuroscience www.frontiersin.org www.frontiersin.org www.frontiersin.org Genetics of autism spectrum disorders Banerjee et al. FIGURE 1 | Synaptic proteins implicated in neurodevelopmental and neuropsychiatric disorders. A schematic illustration of an ensemble of pre- and post-synaptic proteins. The majority of these proteins are highly conserved across species, and thought to confer susceptibility to a host of neurodevelopmental and neuropsychiatric disorders including ASD. The cellular machinery of synapses is comprised of transmembrane heterophilic (such as Neurexin and Neuroligin) and homophilic cell-adhesion molecules (such as Cadherins and NCAM), cytoplasmic scaffolding proteins (such as PSD-95, Cask, and Shank) and cytoskeletal proteins (such as Homer and Cortactin) that link transmembrane and membrane-associated protein complexes with the underlying actin cytoskeleton. One of the emerging models in ASD is based on synaptic dysfunction in a molecular pathway that is orchestrated by trans-synaptic Neurexin–Neuroligin-dependent proteins complexes. This molecular assembly aligns the pre- and post-synaptic apparatus facilitating functional activation and modulation of ion channels that are in proximity to the neurotransmitter containing synaptic vesicles on the pre-synaptic side. These protein complexes recruit other proteins both pre- and post-synaptically and help organize functional neural networks. The cytoskeletal scaffolding protein Cask is one such notable protein which binds to the C-terminus of Neurexin. At the post-synaptic density, Neuroligin binds to PSD-95, other molecules such as PSD-93, SAP97, and the SAPAP family of proteins as well as the Shank family of scaffolding proteins help orchestrate the post-synaptic area. Homer and Shank function is thought to stabilize the post-synaptic density and serve as a platform to incorporate the post-synaptic receptors (such as NMDAR, AMPAR, and mGluR) into the machinery. The synaptic dysfunction in ASD may occur at multiple levels whereby failure to organize proper protein–protein interactions at the synapse may compromise neuronal functions (refer text for more details). NrCAM NrCAM is a CAM that has gene homology to NgCAM and is capable of homophilic cell adhesion as well as heterophilic inter- actions with other non-NrCAM molecules such as Contactin-1, Contactin-2/TAG1, and Neurofascin (Suter et al., 1995; Volkmer et al., 1996; Pavlou et al., 2002). More recently, association analy- sis has linked NrCAM to ASD (Sakurai et al., 2006). This study showed over transmission of particular haplotypes of NrCAM that modulate NrCAM expression in the brain, are associated with a specific subset of autism with a severe obsessive–compulsive behavior. Several single nucleotide polymorphisms (SNPs) in the NrCAM gene were also found to be associated with autism (Marui et al., 2009) further underscoring NrCAM as a strong candidate gene in ASD. Contactins The Contactins (CNTNs) are glycosyl phosphatidyl-inositol (GPI) anchored immunoglobulin (Ig) superfamily proteins with diverse functions ranging from myelination (Berglund et al., 1999; Bhat et al., 2001) to synapse formation and plasticity (Betancur et al., 2009). Disruption of CNTN4 is associated with ASD (Fernandez et al., 2004). Deletion and duplication in CNTN4 and small dele- tions near CNTN3 have been identified in various patients with ASD (Roohi et al., 2009). Since CNTN3 and CNTN4 expression NRXN1 has emerged as a strong candidate in ASD since the identification of overlapping de novo deletions in Nrxn1 in indi- viduals with ASD. Although rare, missense mutations (Feng et al., 2006; Kim et al., 2008) and deletions, and chromosomal aberra- tions in the NRXN1 were also found in ASD patients (Marshall et al., 2008; Zahir et al., 2008; Glessner et al., 2009). Interestingly, NRXN1 deletions also confer risk for schizophrenia pointing to an February 2014 | Volume 8 | Article 58 | 8 Frontiers in Cellular Neuroscience www.frontiersin.org www.frontiersin.org www.frontiersin.org Genetics of autism spectrum disorders Banerjee et al. and localization overlaps with synaptogenesis in the developing brain, it raises the possibility that mutations or genomic rear- rangements in these genes seen in ASD could be attributed to altered synapse formation and function. Reichardt, 2008). CDHs mostly undergo homophilic cell adhe- sion and are involved in intracellular signaling pathways associated with neuropsychiatric disorders. Many of the CDHs have spe- cific spatio-temporal expression patterns in the brain and loss of CDHs leads to altered functional connectivity and neuronal infor- mation processing in human brain (Redies et al., 2012). Recent studies have identified de novo translocation deleting CDH18 in ASD (Marshall et al., 2008). This study also reported CNVs associ- ated with PCDH9 gene inASD. Homozygous deletions in PCDH10 have also been shown in autistic children (Morrow et al., 2008). Other CDHs and PCDHs are disrupted in disorders related to mental retardation and intellectual disabilities (Weiner and Jontes, 2013). ION CHANNELS Ion channels are essential for regulating axonal conduction of elec- trical activity and maintaining the optimum level of excitability within the nervous system. Recent studies linked neuronal exci- tation alterations with ASD pointing to a potential role for ion channels in the etiology of ASD. Mutations in calcium, sodium, and potassium ion channels seem to enhance neuronal excitability. ASD-linked ion channel mutations involve the SCN1A (Nav1.1), CACNA1C (Cav1.2), KCNMA1 (BK Ca2+), and KCNJ10 (Kir4.1) channels (Ji et al., 2009; Liao and Soong, 2010; Li et al., 2011; Sicca et al., 2011). Contactin-associated protein like 2 p Contactin-associated protein like 2 (CNTNAP2) is a member of Neurexin superfamily and is a locus that is significantly asso- ciated with susceptibility for ASD (Alarcon et al., 2008; Arking et al., 2008). CNTNAP2 encodes CASPR2, a multidomain trans- membrane protein that is best known for clustering potassium channels at the juxtaparanodes in myelinated axons (Poliak et al., 2003). CNTNAP2 localizes at high levels in human fetal brain prior to myelination (Abrahams et al., 2007). It also shows a dis- tribution gradient as frontal cortical enrichment in the developing human brain, indicative of a role in patterning circuits that under- lie higher cognition and language. Thus, CNTNAP2 might play a role in the developing brain regions that are likely to be affected in ASD. A recessive frameshift mutation in CNTNAP2 was identi- fied in individuals with cortical dysplasia focal epilepsy syndrome, a congenital disorder, where majority of individuals displayed characteristic features of ASD (Strauss et al., 2006). In addi- tion, other studies that attribute CNTNAP2 to ASD include rare single base pair mutations and common variations in the CNT- NAP2 locus identified in patients with ASD (Alarcon et al., 2008; Arking et al., 2008; Bakkaloglu et al., 2008; Falivelli et al., 2012). Recent phenotypic characterization of Cntnap2 mutant mice revealed deficits in the three core ASD behavioral domains with hyperactivity and epileptic seizures (Penagarikano et al., 2011). These mutant mice also showed neuronal migration abnormal- ities, a significant reduction in the number of interneurons, and abnormal neuronal network activity before the onset of seizures. Most importantly, treatment with the FDA-approved drug risperidone led to amelioration of the repetitive behav- iors in the mutant mice further demonstrating a functional role for CNTNAP2 in neuronal development and opening of new avenues for therapeutic intervention in ASD (Penagarikano et al., 2011). Nav1.1 SCN1A encodes the alpha subunit of the sodium channel type 1 (Nav1.1) which belongs to the voltage-gated sodium chan- nel family necessary for axonal conduction and action potential propagation. These transmembrane proteins possess a large pore- forming alpha subunit and two auxiliary beta subunits. This organization is important for allowing sodium ions to move through the axonal membrane to initiate and propagate action potentials. Recently, SCN1A has emerged as the most impor- tant gene in epilepsy (Mulley et al., 2005). More that 70% of individuals with epileptic encephalopathy posses a mutation in the region encoding SCN1A causing severe myoclonic epilepsy in infancy, also known as Dravet syndrome (DS; Harkin et al., 2007). This disorder is often accompanied by certain behavioral abnormalities such as hyperactivity, sleep-disorder, anxiety, atten- tion deficit, impaired social interactions, restricted interests, and severe cognitive defects (Weiss et al., 2003; Ramoz et al., 2008; O’Roak et al., 2012). Such behaviors are very similar to those observed in patients with ASD and emerging evidence has linked SCNA1 and ASD (Li et al., 2011). Researchers found that mice with a loss of function mutation for SCN1A phenocopy DS and show autistic-like behaviors (Han et al., 2012). It was suggested that the autism-related traits in DS mice might be caused by a decrease in inhibitory neurotransmission in GABAergic interneu- rons due to SCN1A haploinsufficiency providing further evidence that impaired GABAergic signaling may underlie ASD (Chao et al., 2010). Frontiers in Cellular Neuroscience SHANK3 Shank3 is an important member of Shank family of proteins and interacts with NLGN (Gerrow et al., 2006) to play a key role in spine morphogenesis and synaptic plasticity (Sala et al., 2001). Recent studies on Shank3 using knockout mice suggest its involvement in the regulation of glutamatergic synapse size, shape, and structure (Jiang and Ehlers, 2013). In Shank3 knock- out mice, synaptic ultrastructure is compromised. Overall, shank3 loss leads to a reduction in spine volume, decreased PSD thick- ness, and loss of dendritic spines (Bozdagi et al., 2010; Peca et al., 2011a; Wang et al., 2011; Jiang and Ehlers, 2013). Furthermore, Shank3 knockout mice show abnormal social behaviors, com- munication patterns, repetitive behaviors, and impairments in learning and memory (Bozdagi et al., 2010; Peca et al., 2011a; Wang et al., 2011). Shank Shank protein family is one such synaptic scaffolding fam- ily of proteins that includes Shank1, Shank2, and Shank3. They have multiple protein–protein interaction domains and are also known as proline-rich synapse-associated proteins (ProSAPs). Shank proteins are enriched in PSDs and stabi- lize the PSD-95/SAPAP/Shank/Homer complex (Tu et al., 1999; Sala et al., 2001). Additionally, Shank interacts with NMDA receptors/PSD-95/GKAP complex and actin regulatory protein, Cortactin (Naisbitt et al., 1999; refer Figure 1). Strong genetic and molecular evidence has linked SHANK2 and SHANK3 to the development of ASD phenotypes. Cav1.2 Cadherins (CDH) and protocadherins (PCDH) include a large family of CAMs, a number of which are required for synap- tic formation and function (Weiner et al., 2005; Arikkath and The calcium channels, voltage-dependent, L type, alpha 1C sub- unit, also known as Cav1.2 encoded by the gene CACNA1C February 2014 | Volume 8 | Article 58 | 9 Frontiers in Cellular Neuroscience www.frontiersin.org Genetics of autism spectrum disorders Banerjee et al. has been implicated in ASD. Cav1.2 channels are important in the activation of transcription factors and play a key role in promoting neuronal survival and dendritic arborization (Krey and Dolmetsch, 2007). A mutation in the G406R region of the CACNA1C gene is known to cause TS, a rare genetic disorder that results in malformations of multi-organ systems, neurologi- cal and developmental defects, and autism (Liao and Soong,2010). The mutation in this region causes prolonged inward current and has dramatic effects on calcium channel inactivation (Splawski et al., 2004). The cellular and molecular consequences of this mutation are not yet known. Future studies should address the physiological relationship between calcium channel inactivation and ASD. at the synapse, and a decrease in dendritic spine morphology and synaptic transmission (Schmeisser et al., 2012). ProSAP/Shank2 mutants also display behavioral phenotypes that are consistent with those seen in ASD. Shank2 mutant mice are hyperactive, exhibit repetitive grooming, and have impairments in social and vocal behaviors (Schmeisser et al., 2012). Such phenotypic man- ifestations are linked to the reduction of NMDAR function that results from the absence of the Shank2 protein (Won et al., 2012). Human studies using microarray analyses have identified several variants in SHANK2 that are associated with ASD and mental retardation (Berkel et al., 2010). SynGAP1 SynGAP1 encodes the RAS GTPase-activating protein (GAPs) which is a critical component of the PSD. At the PSD, SynGAP1 regulates synapse development and maintenance of proper synap- tic function. It is known to interact with PSD-95 and colocalizes with excitatory NMDA receptor complexes (Chen et al., 1998). SynGAP is shown to play a critical role in the PSD during early Kir4.1 and BKCa Recent studies have linked potassium channel proteins Kir4.1 and BKCa to ASD. Mutational screens identified several missense mutations in the KCNJ10 region encoding the potassium ion chan- nel Kir4.1. This ATP sensitive inward rectifier type potassium channel is characterized by having a greater tendency to allow potassium ions to flow into the cell and is suggested to be respon- sible for the buffering action of glial cells in the brain. Individuals who possess a mutation in the region show symptoms consis- tent with the DSM-IV-TR criteria for ASD along with seizure and intellectual disability (Sicca et al., 2011). BKCa is a potas- sium channel known for its large conductance of potassium ions across cell membranes. The gene KCNMA1 encodes BKCa. which is thought to function as a synaptic regulator of neuronal excitabil- ity which seems to be disrupted in patients withASD (Laumonnier et al., 2006). Disruption of this gene caused a decrease in BKCa channel activity and haploinsufficiency in Autism patients further implicating excessive ion channel activity to ASD (Ji et al., 2009). g There is growing evidence of the involvement of Shank3 in ASD. Molecular characterization of individuals with 22q13.3 dele- tion syndrome that display autism behavior identified a deletion disrupting Shank3 among other genes (Wilson et al., 2003). Hap- loinsufficiency of Shank3 has been confirmed to account for 22q13 deletion phenotype of developmental and speech delays (Durand et al., 2007). Other studies that attributed a role of Shank3 in ASD include identification of de novo splice site mutation in ASD (Gauthier et al., 2009). More recently, Shank3 mutations iden- tified in patients with ASD show a modification in dendritic spine induction and morphology as well as actin accumulation in spines affecting growth cone motility (Durand et al., 2007). Furthermore, a microdeletion in Shank1 locus has been discov- ered using microarray analysis in individuals with ASD (Sato et al., 2012). Recent studies have further uncovered the func- tional role of Shank3 as Shank3 duplication in mice leads to hyperactivity and spontaneous seizures much like human sub- jects who have small duplications in the SHANK3 locus. These recent studies further underscore the function of Shank3 in neu- ronal function and possibly in the maintenance of a balance between the excitatory and inhibitory (E/I) synaptic mechanisms (Han et al., 2013). SCAFFOLDING PROTEINS Scaffolding proteins are essential molecules of the synaptic archi- tecture. They are enriched in post-synaptic densities (PSDs) and function in synapse biogenesis by trafficking and anchoring synaptic proteins and clustering of membrane-associated proteins. Most importantly, the scaffolding proteins serve to link post- synaptic receptors with their downstream signaling components and regulate cytoskeletal dynamics (Verpelli et al., 2012). mTOR PATHWAY Mammalian target of rapamycin is a serine/threonine protein kinase involved in the regulation of cell proliferation, cell growth, cell survival, protein synthesis, and transcription. The mTOR sig- naling cascade plays a very important role in synapse protein synthesis and several studies have linked this pathway to ASD (Hay and Sonenberg, 2004; Hoeffer and Klann, 2010). Many of the signaling components of the mTOR cascade are located at the synapses where they have been shown to regulate dendritic spine morphology and synaptogenesis (Kumar et al., 2005). Muta- tions in the proteins known to inhibit mTOR signaling including NF1, PTEN, TSC1, and TSC2 are all linked to neurological dis- ease and autistic-like behavioral phenotypes (Williams and Hersh, 1998; Butler et al., 2005; Won et al., 2013). Furthermore, muta- tions in the downstream targets of the mTOR signaling cascade have been identified in patients with ASD. The mTOR signal- ing cascade works by the phosphorylation of 4E-BP by mTOR. This causes 4E-BP to dissociate from the eIF4E initiation factor resulting in cap-dependent translation and elongation of mRNA (Laplante and Sabatini, 2009; Wang and Doering, 2013). Genomic sequence analyses of the eIF4E promoter region identified a SNP in autism patients that enhanced the promoter activity of eIF4E (Neves-Pereira et al., 2009). Additionally, 4E-BP knockout mice as well as mice with an overexpression of eIF4E show autistic like behaviors, enhanced translational of Neuroligins, and disrup- tions in the E/I balance (Gkogkas et al., 2013; Won et al., 2013). Analyses of monogenetic sources of ASD found that approxi- mately 8–10% of all ASD are involved in regulation of the mTOR pathway (Moldin et al., 2006; Kelleher and Bear, 2008; Hoeffer and Klann, 2010). Of those, 1–2% of ASD cases result due to a mutation in the gene encoding PTEN, an upstream member of the mTOR pathway (Hoeffer and Klann, 2010). The other upstream members, TSC1 and TSC2, form a heterodimer com- plex. Mutations in the genes encoding this complex cause TSC which is defined clinically by the appearance and growth of benign hamartomas throughout the body and brain (Smalley, 1998). TSC patients suffer from mental retardation and epilepsy. Recent studies show that 25–50% of patients with TSC show behaviors that are consistent with ASD behavioral phenotypes (Hoeffer and Klann, 2010). mTOR PATHWAY Mutations in the genes encoding this complex cause TSC which is defined clinically by the appearance and growth of benign hamartomas throughout the body and brain (Smalley, 1998). TSC patients suffer from mental retardation and epilepsy. Recent studies show that 25–50% of patients with TSC show behaviors that are consistent with ASD behavioral phenotypes (Hoeffer and Klann, 2010). Pharmacological manipulations to identify therapeutic targets that may be enhancers and suppres- sors of mTOR signaling cascades and mRNA translation are currently being explored to combat some of the phenotypic manifestations associated with ASD (Carson et al., 2012). Fur- ther studies into the downstream and upstream targets of the mTOR signaling cascade will provide additional insights into the functional relationship between the mTOR pathway and ASD. SIGNALING PATHWAYS Signaling pathways are a complex system of communication within cells that function to organize cellular activities. Signaling pathways and cascades have long been implicated in many disease models. Understanding signaling pathways may play a central role in developing pharmacological or other agents to better treat dis- ease. Currently, disruptions in signaling pathways are being linked CYTOSKELETAL PROTEINS A set of cytoskeletal proteins is also mutated in individuals with ASD. These include factors regulating dynamics of actin cytoskele- ton, such as GAPs and guanosine exchange factors (GEFs; Newey et al., 2005). Rare non-synonymous variants in cAMP-GEFII are among candidate genes for autism in chromosome 2q (Bacchelli et al., 2003). Mutations in tumor suppressor genes TSC1 and TSC2 are also linked to ASD as the mutant proteins are thought to perturb cytoskeletal dynamics and dendritic spine structure in mutant animals (Folstein and Rosen-Sheidley, 2001). More recently, microtubule associated protein, KATNAL2, has emerged as a credible risk factor for ASD (Neale et al., 2012). Apart from CAMs, scaffolding and cytoskeletal proteins, a host of other receptors, transporters and channel proteins are known to contribute toward the etiology of ASD (summarized in Table 4). Discovery of more and more genes and genetic path- ways are expanding the genetic landscape of ASD. It is interesting to note that these genes include chromatin modifiers, DNA bind- ing proteins, ion channels, and neurotransmitter receptors (State and Sestan, 2012). Recently, CNVs in new candidate genes within GABAergic signaling and neural development pathways associ- ated with ASD were identified using genome wide SNP array (Griswold et al., 2012). These genes include an allosteric binder of GABA receptor, DB1, the GABA receptor-associated protein, GABARAPL1 and a post-synaptic GABA transporter, SLC6A11. Other genes contributing to the genetics of ASD include glutamate receptors, such as GRID1, GRIK2, and GRIK4, synaptic regula- tors, such as SLC6A8 and SYN3, and transcription factors, such as Engrailed 2 (EN2; Griswold et al., 2012). SHANK2 Mutations in ProSAP1/Shank2 gene result in an upregulation of glutamate receptors in certain brain regions, an increase in Shank3 February 2014 | Volume 8 | Article 58 | 10 Frontiers in Cellular Neuroscience www.frontiersin.org www.frontiersin.org www.frontiersin.org Genetics of autism spectrum disorders Banerjee et al. postnatal development as SYNGAP1 knockout mice for die during early development (Kim et al.,2003). Furthermore, mice heterozy- gous for SynGAP1 show impairments in learning and memory consistent with its involvement in NMDA receptor complexes (Komiyama et al., 2002). In humans, sequencing of the SYNGAP1 locus revealed mutations linked to non-syndromic mental retar- dation evidencing the importance of SynGAP in synaptic plasticity and learning (Hamdan et al., 2009). SynGap1 was recently impli- cated ASD because many of its key interacting partners including PSD-95/DLG4, SAP-102/DLG3, PSD-93/DLG2, Neurexins, and Neuroligins have previously been associated with ASD (van de Lagemaat and Grant, 2010). Recent evidence suggests that Syn- GAP may play a crucial role in controlling the E/I balance in cortical neurons through the regulation on ERK signaling path- ways (Wang et al.,2013). This is interesting because the E/I balance seems to be altered in individuals with ASD (Eichler and Meier, 2008; Won et al., 2013). Further characterization of SynGAP as a regulator of synaptic function will provide additional insight into its involvement in ASD. to the development of ASD phenotypes. One such pathway con- nected to ASD is the mammalian target of rapamycin (mTOR) pathway. mTOR PATHWAY Pharmacological manipulations to identify therapeutic targets that may be enhancers and suppres- sors of mTOR signaling cascades and mRNA translation are currently being explored to combat some of the phenotypic manifestations associated with ASD (Carson et al., 2012). Fur- ther studies into the downstream and upstream targets of the mTOR signaling cascade will provide additional insights into the functional relationship between the mTOR pathway and ASD. Mammalian target of rapamycin is a serine/threonine protein kinase involved in the regulation of cell proliferation, cell growth, cell survival, protein synthesis, and transcription. The mTOR sig- naling cascade plays a very important role in synapse protein synthesis and several studies have linked this pathway to ASD (Hay and Sonenberg, 2004; Hoeffer and Klann, 2010). Many of the signaling components of the mTOR cascade are located at the synapses where they have been shown to regulate dendritic spine morphology and synaptogenesis (Kumar et al., 2005). Muta- tions in the proteins known to inhibit mTOR signaling including NF1, PTEN, TSC1, and TSC2 are all linked to neurological dis- ease and autistic-like behavioral phenotypes (Williams and Hersh, 1998; Butler et al., 2005; Won et al., 2013). Furthermore, muta- tions in the downstream targets of the mTOR signaling cascade have been identified in patients with ASD. The mTOR signal- ing cascade works by the phosphorylation of 4E-BP by mTOR. This causes 4E-BP to dissociate from the eIF4E initiation factor resulting in cap-dependent translation and elongation of mRNA (Laplante and Sabatini, 2009; Wang and Doering, 2013). Genomic sequence analyses of the eIF4E promoter region identified a SNP in autism patients that enhanced the promoter activity of eIF4E (Neves-Pereira et al., 2009). Additionally, 4E-BP knockout mice as well as mice with an overexpression of eIF4E show autistic like behaviors, enhanced translational of Neuroligins, and disrup- tions in the E/I balance (Gkogkas et al., 2013; Won et al., 2013). Analyses of monogenetic sources of ASD found that approxi- mately 8–10% of all ASD are involved in regulation of the mTOR pathway (Moldin et al., 2006; Kelleher and Bear, 2008; Hoeffer and Klann, 2010). Of those, 1–2% of ASD cases result due to a mutation in the gene encoding PTEN, an upstream member of the mTOR pathway (Hoeffer and Klann, 2010). The other upstream members, TSC1 and TSC2, form a heterodimer com- plex. Frontiers in Cellular Neuroscience CONCLUDING REMARKS Current efforts to identify the constellation of genes that confer the characteristic phenotypic manifestations within the autism spec- trum have improved our understanding of this complex disorder. February 2014 | Volume 8 | Article 58 | 11 Frontiers in Cellular Neuroscience www.frontiersin.org www.frontiersin.org www.frontiersin.org Genetics of autism spectrum disorders Banerjee et al. While modeling mutations in experimental animal model systems will highlight the underlying disruptions in conserved signaling pathways, the daunting task will still be to establish ASD-specific phenotypes at the molecular, cellular and neural circuit levels. The staggering number of genes already discovered in ASD holds the promise to translate the knowledge into designing new therapeutic interventions. The very interesting and equally challenging obser- vation from the recent genetic studies has been a high degree of overlap of risk factors for various neurodevelopmental disorders, such as ASD, epilepsy, and schizophrenia. This pattern of overlap provides the feasibility to address which genes and genetic path- ways intersect and specify the spatio-temporal sequence of events that occur within the developing human brain. The recent advent of comprehensive maps of spatio-temporal gene expression in the human brain (Kang et al., 2011) will greatly help toward provid- ing a powerful developmentally informed approach to studying disorders such as ASD. Arikkath, J., and Reichardt, L. F. (2008). Cadherins and catenins at synapses: roles in synaptogenesis and synaptic plasticity. Trends Neurosci. 31, 487–494. doi: 10.1016/j.tins.2008.07.001 Arking, D. E., Cutler, D. J., Brune, C. W., Teslovich, T. M., West, K., Ikeda, M., et al. (2008). A common genetic variant in the neurexin superfamily member CNTNAP2 increases familial risk of autism. Am. J. Hum. Genet. 82, 160–164. doi: 10.1016/j.ajhg.2007.09.015 CNTNAP2 increases familial risk of autism. Am. J. Hum. 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ACKNOWLEDGMENTS The work in our laboratory has been generously supported by the grants from the Simons Foundation Autism Research Initiative (SFARI-177037), the National Institutes of Health (GM063074, NS050356), and the University of Texas Health Science Center, San Antonio, TX, USA. We sincerely regret that the work of many authors related to the topic could not be cited here due to space limitations. Barnby, G., Abbott, A., Sykes, N., Morris, A., Weeks, D. E., Mott, R., et al. (2005). Candidate-gene screening and association analysis at the autism-susceptibility locus on chromosome 16p: evidence of association at GRIN2A and ABAT. Am. J. Hum. Genet. 76, 950–966. doi: 10.1086/430454 Baudouin, S. J., Gaudias, J., Gerharz, S., Hatstatt, L., Zhou, K., Punnakkal, P., et al. (2012). Shared synaptic pathophysiology in syndromic and nonsyndromic rodent models of autism. Science 338, 128–132. doi: 10.1126/science.1224159 Beaudet, A. L. (2007). Autism: highly heritable but not inherited. Nat. Med. 13, 534–536. doi: 10.1038/nm0507-534 CONCLUDING REMARKS doi: 10.1038/sj.mp.4001340 Bader, P. L., Faizi, M., Kim, L. H., Owen, S. F., Tadross, M. R., Alfa, R. W., et al. (2011). Mouse model of Timothy syndrome recapitulates triad of autistic traits. Proc. Natl. Acad. Sci. U.S.A. 108, 15432–15437. doi: 10.1073/pnas.1112667108 Although concerted efforts from studies of human clinical ASD populations and various ASD-related animal models have provided a better understanding of the genetic, molecular, and circuit level aberrations in ASD, several intriguing, yet signifi- cant questions still remain. For instance, how can the compound effects of genetics, epigenetics, and environment be consolidated in understanding ASD pathogenesis? What other events play a role in determining the appearance and trajectory of ASD symp- toms? How do the majority of the genetic susceptibility loci in ASD affect synapse assembly, maintenance and functional modu- lation? 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The use, dis- tribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. This article was submitted to the journal Frontiers in Cellular Neuroscience. Copyright © 2014 Banerjee, Riordan and Bhat. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, dis- tribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Zeng, X., Sun, M., Liu, L., Chen, F., Wei, L., and Xie, W. (2007). Neurexin-1 is required for synapse formation and larvae associative learning in Drosophila. FEBS Lett. 581, 2509–2516. doi: 10.1016/j.febslet.2007.04.068 February 2014 | Volume 8 | Article 58 | 18 Frontiers in Cellular Neuroscience Frontiers in Cellular Neuroscience www.frontiersin.org
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Threat Poker: Gamification of Secure Agile
IFIP advances in information and communication technology
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To cite this version: Audun Jøsang, Viktoria Stray, Hanne Rygge. Threat Poker: Gamification of Secure Agile. 13th IFIP World Conference on Information Security Education (WISE), Sep 2020, Maribor, Slovenia. pp.142-155, ￿10.1007/978-3-030-59291-2_10￿. ￿hal-03380689￿ Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-03380689 https://inria.hal.science/hal-03380689v1 Submitted on 15 Oct 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Threat Poker: Gamification of Secure Agile Audun Jøsang, Viktoria Stray, and Hanne Rygge University of Oslo, Norway, {josang,stray}@ifi.uio.no Norsk Tid AS, hanne.rygge@gmail.com Abstract. Agile software development is practiced in most software de- velopment projects around the world. To explicitly consider and include security requirements as part of agile software development is referred to as ‘secure agile’. To include security will naturally require additional time and effort, with potentially reduced agility as a consequence. To main- tain agility, it is important to have efficient methods to include security in the development process. In this study, we describe enhancements to Threat Poker, which is a game designed for the software development team to deal with security threats identified during the agile development project. Games can be valuable educational tools for actively engaging students and practitioners alike. An experiment with students indicates that playing Threat Poker increases security awareness and that it is a fun and simple way to discuss identified security threats and how to remove security vulnerabilities during the software development process. 1 Introduction Agile software development, currently the most common approach to software development, was not originally conceived with security in mind. As such, many agile projects do not have built-in steps for dealing with security issues. However, with the introduction of new laws and regulations for security, such as GDPR in the EU, design principles for ‘security by design’ and ‘privacy by design’ must be followed in order for the developed systems to be legally compliant [1]. ‘Security by design’ and ‘privacy by design’ are relatively general concepts for describing the integration of security and privacy considerations throughout the life-cycle of ICT systems. The term ‘by design’ simply means that threat, vulnerability and risk assessment related to information security and privacy must be included in the requirements specification, architecture design, coding, testing, deployment and management of all systems, products and services that involve ICT components. This paper focuses on using gamification to strengthen security and privacy by design during the steps of architecture design and coding. Gamification [2] is defined as “The application of typical elements of game playing (e.g., point scoring, competition, rules of play) to other areas of activity, typically as an online marketing technique to encourage engagement with a prod- uct or service”. The principle of gamification is to use various aspects of games to engage the ‘players’ in the task at hand. Students of software engineering are generally positive to using games to stimulate learning [3]. A recent systematic Jøsang, Stray and Rygge 2 literature review concluded that gamification in software engineering education is still in its infancy [4]. The authors encourage researchers to explore this topic further since gamification is shown to motivate students and stimulate learning. Several researchers have introduced games to increase awareness of secu- rity in software development projects. For example, Denning et al. proposed a tabletop card game called Control-Alt-Hack to generate awareness of security issues and increase the accuracy of people’s perceptions of computer security as a discipline [5]. They found that students enjoy playing the game and that it in- creases their security awareness. Another game called [d0x3d!] is an open-source content-license game (allowing free distribution and adaptation) that provides an artificial context for discussing real ideas in network security [6]. 2.1 Threat Modelling Threat modelling is to identify how systems can be attacked, which is an es- sential step in identifying vulnerabilities and risks. “Threat modelling works to identify, communicate, and understand threats and mitigations within the context of protecting something of value.” [13]. The main purpose of doing threat modelling is to identify relevant threat scenarios with the aim of understanding how they can be mitigated or blocked. Threat modelling is a component of risk management. The steps of risk man- agement are typically to define the target scope of the risk management, iden- tify the threat agents and possible threat/attack scenarios, understand existing countermeasures and their limitations, identify remaining exploitable vulnerabil- ities, estimate and prioritize identified risks, and last to propose and implement countermeasures to mitigate and remove vulnerabilities which in turn results in strengthened security, and hence in reduced risk. 1 Introduction The Security Requirements Educational Game (SREG) is a card-based game that is intended to be used for security-requirements education and that has shown to increase the understanding of security issues and vulnerabilities [7]. Williams et al. introduced Protection Poker as a game for estimating security risk during software development projects [8, 9]. Protection Poker is inspired by Planning Poker (see Sec.3.3 below) and uses special cards to express levels of risk resulting from a specific threat. The effect and usability of Protection Poker were further studied in [10, 11] where it was found that challenges for its adoption are the overhead in terms of time spent on playing Protection Poker, as well as the difficulty of transforming results from playing Protection Poker into more secure code. This calls for the need to make the game light-weight and to focus on solutions that have a positive impact on making the code more secure. Threat Poker introduced in 2018 by Rygge and Jøsang [12] is inspired by Protection Poker in the sense that it also focuses on estimating risks related to threats, similarly to Protection Poker. In addition, Threat Poker also focuses on estimating the effort of implementing security solutions during agile software development. An important goal of Threat Poker is to make the game easy to learn and play for students, and for that reason it only uses standard playing cards. Using a deck of familiar playing cards (instead of unfamiliar specialized cards) is a good way of giving an intuitive feeling of playing a game during learning, and during practice in a software development team. This study proposes ways to improve Threat Poker and investigates how it fits into an agile development process. For this purpose we investigated the following research questions: RQ1: How can threat modelling and risk assessment be included in agile software development? RQ2: How can Threat Poker be adapted and utilized in agile projects? The remainder of the paper is organized as follows: Risk management and threat modelling are briefly described in Section 2, then the principles of agile software development are described in Section 3. In Section 4, we describe our research method and results of adapting Threat Poker. In Section 5, we discuss our findings, conclusion and suggest future work. 3 Threat Poker: Gamification of Secure Agile 2.2 Threat Actors and Threat Scenarios It is important to distinguish between the concepts of ‘threat actor’ and ‘threat scenario’, where the former denotes active agents (e.g., persons or organisations), and the latter denotes what they do as a series of steps during an attack. When simply using the term ‘threat’ in this paper, it should be understood as a ‘threat scenario’. Many threat actors have the motivation and capacity to attack digital sys- tems and ICT infrastructures. It is crucial to understand and attempt to predict how threat actors will attack, which is done by threat modelling. Identifying threat scenarios is a prerequisite for knowing how to stop them. It is, of course, impossible to identify all relevant threat scenarios. However, the designers and developers of software products must use the security skills and experience they have to do the best they can. Additional training or the inclusion of more expe- rienced persons can be required if the software development team members feel that they have insufficient security skills and experience. The identification of threat scenarios requires the team members to think like an attacker, and try to come up with a method to attack the system. Whenever the team sees an opportunity to successfully attack the system they have iden- tified a threat scenario. The difference between ‘threat scenario’ and ‘attack’ is that an attack can be seen as the instantiation of an abstract threat scenario. There can be an infinity of different threat scenarios, but only those that are practical and realistic should be considered. When a threat scenario has been identified which realistically could be executed, it means that there are vulnera- bilities in the system or in the surrounding infrastructure that can be exploited by potential attackers to execute the threat scenario. Blocking or mitigating a threat scenario is equivalent to removing the corresponding security vulnerabili- ties, and this is precisely what has to be done by the design team during software development. 4 Jøsang, Stray and Rygge 3.1 Scrum Scrum is the most widely used agile development methodology [16] and is set up by fixed rules and roles for the team members [17]. The Scrum process consists of several different components such as the prod- uct backlog, sprint planning, daily Scrum meetings, review meetings and retro- spective meetings. Like most development methodologies, Scrum originally was not designed with a specific integrated way to deal with security and privacy concerns, and because of this it does not contain a guide on how to best imple- ment security in the method. There have been developed several methods to try and solve this problem like ‘Secure Scrum’, and ‘Security Backlog’ in Scrum. Au- thors have also developed games that can be useful for dealing with the security shortcomings of Scrum. Examples of games are Protection Poker [9], Microsoft’s Elevation of Privilege [18], as well as Threat Poker described here. 3 Agile Software Development Agile software development is an umbrella term which covers different frame- works and practices which can be applied by software developers to deal with a continuous changing environment, based on specific principles that were outlined in the Agile Manifesto [14]. The main principles consist of early and continuous delivery, welcoming changes, frequent delivery of working software, business peo- ple and developers working together, building projects around motivated people, and giving them the resources to succeed [15]. Agile focuses on face-to-face meetings, seeing working software as the mea- surement of progress and also on promoting sustainable development. Agile also gives attention to technical excellence and good design, simplicity, and assumes that self-organizing teams help develop good architectures, requirements and design. It also has a focus on reflection and on adjusting the process accordingly. 3.4 Kanban Kanban was developed for Toyota’s Lean Production System as a specific ap- proach to agile software development and is inspired by the requirements for just-in-time production systems. Kanban focuses on visualizing the ‘what’, the ‘when’ and the ‘how’ of the production process, or development process when talking about software development. There are four major principles that make up the Kanban framework. These consists of visualizing work to increase commu- nication and collaboration, avoiding an unmanageable amount of non-prioritized open tasks, measure and optimize the flow, gather information, and predict fu- ture problems, and lastly, to aim for continuous improvement by analysis [21]. Teams that employ Kanban often use practices such as daily meetings and ret- rospective meetings, but rarely Planning Poker as they do not have to estimate the backlog items the same way as is needed in Scrum (when planning the next iteration). 3.3 Planning Poker Planning Poker [20] is a card-based method for time estimation and planning, often used when following the Scrum process. Planning is started by reading a user story or description of the feature to be developed. Each of the team members will have a deck of special cards, Planning Poker cards, which are cards with increasing values, often the sequence: 0, 1, 2, 3, 5, 8, 13, 20, 40 and 100. The team members are then able to ask questions of the product owner and discuss the feature internally. When the team finishes the discussion, each member will use the cards for estimation, and if the team members are in agreement, the estimate becomes the estimate for the feature. If there are discrepancies in the estimation, a second round will commence, with new discussions and estimations, and this pattern will continue until a relative consensus is reached. 3.2 Secure Scrum The Secure Scrum Model [19] is an example of how to consider security in Scrum. Its four main components are: 1) the Identification component, 2) the Implemen- tation component, 3) the Verification component, and 4) the Definition-of-Done component. These components have the effect of influencing the different stages in the Scrum process. Security concerns are identified and marked in the Product Backlog as a result of the Identification component. The Implementation compo- nent is used in Sprint Planning and the Daily Scrum meetings and is focused on the awareness of the security concerns. The possibility of testing with focus on security is done in the Verification component. Finally, the Definition-of-Done component defines what Definition-of-Done for security related issues are. When dealing with the security backlog of Scrum in Secure Scrum, a new backlog can be added to deal with all the new security concerns the occurs in the software or due to new features added to the software solution [19]. The purpose of the security backlog is to implement different design principles based Threat Poker: Gamification of Secure Agile 5 on security and privacy to limit the numbers of vulnerabilities and to reduce the risk to the software that is being developed. In addition to the new backlog, another role can be added, called the Security Master, whose role is to manage the security backlog and also decide which security or privacy features require the most attention, and then add it to the sprint backlog which is then completed by the developers. 4.1 Equipment In Threat Poker, the team members use regular decks of playing cards. Most people are familiar with traditional playing cards, and sitting around a table with playing cards in the hand typically triggers the positive feeling of playing a game. People’s familiarity with playing cards also gives an intuitive interpretation of the cards’ face values with regard to threats and their solutions. Low cards intuitively represent low threats or effort estimations, and picture cards intuitively represent high threats and effort estimations for solving the threat scenarios. 4 Threat Poker Threat Poker is an efficient method for discussing threats in agile software projects and how to remove or mitigate vulnerabilities in the developed soft- ware [12]. Two essential elements of security by design are that the designers identify relevant threat scenarios, and then take the necessary steps to block or mitigate the said threat scenarios, i.e., to solve the threats. In this paper we focus on Threat Poker as a form of ‘gamification’ of the process for dealing with threats and vulnerabilities during agile software development. Jøsang, Stray and Rygge Jøsang, Stray and Rygge 6 4.4 Vulnerability Management The Open Web Application Security Project (OWASP) describes the ‘Top 10 Security Risks’ [23]. These risks involve well-known security threats and corre- sponding vulnerabilities that (unfortunately) are typically found in web appli- cation software. Similarly, the Common Weakness Enumeration (CWE1) is a community-developed list of common software security weaknesses (vulnerabili- ties). CWE serves as a common language, a measuring stick for software security tools, and as a baseline for weakness identification, mitigation, and prevention ef- forts. CWE and OWASP Top 10 are a good starting point for discussions around threats and vulnerabilities during software development. Security vulnerabilities in software can also be identified and assessed without reference to specific threat scenarios. For example, CVSS (Common Vulnerability Scoring System) provides a method for estimating the severity of specific security vulnerabilities. The severity level can be translated into a qualitative representation (such as low, medium, high, and critical) for prioritization in vulnerability management. 4.2 Adaptation to Agile Methods Depending on the development method, there are several stages in the used methodology where Threat Poker can be implemented. When using Scrum, sev- eral specified meetings are used for different purposes. There are daily Scrum meetings, planning meetings, and also retrospective meetings. When using Scrum, Threat Poker is most useful in the planning stages of the process where the sprint iteration is planned out, what feature to work on, where security and privacy risks can be assessed and evaluated and where the solution can be discussed, found and solved, if needed, during each sprint. Kanban, on the other hand, does not have specified meetings set up in the methodology, but a common practice is to use retrospective meetings to improve the team’s way of working, and participants often discuss past challenges and how to overcome them to work better together in the future. We suggest that a retrospective in agile software development could be a good meeting to play Threat Poker in since it is meant to be a positive team meeting which would fit with playing a game. We suggest that Threat Poker can be used as a means to identify and discuss threats to be worked on, and that the next step would be to make these threats visible to the whole project, for example by introducing a separate ‘Security board’ or ‘Threat board’ or as tasks added to the backlog or the ‘todo’-column. The idea is that when, during the development process, a security or privacy concern is discovered, it could be added directly to the security board and then, during either the planning meeting or the retrospective meeting, could be dis- cussed and made into a features and added to the ‘To-Do’ column of the Kanban board. Agile teams also rely on daily meetings to coordinate their work and make decisions [22], we therefore suggest that identification of threats could be part of the daily meetings. If a person has identified a threat, this can be discussed in the daily team meeting. It could be added to the board or backlog. Either as a security item to be investigated or the team could have a designated Security or threat board were they could collect possible threats and discuss these when playing Threat Poker. Discussing security threats would then be an iterative process, which fits well with agile methods. 4.2 Adaptation to Agile Methods Threat Poker: Gamification of Secure Agile 7 4.3 User Stories and Use Cases vs. Attacker Stories and Threat Scenarios A user story describes the user, what the user is supposed to do as well as what the user wants to achieve. A user story typically is in the format: “As a user, I want action so that effect”. This approach can also be applied to the attacker which can be expressed as an ‘attacker story’ e.g. expressed as “As an attacker, I want to execute the threat scenario so that I reach my attack goals”. A use case is a more detailed description of a function or application than a user story. A use case includes actions or event steps typically defining the interactions between roles (actors) and system components to accomplish a goal. The negative version of a use case is a ‘threat scenario’ which includes actions or event steps typically executed by the threat actor (attacker) who misuses system and network components and interactions between them to accomplish the attack goal. A threat scenario can thus be seen as a ‘misuse case’. The features to be discussed during Threat Poker can be presented either as an attacker story, as a threat scenario, or as a specific vulnerability without any specific attacker story or threat scenario associated with it. 1 https://cwe.mitre.org/ – the estimated effort level to solve the specific threat scenario or vulnerability. 4.5 Estimation of Risk and Effort Threat Poker focuses on evaluating the risks from relevant threat scenarios or other vulnerabilities that the team is able to identify, and on estimating the efforts needed to solve those threats and vulnerabilities. The face values of the playing cards are thus used to represent these two things: – the estimated risk level of a specific threat scenario or vulnerability, – the estimated effort level to solve the specific threat scenario or vulnerability. Jøsang, Stray and Rygge 8 Risk-Level Estimation. There is a distinction between security risk as seen from the point of view of the organisation, and the privacy risk as seen from the user (data subject in the terminology of GDPR). Threat Poker considers both types of risk together. Two main factors or principles are considered for estimating security/privacy risk levels. The first principle states that risk increases as a function of the prob- ability of attack, i.e. the ease with which the attacker can successfully execute the threat scenario. The second principle states that risk increases as a function of the negative security/privacy impact resulting from an attacker’s successful execution of the threat scenario. Security risk is the negative impact affecting the owner of the information assets that are being attacked. Privacy risk is the negative impact affecting a person whose personal information has been misused with reference to relevant data protection principles such as those of GDPR. This can be expressed as:  Security risk = (ease of executing threat) × (potential security impact) ( )    Security risk = (ease of executing threat) × (potential security impact) Privacy risk = (ease of executing threat) × (potential privacy impact) (1) (1)  Privacy risk = (ease of executing threat) × (potential privacy impact) ( Given the different victims of security risk and privacy risk respectively, there can be different threat scenarios causing these different risks. To consider these two types of risks separately might help the team analyze and prioritize the most important and pressing features to implement and how to best deal with the security and privacy concerns regarding the features. Note that the system owner can represent a privacy threat actor in case it collects and processes personal information in breach of data protection principles. 4.6 Card Values Figure 1 illustrates suits of cards used for playing Threat Poker. Each player (member of the Scrum or Kanban team) gets an entire suite from the deck, i.e. of Hearts, Spades, Diamonds or Clubs. With four suits one deck is sufficient for four players. For more than four players, two or more card decks are needed. The suit colour has no meaning other than separating the players from each other. Fig. 1. Card Suits Fig. 1. Card Suits Fig. 1. Card Suits Each player sorts out the cards, odd-numbered cards and even-numbered cards. The security and/or privacy risk is represented by odd values: 3, 5, 7, 9, Jack (11), King (13) and A (Ace). The solution-effort level is represented by even values: 2, 4, 6, 8, 10 and Queen (12). As a mnemonic, players of Threat Poker can see risk as an ‘oddity’ (represented by an odd-value card) which must be ‘evened out’, i.e. fixed (represented by an even-value card). ( ) Table 1 gives the interpretation of each card value in terms of security/privacy risk and effort level to solve the threat. Table 1. Risk and Effort Levels Odd Values: Risk Levels Even Values: Effort Levels 3 Insignificant risk, can be ignored unless the effort is minimal. 2 Minimal effort, can be solved quickly and easily. 5 Very low security and/or privacy risk, only solve if the effort is (very) low. 4 Very low effort, very easy and quick to solve. 7 Low security and/or privacy risk, should be solved if the effort is low/moderate. 6 Low effort, relatively easy to solve 9 Moderately high risk, should be solved even if (moderately) high effort needed 8 Moderately high effort, solve threat in this sprint if time, or else in other sprint J High risk, should be solved even when high or very high effort needed 10 High effort, the solution to this threat will be a major part of this/other sprint K Very high risk, with potentially very se- vere consequences. Must be solved. Q Very high effort, a separate sprint might be needed to focus on solving this threat A (Ace) – Extreme risk. Must be solved, or else reconsider the viability of user story. Table 1. Risk and Effort Levels Threat Poker described in [12] used a different interpretation of card values, and had two different types of rounds for playing a game. 4.5 Estimation of Risk and Effort Identifying and estimating these risks can be difficult, especially when the team consists of developers who may not have much experience with software development that focuses on security. There are several standards that can aid the team in finding risks or give an indication of where the most common security and privacy risks occur and also how to deal with them. In software and system development, the principle of ‘security by design’ means to adequately consider security during every step of the development process, and when working with this, it can be helpful to consider common de- sign principles. Several principles are defined by OWASP (Open Web Applica- tion Security Project) in its ASVS (Application Security Verification Standard) [24], and these can give an indication of most common vulnerabilities, and also guidelines on how to prevent these risks. Effort-Level Estimation. The estimation of the effort needed to solve the threat or vulnerability is similar to the estimation of effort levels in Planning Poker described in Section 3.3 above. The solution to the identified threat can be seen as a new item to be added to the backlog in Scrum, or to the board in Kanban. This item can be taken into the current development sprint, or be delayed to a subsequent sprint. In any case, the effort to implement the idem should be estimated by the members playing Threat Poker. 9 Threat Poker: Gamification of Secure Agile 4.6 Card Values First there was a risk estimation round which was followed by a solution-effort estimation round. We found that the relative complexity of this scheme in itself was a barrier to learning and for successfully practicing secure agile. 10 Jøsang, Stray and Rygge Jøsang, Stray and Rygge We simplified the original Threat Poker from [12] so that participants are playing integrated risk and solution rounds instead of separate rounds for the risk level and the effort level respectively. In the integrated version, odd-numbered cards now indicate the (security and privacy) risk level associated with the threat, while the even-numbered cards indicate the estimated effort required to solve the threat, i.e. to remove or mitigate the corresponding vulnerability. 4.7 Playing Threat Poker For each user story to be implemented the team must do threat modelling as described in Section 2.1. Threat modelling is a prerequisite for secure agile, and for applying Threat Poker. A game of Threat Poker starts by discussing a specific threat scenario or vulnerability which has been identified. This discussion can continue during the repeated rounds of the same game. For each threat or vulnerability that the team has identified and wants to consider, they play a game of Threat Poker as illustrated in Figure 2. Fig. 2. Integration of Threat Poker in secure agile Fig. 2. Integration of Threat Poker in secure agile The game starts by discussing the security and privacy risks involved with the threat, as well as the best solution to solve the threat, i.e. to remove the vulnerability. When the preliminary discussion is completed, each player must make two subjective estimations; one for security/privacy risk and one for the effort/time needed to solve it. Then they play out cards for the first round. / Each player puts down two cards facing down; an odd-value card for the risk level and an even-value card for the solution effort level. Low cards express low risk/effort, and high cards express high risk/effort. Then the cards are turned to show their values. In the case of significant deviations between card values, a discussion follows where each player explains the reasoning behind the risk and/or effort assessment. During the discussions, the players typically influence each other’s estimations. Then a new round is played. 11 Threat Poker: Gamification of Secure Agile Repeated rounds are played, with discussions in between, until an approxi- mate consensus is achieved, for both risk and effort levels. After a consensus is reached, the estimated risk and solution effort levels are used as parameters in the simple decision rule of Eq.(2) as an approximate guideline to decide whether the team should spend time and effort to solve the threat. IF Risk level −Effort level > 0 THEN Solve threat ELSE Ignore threat (2) (2) (2) Threat Poker is thus also an instrument to assist the team in making the decision whether a threat should be solved. The team must decide if the solution shall be implemented in the current iteration or be put into the backlog, or even if it is better to reconsider the viability of the whole user story. We had students at the University of Oslo trying out Threat Poker. The course was a 20 ECTS course involving a major project in software engineering. During a project period of 13 weeks, students were assigned to develop an app and using agile methods and practices. We had student teams play Threat Poker and report how they experienced it. The teams participating were comprised of between 2-6 students. The simplification of Threat Poker by integrating separate rounds of risk and effort estimation into one round also meant that the participants just played a single odd-valued card to represent a combined risk level for both security and privacy, and a single even-valued card to represent the effort for solving the risk. At this point in the game, the specific type of risk (security or privacy) is considered irrelevant. The important point is that threats and risks should be solved anyway, regardless of whether it is a security risk or a privacy risk. When revealing the card values, each developer explained whether the risk value represented by the odd-valued card was a security risk, a privacy risk, or both. 4.9 Variations of Threat Poker Another way of playing Threat Poker is as a board game. Some modifications are required to the method on how to play, but not on the fundamental principles of the game. The team will still use the same user stories and discussion techniques, but will forgo the deck of cards in favor of a created 2x2 grid that can easily be drawn up before the game is stared. The grid is drawn up and each sector will represent a different level of risk and effort. One sector will be high risk and difficult to solve/long time to solve, another will be high risk but easy to solve/short time to solve, the third will be low risk and difficult to solve/long time to solve and the last is low risk and easy to solve/short time to solve. Each player will use some form of tokens, to place on the board, and after each discussion or round, will move their tokens to represent their opinions on the feature that is discussed. When all players are in agreement with tokens in the same section of the grid, the team has reached consensus and can move on. 4.8 Traditional Playing Cards vs. Planning-Poker Cards While specially designed cards, such as the Planning Poker cards, and the cards used in Microsoft’s Elevation of Privilege can be difficult and time consuming to get hold of, normal playing cards are easy and cheap to buy anywhere. Getting regular playing cards does not require special ordering or manufacturing, and most offices will have at least one deck lying around, ready to be used. Another reason for using regular playing cards is their availability. While specially designed cards, such as the Planning Poker cards, and the cards used in Microsoft’s Elevation of Privilege can be difficult and time consuming to get hold of, normal playing cards are easy and cheap to buy anywhere. Getting regular playing cards does not require special ordering or manufacturing, and most offices will have at least one deck lying around, ready to be used. 4.8 Traditional Playing Cards vs. Planning-Poker Cards There are several different games used for development purposes, and many of them card games. Common among them is that they often require or encour- age the use of specially developed cards to play the game. Planning poker use planning poker cards with values based on a Fibonacci sequence, Microsoft’s Ele- vation of Privilege also use specific cards to describe the risk to be played about, Threat Poker, on the other hand, when under development, it was decided to use regular playing cards. There exists several different reasons why this was thought the best tool to use for estimation in this game, but reasons that could also be applied to other games intended for development teams. By using playing cards, the team members are provided with cards they are already familiar with, and with face values that are intuitive to the players because they already know how the cards are used for playing. The use of regular playing cards with familiar face values give better intuitive meaning than just a Jøsang, Stray and Rygge 12 set of values in a number sequence. Based on the intrinsic value of the different types of cards, the risk severity expressed by the value of a king which is a high value card in a regular card deck, might be easier to understand than the value 50 in a number sequence, because familiar playing cards provide more of a context to the values than what unfamiliar cards would provide. Using regular playing cards also helps creating an association between tradi- tional card games and Threat Poker, and as such can help make the relatively abstract tasks of risk evaluation and effort estimation into something fun and tangible. Gamification around the discussion and estimation of threats and solu- tions will also stimulate learning among the members of an agile software team. Using regular playing cards also helps creating an association between tradi- tional card games and Threat Poker, and as such can help make the relatively abstract tasks of risk evaluation and effort estimation into something fun and tangible. Gamification around the discussion and estimation of threats and solu- tions will also stimulate learning among the members of an agile software team. Another reason for using regular playing cards is their availability. 5 Discussion and Conclusion Threat Poker is a card-based method to help development teams focus on secu- rity and privacy by design which is becoming a standard requirement for software development, as e.g. required by law in the EU due to GDPR. Threat Poker helps guide developers into considering security and privacy threats and vulnerabilities during the entire development process and also in producing documentations for privacy and security audit. Using Threat Poker helps estimating the severity of security risks and/or privacy risks identified during software development, as well as estimating the effort to solve those risks. This can be seen as an important element of privacy- by-design and security-by-design which are now required for the development of computer software. 13 Threat Poker: Gamification of Secure Agile We had student teams in a software engineering course at the University of Oslo play an adapted version of Threat Poker. Our results show that even though the students shared a lot of the same general knowledge, and none of them were security experts, Threat Poker helped articulate thoughts and opinions about possible threats to the system. During the session in which Threat Poker was played and practiced, all the participants took part in the discussion, asking questions, sharing knowledge, trying to think what could go wrong with the user story in case of an attack, how this could be exploited and how best to protect against the threat scenario. The general feedback from all the student teams was that Threat Poker is a useful technique, and helpful to generate a discussion about security that should be a mandatory part of the development process. Using Threat Poker forced the teams to consider different threat scenarios and how to deal with them. We believe that the simplified version of Threat Poker described in this paper, a threat and its solution are discussed and evaluated in the same round, is better than the original version of Threat Poker where threats and solutions were discussed in separate rounds. Future research should investigate how Threat Poker encourages and sup- ports teams to solve security threats, compared to other games that exist such as the Security Requirements Education Game [7], [d0x3d!][6] and Control-Alt- Hack [5]. 5 Discussion and Conclusion Furthermore, since we have only tested Threat Poker on students, future research should investigate how agile software development projects in companies would benefit from Threat Poker, and in what way the developed software becomes more secure as a result of playing the game. References 1. Colin Tankard. What the GDPR means for businesses. Network Security, 2016(6):5–8, 2016. 2. Definition of gamification. https://en.oxforddictionaries.com/definition/gamification. 3. G Ivanova, V Kozov, and P Zlatarov. Gamification in Software Engineering Ed- ucation. In 42nd International Convention on Information and Communication Technology, Electronics and Microelectronics (MIPRO), pages 1445–1450. IEEE, 2019. 4. Manal M Alhammad and Ana M Moreno. Gamification in software engineering education: A systematic mapping. Journal of Systems and Software, 141:131–150, 2018. 5. Tamara Denning, Adam Lerner, Adam Shostack, and Tadayoshi Kohno. Control- Alt-Hack: the design and evaluation of a card game for computer security awareness and education. In Proceedings of the 2013 ACM SIGSAC conference on Computer & communications security, pages 915–928. ACM, 2013. 6. Mark Gondree, Zachary NJ Peterson, and Tamara Denning. Security through play. IEEE Security & Privacy, 11(3):64–67, 2013. 7. Affan Yasin, Lin Liu, Tong Li, Jianmin Wang, and Didar Zowghi. Design and pre- liminary evaluation of a Cyber Security Requirements Education Game (SREG). Information and Software Technology, 95:179–200, 2018. Jøsang, Stray and Rygge 14 8. L. Williams, M. Gegick, and A. Meneely. Protection Poker: Structuring Software Security Risk Assessment and Knowledge Transfer. In International Symposium on Engineering Secure Software and Systems, pages 122–134. Springer, 2009. 9. Laurie Williams, Andrew Meneely, and Grant Shipley. Protection poker: The new software security” game”. IEEE Security & Privacy, 8(3):14–20, 2010. 10. Inger Anne Tøndel, Martin Gilje Jaatun, and Daniela Soares Cruzes. ”collabo- rative security risk estimation in agile softwarer development”. Information and Computer Security, 27, 2019. 11. Inger Anne Tøndel, Martin Gilje Jaatun, Daniela Soares Cruzes, and Tosin Daniel Oyetoyan. Understanding challenges to adoption of the protection poker soft- ware security game. In Katsikas. S. et al., editors, Computer Security, ESORICS 2018 International Workshops: SECPRE 2018, CyberICPS 2018, LNCS 11387. Springer, Cham, 2018. 12. Hanne Rygge and Audun Jøsang. Threat Poker: Solving Security and Privacy Threats in Agile Software Development. In The 23rd Nordic Conference on Secure IT Systems (NordSec 2018), Oslo, 2018. 13. Adam Shostack. Threat modeling: Designing for security. John Wiley & Sons, 2014. 14. Kent Beck, Mike Beedle, Arie Van Bennekum, Alistair Cockburn, Ward Cunning- ham, Martin Fowler, James Grenning, Jim Highsmith, Andrew Hunt, Ron Jeffries, et al. Manifesto for agile software development. 2001. 15. Kent Beck, Mike Beedle, Arie van Bennekum, Alistair Cockburn, Ward Cunning- ham, Martin Fowler, James Grenning, Jim Highsmith, Andrew Hunt, Ron Jef- fries, Jon Kern, Brian Marick, Robert C. References Martin, Steve Mellor, Ken Schwaber, JeffSutherland, and Dave Thomas. Principles behind the Agile Manifesto. http://agilemanifesto.org/iso/en/principles.html, 2001. 16. VersionOne. 12th state of agile report. https://www.infoq.com/news/2018/04/state- of-agile-published. 17. Ken Schwaber and Mike Beedle. Agile software development with Scrum, volume 1. Prentice Hall Upper Saddle River, 2002. 18. Adam Shostack. Elevation of privilege: Drawing developers into threat modeling. In 2014 {USENIX} Summit on Gaming, Games, and Gamification in Security Education (3GSE 14), 2014. 19. Sven T¨orpe and Andreas Poller. Managing security work in Scrum: Tensions and challenges. In Martin Gilje Jaatun, editor, International Workshop on Secure Soft- ware Engineering in DevOps and Agile Development (SecSE 2017), Oslo, 2017. ( ) 20. James Grenning. Planning Poker or How to avoid analysis paralysis while release planning. Technical report, Wingman Software, 2002. 21. H Kniberg and M Skarin. Kanban and Scrum making the most of both. http://www.infoq.com/minibooks/kanban-scrum-minibook, 2010. 22. Viktoria Stray, Nils Brede Moe, and Dag IK Sjøberg. Daily Stand-Up Meetings: Start Breaking the Rules. IEEE Software, 2018. 23. OWASP (Open Web Application Security Project). https://owasp.org/www-community/Threat Modeling, 2020 24. OWASP. ASVS - Application Security Verification Standard v.4.0, 2019.
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Business School Professors’ Perception of Ethics in Education in Europe
Sustainability
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Edinburgh Research Explorer Edinburgh Research Explorer General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Business school professors' perception of ethics in education in Europe Citation for published version: Gottardello, D & del Mar Pamies, M 2019, 'Business school professors' perception of ethics in education in Europe', Sustainability, vol. 11, no. 3, 608. https://doi.org/10.3390/su11030608 Citation for published version: Gottardello, D & del Mar Pamies, M 2019, 'Business school professors' perception of ethics in education in Europe', Sustainability, vol. 11, no. 3, 608. https://doi.org/10.3390/su11030608 Received: 30 November 2018; Accepted: 22 January 2019; Published: 24 January 2019 Received: 30 November 2018; Accepted: 22 January 2019; Published: 24 January 2019 Abstract: This qualitative study aims to investigate business school professors’ perception of ethics in business education, and their possible role in achieving ethical awareness in these schools. Data were collected through semi-structured interviews with 59 professors from four business schools, each from a different European country. The results show that participants define ethics along four dimensions, and express two divergent forms of implementing it. These differ by the country in which the business school is located. The findings shed light on the issues of ethics and sustainability in business education, and the importance of preparing students to become responsible leaders. For that purpose, we develop recommendations to foster ethics and sustainability in education in business schools in order to develop more socially responsible citizens. Keywords: ethics in business education; sustainability education; students’ development; business school; integrity Sustainability 2019, 11, 608; doi:10.3390/su11030608 Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 sustainability sustainability www.mdpi.com/journal/sustainability Debora Gottardello * and Maria del Mar Pàmies Debora Gottardello * and Maria del Mar Pàmies Department of Business Management, Faculty of Business and Economics, University Rovira i Virgili, 43205 Reus, Spain; mar.pamies@urv.cat * Correspondence: debora.gottardello@urv.cat   Received: 30 November 2018; Accepted: 22 January 2019; Published: 24 January 2019 1. Introduction In recent years, socioeconomic factors, such as the continuous evolution of technological factors and the massification and internationalisation of higher education, have resulted in major changes within the higher education sector. They have presented multiple challenges for universities, who have found ways to offer not only more education, but also better quality education, while meeting the needs of a diverse and constantly changing society [1,2]. For most countries it is difficult to expand the number (and size) of universities and increase participation in higher education while ensuring the highest standards and quality [3]. The quality of education is based, not only on the dissemination and production of new and important knowledge and innovations, but also on the implementation of economic, social and cultural developments [4]. To that end, the Bologna Process was launched by the Bologna Declaration in 1999 and, during the intervening years, the European Higher Education Area (EHEA) has progressively adopted a series of reforms and highlighted the need to improve the quality of education and encourage the development of educational curricula that promote ethical values essential for the cultural and personal development of individuals within society [5]. This cannot be done without a clear commitment to ethical and moral values. Training students how to be responsible citizens and make equitable and sustainable choices requires the commitment of academic staff to teach them good habits, good judgment, and how to become socially responsible [6]. This is even more relevant in the context of business schools as they represent the cradle where the business leaders of the future will gain their formal education. Indeed, some studies have shown a positive relationship between teaching ethics in university and sustainable business practices among global companies [7]. The phenomenon of globalization has thrown up multiple contradictions inasmuch as it can produce reasons for distancing from and, for drawing closer to, the topic of ethics [8]. Increased economic globalization and competitiveness requires more cooperation Sustainability 2019, 11, 608; doi:10.3390/su11030608 www.mdpi.com/journal/sustainability Sustainability 2019, 11, 608 2 of 19 and transparency, while simultaneously encouraging a cavalier attitude to ethical behaviour and acting for the protection of the particular benefit to the detriment of the collective interest [9]. and transparency, while simultaneously encouraging a cavalier attitude to ethical behaviour and acting for the protection of the particular benefit to the detriment of the collective interest [9]. 1. Introduction Enterprises are embedded in a cultural context with specific moral standards that define what is forbidden, permitted, encouraged or mandatory, and which are shared by society or a group of equals. But, in the global business environment, not all individuals make moral decisions [10]. Thus, a potential practical approach to influencing moral decisions is through, including ethics education. This is where the relationship between morality and ethics arises and, in this sense, it is important that business schools are run on ethical principles. Future leaders need to know how to adapt to different cultural contexts in which different moral norms exist. Ethics may help in the requisite interpretation and adaptation [10,11]. Consequently, ethics has become an essential element especially in business schools, and has led many of them to begin to reflect on ways to teach essential values in order to create future business leaders who act sustainably and responsibly [7], in accordance with ethical and moral principles. Educating in regard to morality means teaching the rules, codes of conduct and how to avoid potentially harmful actions for society [12,13], thus providing ethics education has to do with good actions and good practices. Despite the efforts made by different universities to improve educational quality and promote a sustainability education geared towards the ethical and social considerations essential for business leadership [14], little is known about the role of the teacher, their perception and awareness of business ethics and how these relate to sustainability. The majority of sustainability research has focused on investigating how environmental sustainability is taught in the university, or on ways of educating students about prototypical sustainability issues, such as responsible consumption. Sustainability education is not only concerned with the welfare of the environment, but also with the well-being of the society, culture and the economy. It is about educating students on how companies can compete while respecting ethical values. Its main objective is to reorient education and learning so that students have the opportunity to acquire knowledge, skills, values and attitudes with which they can contribute to sustainable practices in the future. Sustainability education makes it possible for students to learn the possible negative impacts a business might have and helps them make ethical and moral business decisions. 1. Introduction Given the central role of professors in in promoting ethics in education, and the cultural embeddedness of morals and ethical standards, the current study focuses on these aspects and explores them in a comparative way. Our motivation here is that a comparative study can help in understanding whether the ways of perceiving ethics depend, not only on the university’s internal context, but also on the cultural context (as affirmed, for example, by Hofstede [15,16]. We have found no such comparative studies using a qualitative methodology in the literature. Specifically, we examine professors’ perceptions regarding ethics in business education in four different European countries. The aim of our analysis is to better understand what ethics in education means for business professors, and what might be done to develop it in different international environments. Following a review of the literature on ethics education and sustainability education [4,17] (or, as some authors term it, “education for sustainability (EfS)” [17]), as an education that is intended to develop an understanding of ethics and values, which in turn needs to be included in business schools [18,19], we propose two research questions. Next, we present the methodology used, including information on the sample and the procedure employed for data collection and analysis. The Findings section identifies the topics that emerged from the data analysis, and the article ends with a discussion of the theoretical and practical implications of the study. 2. Literature Review The incorporation of ethics in education has commonly been carried out through specific courses (normally called business ethics) and it is considered important in business schools in order to promote a responsible and sustainable ethics education [24]. However, the integration of ethics in education (ethics education) throughout all business disciplines is considered even more valuable, as it allows a broad spectrum of concepts and topics to be viewed under an ethical lens [25]. Ethics in business education can explain the importance of human behaviour, of what is good or bad, and the standards of behaviour adapted by organizations, helping students (future leaders) to make a moral reflection on their actions [26]. The reconciliation between reason and morality, so that individuals and organizations are responsible for their actions and committed to society, both in the present and the future, underlines the relationship between ethics and sustainability [14,18]. Thus, teaching ethics in business schools involves forming organizational leaders who understand the need to take honest, responsible, and sustainable actions and for the generation of value as a management approach without prejudice to different stakeholders [14,26]. Ethics in education begins with teaching values, with what is good and bad, with the impact of any irresponsible behaviour as students on their subsequent careers. In this way, it is possible to convey to students the importance of being reflective professionals capable, as a routine aspect in the exercise of the profession, of analysing and criticizing their actions. Here, as the student learns to detect the ethical dimensions of business situations and a habit of constructive analysis, the ability to empathize with stakeholders is fostered and a relationship with sustainability arises. Ethics in education embraces the main objective of increasing students’ ability to make ethical and responsible decisions and thus contribute to sustainable development [4]. The literature claims that sustainable development requires individuals to have an ethical conscience and highlights the importance of including topics of ethics, corporate social responsibility and sustainability especially in business schools [4,27]. Integrating these principles within the curricula allow students to develop a set of key competencies that can guide them in their future decisions as managers or employees. Authors, such as Leal Filho et al. 2. Literature Review This section introduces the concept of ethics in education in order to differentiate it from ethics education as a discipline. We analyse the literature on ethics in education in business schools (henceforth, ethics in business education) and the important role that professors play in the education of future business leaders. Afterwards, to contribute towards filling the perceived gap in the literature, we introduce the research questions that guide the present study. Sustainability 2019, 11, 608 3 of 19 2.1. Ethics in Business Education and Its Relationship to Sustainability Education .1. Ethics in Business Education and Its Relationship to Sustainability Education Ethics (or moral philosophy) is a discipline that has to do with principles and moral reasoning [20] regarding what is good or bad, right or wrong. The function of ethics education (as a discipline) is to explain adequate ethical behaviour (acts and decisions) through rules that are in accordance with social and/or psychological laws, that is, rules for ethical decision-making [21]. Ethical attitudes begin to develop in the home, then through interpersonal relationships, and are influenced by the behavioural norms of a society and the surrounding context [22]. Any improper, or even deviant, behaviour by other community members may affect the individual attitudes learned and have an impact on the social and business context. Therefore, despite the fact that students have certain ingrained behaviours from their earlier education, it is possible to train them at university to behave ethically, and to resist the influence of others who behave unethically at work [23]. y Higher education has a key function in providing students with the means to achieve their degree, and at the same time to create influential citizens capable of valuing and respecting their society and the environment. For business schools, the task of creating respectful managers and employees able to work for the development of society and the environment has been identified as a key aspect of business Higher education has a key function in providing students with the means to achieve their degree, and at the same time to create influential citizens capable of valuing and respecting their society and the environment. For business schools, the task of creating respectful managers and employees able to work for the development of society and the environment has been identified as a key aspect of business education. 2. Literature Review [28] cited by Straková, and Cimermanová [29], point out that an education towards sustainability should integrate theory and practice, and engage people in activities that make them reflect about ethics so that they are encouraged to think critically. According to Straková, and Cimermanová [29], this implies the need to develop individuals with “creative problem-solving skills, scientific and social literacy, and a commitment to engage in responsible individual and cooperative actions”, to improve their personal and emotional skills and to act in a sustainable way [30] These emotional skills include the ability to interact and listen to others, to assess, commit and review their behaviours, and to act ethically. In order to achieve these skills, people need to be trained to recognize and solve common ethical problems in certain professions [4]. Sustainability 2019, 11, 608 4 of 19 Teaching business students to take into account the interest of various stakeholders, and about ethical values such as integrity and honesty, may help them to act responsibly and with integrity in the future. In organizations, behaving with integrity means not deliberately harming customers, employees or even competitors, through deception or misrepresentation. Integrity and honesty are related. They represent the glue that holds business relationships together and allows everything to be more effective and efficient. Lying about a product, cheating or stealing for the achievement of business objectives, are actions usually associated with dishonest behaviour. Integrating ethics in business must be a lifetime commitment for universities and needs not only business leaders with an ethical vision, but also educators capable of balancing individual economic objectives with the social responsibility that our society requires. Through a sustainability education it is possible to help students develop moral reasoning, and prepare them for their future roles in the business environment [9]. In these moments of crisis of ethical values within corporations and within educational institutions, it is important to intervene and involve students. Encourage them to explore the environment, and the ways of working, and learn important life lessons while applying the theory to practice. Furthermore, the literature has shown how the moral values of a profession are learned and internalized for the first time in the course of higher education [31,32]. However, there is little evidence that business schools are implementing ethics in education focused on highlighting the importance of high standards of professional conduct. 2. Literature Review Although the importance of including this training has emerged in recent years, business schools have been accused of being “irrelevant to business” [33]. The globalization and the massification of universities have made problems greater, sparking an identity crisis [34]. Business schools have been accused of being more interested in the number of enrolments than in the quality of their students [34]; of not sufficiently preparing them for management practice [35]; and of failing to teach the importance of ethical and professional standards for a sustainable environment. In fact, some authors suggest that business schools are implicitly conveying to their students the message that unethical behaviour is acceptable. This might be corroborated by the prevalence of academic dishonesty among these students [34]. Authors, such as McCabe et al. [36,37], have stated that the incidence of cheating was higher in business students than in those who studied other subjects, such as Law or Science. Similarly Nonis et al. [38] pointed out that the probability of acting dishonestly in the workplace was more closely linked to those people who were involved in unethical practices at school. Carpenter, et al. [39], suggested that when students engaged in academic dishonesty in college, there was a greater chance that they might behave in an unethical way in their professional practice. These authors show how a comprehensive education in ethics is imperative in enhancing students’ critical thinking skills and in enabling them to grasp the impact that their attitude while in university can have on the exercise of business activities. Organizations at the global level need to take a major step along the road towards sustainability. However, without the collaboration of the universities in effectively training professionals and future decision makers, it may not be achieved. The widely-reported cases of bad business practices suggest that organizations are more focused on obtaining profits than acting responsibly. Therefore, business schools should reflect on their current task and train future managers in responsible management practice. 2.2. The Role of the Teacher in the Education of Future Leaders with Ethical Consciences In this context, professors hold a unique position. Through their role, they can heighten their students’ abilities, give clear instructions, and educate not only on issues related to the subject, but also on any matters that have a close relationship and are linked with the professional development of the students, thereby becoming “Moral educators” [6]. As agents of effective change, they can generate commitments and civic leaders, preparing students for future sustainable decision-making [32]. According to Giacalone and Thompson [40], the primary responsibility to assist and encourage students to become ethically sensitive falls, of course, first and foremost on professors, since they are in charge of “preparing a new generation of business Sustainability 2019, 11, 608 5 of 19 professionals”. Professors must improve their students’ understanding of the ethical component, help them to achieve their goal in both their professional and personal lives. professionals”. Professors must improve their students’ understanding of the ethical component, help them to achieve their goal in both their professional and personal lives. Being educators of future business leaders, it is important to investigate what professors think about the topic of ethics in business schools, and to understand what is important for them to do in order to better prepare future managers and leaders. The literature is clear that the willingness of professors to include ethics in education can be altered by factors, such as the lack of qualification or training to teach in this area [41], the lack of time, or work overload, that make it impossible to incorporate materials on moral or ethical issues into their courses [9], or even the lack of interest or the perceived low value in teaching ethics. The latter situation, according to Adkins et al. [42], arises when teachers perceive that ethical or unethical behaviour depends on the values developed through life, through culture or family, and is separate from the university education. The authors add that, although faculty members may experience these doubts, they should assume responsibilities and provide their students with the necessary means to learn the possible ethical problems of their behaviour. It would be useful for them to know how to educate their students about the possible ethical situations they may face in the workplace and the repercussions for society. Our literature review did not find any empirical studies that analysed in depth the perception of business school professors about developing ethics in business education. 2.2. The Role of the Teacher in the Education of Future Leaders with Ethical Consciences Therefore, there are potential benefits to research in this area; further study would give us some idea of how professors of business schools, not only understand, but also how they approach ethics and responsible management by exploring their perceptions in a broad sense. Little research has been done on the role that professors can have in ethics and sustainability education and their perception. Those who have researched this topic have focused mainly on countries, such as the United States and other Anglo-Saxon countries [9], or have studied the perception of students [43,44] or deans [4]. Furthermore, those who have studied professors’ perceptions, have used quantitative methodologies [42] that have not allowed for an in-depth understanding of the topic.Consequently, this study focused on the following two research questions: RQ1: What are professors’ perceptions about what ethics in business education is and includes? RQ2: What could be done to develop ethics in business education? We wished to carry out a cross-cultural comparison to see to what extent national culture explains the perception about the importance of teaching ethics. According to the literature, when students are educated about ethics, ethics is more likely to be properly managed, and global business practices achieve better results [45]. Globalization has meant that organizations have to face increasing challenges to adapt effectively to different cultures and understanding that some cultures have strong ethical principles is essential. According to Hofstede [15], the most individualistic cultures, unlike the collectivists, have “strong moral connotations.” Thus, in collectivist cultures, individuals display fewer ethical behaviours because they try to do what is best for the organization; leaders in organizations demand greater emotional dependence of members, and essential values are not emphasized. However, in more individualistic societies, organizations assume broad responsibility for their employees and try to inculcate moral values [46]. 3.1. Research Design The present study uses a qualitative research approach to answer our research questions, this methodology is considered the most appropriate when exploring an unknown research topic, for the development of a theory, or to add a new perspective to a subject already investigated [4,30]. The objective of the present study was not to test predetermined hypotheses and produce generalized results, as would be typical of quantitative methodologies, but rather to gain a deeper understanding of the phenomenon. An important advantage of the method chosen is that it allows us to hear professors’ perspectives and “to capture the voice and way they make meaning of their experience” [47] thus, gathering rich information, 6 of 19 Sustainability 2019, 11, 608 thick description and thick meaning [48,49], about what is being observed. To date, professors’ own perspectives about ethics in business education have largely been overlooked. Since, more than ever, they play a key role in developing students’ knowledge and increasing their competency and skills [50], in-depth research under these tenets is of paramount importance. Despite the advantages of this methodology in terms of its relevance to the research questions posed, there were several disadvantages. In particular, it required intensive and prolonged work, such as translating from the original language to English and transcribing all the interviews, categorizing, codifying and recodify texts until an agreement was reached among the authors [33,36]. Results from qualitative methodologies also aid in highlighting the findings in the specific context where they were extracted, but cannot be extrapolated to a whole population. Notwithstanding this, interviews allow us to better understand the role that professor play in the training of future ethically- and sustainably-aware entrepreneurs and to see what is common among different countries and economies and the critical differences emerging from the way in which sustainability issues are presented and addressed. Most of the existing studies in this area have used a quantitative methodology or have explored students’ perceptions, rather than professors’. In addition, exploring how professors transfer sustainability education to their students in business schools is essential in order to be in a position to analyse possible corrective educational policies. 3.2. Sample 7 of 19 Sustainability 2019, 11, 608 Table 1. Hofstede’s Country Comparison. Table 1. Hofstede’s Country Comparison. Ireland Italy Spain Sweden Power Distance 28 48 57 31 Individualism 70 76 51 71 Masculinity 68 70 86 5 Uncertainty Avoidance 35 75 48 29 Thus, Sweden was chosen is because it is the country in Europe with the lowest score in uncertainty avoidance and masculinity while, at the same time, it has developed a law on the inclusion of sustainability issues in its universities. This may imply a strong commitment to ethical issues. On the contrary, Spain is one of the countries in Europe with the highest levels in those two dimensions. Ireland and Italy are countries whose Hofstede dimensions lie somewhere between Sweden and Spain. Ireland is similar to Sweden in uncertainty avoidance and power distance, but scores higher than Sweden on masculinity, which may mean that, despite having an interest in themes of ethics and sustainability; it does not have sufficient capacity to seriously address toward these issues. Italy is a country whose uncertainty avoidance and power distance are similar to those of Spain, but which has high levels of individualism and may consequently have a greater ethical conscience than Spain. The significant range from Spain, through the intermediary levels of Ireland and Italy, to Sweden that we find across each of Hofstede’s dimensions, allows us a more thorough examination of behaviours and perceptions and how these are subject to cultural differences. Purposive sampling was used to identify and choose cases rich in information. This let us analyse the differences between the participants and gather key information. In addition, selected participants were invited to identify other information-rich teachers who could participate in the study (snowball technique). As qualitative research, there were no defined rules to determine the number of participants so sampling was continued until the point of data saturation, where the authors were sure that no new information could be obtained [59]; thus, when the comments continued being the same and new data replicated that already collected, the interview was stopped, considering that the study had enough data to illustrate the phenomenon [60]. As pointed out by Creswell [61] for qualitative research studies, the range of participants between 5 and 25 individuals, who share similar experiences, is often considered adequate. Kazley et al. 3.2. Sample The population of the research was composed of business school professors in four countries, Ireland, Italy, Spain and Sweden. The study focused on professors, because their voice on ethics subject and sustainability is heard less frequently, while the opinions of students and other stakeholders have been extensively explored. While academics may have shared their views regarding sustainability and ethics in the form of a survey, this qualitative study allows a broader understanding of perceptions. These four countries were chosen because, as stated previously, they have different cultures. Comparing different countries according to the Hofstede dimensions [15,51] of national cultural values, could help to understand the existing connection between cultural context and ethical perception. He developed four dimensions that characterize different cultures around the world and which are applicable to a wide range of studies in social sciences. Specifically, he shows how power distance, uncertainty avoidance, masculinity and individualism help explain the beliefs and values shared among the members of a community. The European cultural context is not homogenous, and some of the cultural dimensions, being value-oriented, may be related to a country’s capacity to engage with ethics and sustainability [52]. In the same way, they might influence personal perceptions of ethical values and behaviours [53] and sustainability [54–56]. Accordingly, countries with a greater level of power distance and uncertainty avoidance and masculinity may have a lower social and institutional capacity to progress toward ethics and sustainability [57]; high levels of individualism appear to be associated with lower rates of unethical behaviour and more ethical commitment. The countries selected for this study have significantly different scores in the four underlying cultural dimensions (see Table 1). Specifically, Spain and Italy have very high scores (over 50%) in the dimensions of power distance and uncertainty avoidance and masculinity. Spain is the country with the lowest percentage in individualism (the other 3 countries score more than 70% in this dimension). On the other hand, Sweden and Ireland are in low positions in power distance and uncertainty avoidance (less than 35%), while Sweden scores a mere 5% with respect to masculinity. A study by Ringov and Zollo [58], highlights how low levels of uncertainty avoidance and masculinity are particularly related to the propensity to guide the members of this society towards responsible and ethical attitudes; collectivistic societies, on the contrary, are less inclined towards an ethical orientation. 3.3. Data Collection Each interview lasted between 45 and 90 minutes. The interview guide addressed the topics posed by the two research questions, namely, professors’ perception about what ethics in business schools entails, and the possible initiatives they could undertake to develop ethics in the context of business education. Before carrying out the fieldwork, the researchers tested the interview. Following the qualitative research procedures of Corbin and Strauss [59], the script of the interviews was adjusted during the research process, thus reformulations were introduced to improve the understanding of the subjects interviewed. The questions were reordered so that the interviews were more fluid, and questions were added to include the questions that had been raised in previous interviews. One researcher conducted all the interviews and moved to the university where the interviewees worked. With the respondents’ permission, the interviews were recorded with the help of a digital recorder and successively transcribed. 3.2. Sample [62] suggested that saturation can be counterproductive in cases where the new does not expand or extend the information to the general investigation. In light of the above, we assume that the sample size in the present study is adequate for the design and purpose. D t t ti hi d ith 17 f i I l d 10 i It l 11 i S d d 21 i S i p y p The interviews were organized in advance with the professors to ensure convenience in their participation. To obtain rich and diverse information, participants were selected according to age and years of education. The inclusion criteria were that (at the time of the interview) they worked in a business school and had a minimum of 4 years of experience in the university. Both men and women needed to be represented and reluctance to participate was an exclusion criterion. Within these categories, the teachers were selected randomly. In Spain the sample was 57% female and 42% male, in Italy it 50% male and female, in Ireland it 42% male and 58% female, in Sweden 30% male and 70% female. The average number of years of teaching in our sample was approximately 11 years in Spain, 13 years in Ireland, 12 years in Sweden and 12 years in Italy. Our final sample had an average participant age of 44 years in Spain and Ireland, 42 in Italy and 43 in Sweden (Table 2). In order to protect the identities of the participants and guarantee their confidentiality and anonymity, we decided to use a code for each participant as this is one of the key principles of qualitative research [63]. 8 of 19 Sustainability 2019, 11, 608 Table 2. Interviewees’ profile. Table 2. Interviewees’ profile. Profile Elements Country Spain Italy Ireland Sweden Female 57% 50% 58% 70% Male 42% 50% 42% 30% Average number of years of teaching 11 12 13 12 Average age 44 42 44 43 Min-Max [33, 61] [34, 55] [32, 62] [34, 60] Standard deviation 7.17 6.06 7.38 7.09 3.3. Data Collection 4. Results This section presents the results of the analysis of the interviews. It is structured according to the two research questions posed in the study. Table 3 summarizes the dimensions emerging from the analysis of the data (which are then described in each corresponding subsection below), alongside the indication of their coding total intensity (column “all”) broken down by country. Table 3. Dimensions identified in the exploration of professors’ perception of what ethics is and what they could do to develop it in the context of business education. Table 3. Dimensions identified in the exploration of professors’ perception of what ethics is and what they could do to develop it in the context of business education. Table 3. Dimensions identified in the exploration of professors perception of what ethics is and what they could do to develop it in the context of business education. Countries IRL SW IT SP All 1. What are ethics in business education? 1.1. Morality: Bad and good 15 8 8 16 48 1.2. Code of conduct, rules and values 13 7 7 14 43 1.3. Integrity and honesty 16 9 9 14 48 1.4. Sustainability 5 9 2 0 14 2. What could be done to develop ethics in business education? 2.1 Teach ethics and integrity and give examples 14 9 8 4 35 2.2. Not my role 0 0 0 13 13 4.1. Research Question 1: What Are Professors’ Perceptions about What Ethics in Business Education Is and Includes? 4.1. Research Question 1: What Are Professors’ Perceptions about What Ethics in Business Education Is nd Includes? The analysis of RQ1 revealed that the concept that professors had about ethics was not a simple one. Instead, their ideas ranged across four dimensions that make up the conceptual map of what they consider to be key elements of ethics in the context of business education, namely: Morality, good or bad (sub-code 1.1.); Codes of conduct and rules (sub-code 1.2.); Integrity and honesty (sub-code 1.3.); Sustainability (sub-code 1.4.). Some similarities and differences among professors from different countries in their manner of perceiving and understanding ethics in business education were observed, we will address each of these in turn. Importantly, three of them were of similar strength across countries, but the fourth dimension was specific to just one cultural context (that of Sweden). However, Hofstede’s cultural dimensions emerge and influence the perception of how to develop these ethical consciences. 4. Results As will be discussed later, the regulation and awareness of the need to guide in order to strengthen ethics depend, as confirmed by the literature, upon the culture of a given society. In the following sections, we discuss these dimensions, which are summarized in Table 3 3.4. Data Analysis The qualitative data collected through the semi-structured interviews were organized and prepared for analysis. Transcripts were analysed and reviewed to search for affirmations and relationships between data categories [64] and to establish themes and thus transform the data into findings [61]. For data analysis, the Nvivo 11 software was used due to its ability to combine the interpretation and codification of the text, the relations of categories and subjects, and the search and retrieval of coded units, thus increasing the transparency of the analytical process [65]. The information found at the beginning was compared with the information found successively to discover new topics or improve the understanding of the previous ones. Ultimately, the procedure led to the identification of different patterns among professors in different countries. Content analysis is a useful research method to make replicable and valid inferences from the data to its context, for the purpose of providing new insights, more knowledge about the facts and a practical guide for action [66], the objective being to achieve a condensed and comprehensive description of the phenomenon. The most important steps in our content analysis were the creation of codes and the establishment of categories and definitions (Table 3). In open coding, concepts and text fragments were tagged, and defined. A defined code allows one to identify the main thought behind each piece of text. Both categories and codes were assigned a name and definition—the definition of the categories, unlike the definition of the codes, taking into account not only a key thought, but all the codes included and their definitions thus generating a complete map. This map was validated by three different researchers until a final agreement was reached. To understand the phenomenon under study in a more comprehensive way, the initial configuration was slightly modified by the adjustments derived from the intensity of the coding and the possible variants or the appearance of nuances of meaning [67]. Finally, emerging codes were articulated in such a way to provide an organized and rigorous structure within the conceptual framework of the established research theory [68]. The research questions were used as the guiding framework for the first stage of the data analysis, from there more codes emerged, and different sub-codes were identified. Sustainability 2019, 11, 608 9 of 19 4.1.2. Code of Conduct, Rules and Values The second generalized aspect among the professors was to consider ethics in business education as not only governed by principles of morality, but also by a respect for rules, code of conduct and values. (see Table 3, sub-code 1.2.: Code of conduct, rules and values). A number of professors consider that it involves teaching codes of conduct, rules and values. Educating about ethics means understanding that there are values and that these values are included in codes of conduct that guide all people regardless of the status they hold within the organization. Students should learn that every organization must have codes of conduct respected by all members. One participant said: (SP 01): “We have to teach them that the codes are insufficient if they only focus on ensuring that everyone respects the rules.” And some added that it is important to (IT 01) “educate professionals to know not only which codes of ethics and conduct to apply but also how to apply them in practice, to give them knowledge of how it is done”. In general, professors believe that educating about the importance of professional codes of conduct is essential. However, this should be taught as complementary to the teaching of values and indeed values specify what society expects companies to take into account when making decisions. They need to convey to students that the rules cannot be applied mechanically, they need to be shown how to develop a practical judgment so that, when applying the rules, they take into account the values. According to professors, ethics in business education has to relate standards, values and practices and, as another professor said: (SW 01): “Makes, that not only ethical dilemmas are included but also concrete situations and ways of approaching them, relating people, environment and norms”. 4.1.1. Morality, Good or Bad As can be seen in Table 3 (sub-code 1.1. Morality, bad and good), the professors perceive ethics in business education as a moral education regarding Good and Bad. It concerns educating students in regard to what is morally good and morally bad; more than half of the professors in the four countries said that it has to do with teaching what is right, and what is not right. For example, one respondent said: p (IRL 01): “Ethics in business education has to do with morality, teaching what is good and what should not be done, what can or cannot be done”. Participants stated that it responds to the need to maintain and uphold ethical principles within the organization, to carry out ethical and moral actions, to and assess whether those actions can be harmful to others. Ethics in education guides the decision that future managers will take and teaches them how to discern right from wrong, good from bad, and the appropriate from the inappropriate. It also implies a commitment to do what is right, appropriate, and good for people. Thus, ethics and morality are be related, since morality says what to do and what not to do when my interest is to maintain the coexistence, and ethics explains why you should follow the dictates of morality. Sustainability 2019, 11, 608 10 of 19 4.1.3. Integrity and Honesty As shown in Table 3 (sub-code 1.3.), according to most professors, ethics in business education should not be skewed towards the fields of peoples’ rights, the observance of rules and morality, but should also address behaviour, expectations, honesty and integrity. This includes teaching students’ social skills, how to listen to others, and how to respect their integrity. The interviewees affirm that teaching ethics in business addresses issues of corporate responsibility for decisions and actions taken by organizations from the grassroots level to the macro level, in a sincere and honest manner; decision-making must combine reason and emotion, self-interest and caring for others. One participant said: (IT 05): “Educate about ethics is to educate about integrity which in turn means, essentially honesty”. Another (IRL 06) affirmed: “[T]o teach that to be successful in business it is not enough to earn money but it is important to be honest”. (IT 05): “Educate about ethics is to educate about integrity which in turn means, essentially honesty”. Another (IRL 06) affirmed: “[T]o teach that to be successful in business it is not enough to earn money but it is important to be honest”. Teaching this means making students understand that tomorrow they will be required to improve the standards of truthfulness, integrity, and honesty in their businesses in order to achieve business development. It also means understanding that their commercial actions may violate the social values of transparency and cause economic losses of unimaginable dimensions. Companies have become increasingly competitive and, to achieve profits, sometimes act dishonestly. Thus, professors argue that teaching ethics in business education can help students grasp that they have to compete honestly, since future unethical business behaviours hurt productivity, living standards, and therefore integrity. It is imperative to encourage students to perceive the ethical problems of their actions by comparing their unethical behaviour in academia with their unethical behaviours in the business world. In this sense, one professor provided a comparison of academic integrity and business integrity and said: (IRL 13): “Teaching ethics in business is like teaching integrity in the classroom. For example, they cannot appropriate the knowledge of other people, and that tomorrow nor can they can violate the copyright of other companies. They cannot steal the business ideas of others”. (IRL 13): “Teaching ethics in business is like teaching integrity in the classroom. 4.1.3. Integrity and Honesty For example, they cannot appropriate the knowledge of other people, and that tomorrow nor can they can violate the copyright of other companies. They cannot steal the business ideas of others”. Regarding these first three dimensions, as far as we can see there is a widespread understanding of what ethics in business education means. The majority of professor in the four countries relate ethic Regarding these first three dimensions, as far as we can see there is a widespread understanding of what ethics in business education means. The majority of professor in the four countries relate ethic 11 of 19 Sustainability 2019, 11, 608 with morality, the codes of conduct and the integrity and honesty. The findings regarding Spain and Italy seem to contradict the previous research carried out by Hofstede, who considered that countries with a high level of power distance and uncertainty avoidance and masculinity and low individualism are less likely to assume ethical commitments. However, we should emphasize that the first research question focuses on business school professors’ understanding (not on acts) of ethics in business education, thus their knowledge about ethics seems to derive from the fact that they taught in business school and thus are familiar with the topic. Nevertheless, we need also to understand if what they think is congruent with what they think could be done. 4.1.4. Sustainability When comparing the cases in the four countries, it is evident that a different dimension emerged in Sweden as opposed to the other countries. Specifically, as shown in Table 3 (sub-code 1.4) most of the participants reported that ethics in business education has to do with sustainability education. Respondents perceive that sustainability education is teaching students skills and abilities that will be necessary for tomorrow, the aim being to give them different stimuli so that they can develop critical thought processes and problem-solving strategies. Teachers relate this to the importance of business sustainability, since one needs to emphasize not simply the importance of creating values and generating competitive advantages, but also respecting the rights of others and treating the environment in a sustainable manner. The results are consistent with Hofstede and other literature [69] according to which countries with high individualism, low uncertainty avoidance and low masculinity (Sweden is the only country that meets this three dimensions) are more concerned with ethical issues and also demonstrate more capacity to promote sustainability and responsibility to protect the interests of stakeholders. Specifically, these dimensions underscore the need to harmonize individual interests with the broader demands of society highlighting the facet of sustainability [70]. (SW 07): “I believe that ethics in business means teaching about the environmental and social responsibility, that is, tomorrow they have some challenges and they have to balance the economic and social impacts of their companies. But the sustainability of business is viable only if you start teaching at the university in an environment of trust . . . is to teach students skills and values through real action and show that these real actions benefit society and the environment”. Swedish professors stress the importance of teaching students to reflect and develop an ethical culture that contributes to educating responsible citizens. One participant said: Swedish professors stress the importance of teaching students to reflect and develop an ethical culture that contributes to educating responsible citizens. One participant said: (SW 08): “Ethics in business education is about sustainability environment etc., and it is obligatory to teach it from the school, teach that when they become employees, they should respect standards of ethics and commitments with the society”. 4.1.4. Sustainability (SW 08): “Ethics in business education is about sustainability environment etc., and it is obligatory to teach it from the school, teach that when they become employees, they should respect standards of ethics and commitments with the society”. y Moreover, in Ireland, some teachers (5 in all) spoke of ethics in business as teaching sustainability but they were not the majority as in Sweden. In that way, a participant commented as to why students should be taught about sustainability: Moreover, in Ireland, some teachers (5 in all) spoke of ethics in business as teaching sustainability but they were not the majority as in Sweden. In that way, a participant commented as to why students should be taught about sustainability: (IRL 04): “Ethics in business education means explaining to students that unethical commercial behaviours damages productivity and society. Nowadays there is a major concern on sustainability and this must be taught so that our practices do not affect society and the environment where society lives”. According to participants in the study, the work carried out by professors is of paramount importance for the development of students and society, and this “goes beyond that of simple instruction of program themes, it is instruction about a world in which Sustainable Development becomes a reality for all students” (SW 09). Students must know how to responsibly manage the companies of tomorrow. On the one hand, future entrepreneurs have the prime economic responsibility for generating profits, but they also have the responsibility to act as responsible citizens in a complex and continuously evolving environment. (SW 03): “It is for your future, you have to teach ethics and sustainability go hand in hand. It is teaching how to dialogue with stakeholders, and be responsible, how to generate long-term sustainable (SW 03): “It is for your future, you have to teach ethics and sustainability go hand in hand. It is teaching how to dialogue with stakeholders, and be responsible, how to generate long-term sustainable Sustainability 2019, 11, 608 12 of 19 wealth, act with honestly and integrity and, not to cheat because they will deceive not only others but also themselves”. Thus, ethics education about can open students’ minds by teaching them the importance of being honest in society and of becoming business leaders who are capable of making ethical and sustainable decisions. 4.2.1. Teach Ethics and Integrity and Give Examples In Table 3, we can see that the first sub-code (2.1.) that emerged in the interviews was “Teach ethics and integrity and give examples”. In Sweden, Ireland and Italy, professors stated that there is a specific course on Business Ethics in their universities, but they also add that they provide additional emphasis within their own classes. Thus, they include a module or a class on careful teaching of ethics and responsibility in the university. Teachers, especially in Sweden (9 teachers out of 10), consider it necessary to teach students present and future needs of their own and society, incorporating issues of ethics and integrity and sustainability in the modules. They emphasize the importance of teaching students how to solve the ethical problems they face in their courses so that they learn the failures directly from the practice. They also explain what has to be done to develop the students, allowing them to build their personal knowledge, without appropriating the knowledge of others. They stress personal and professional development, that is preparing their students for the future and in how to resolve potential conflicts and problems in their lives. They believe that students need models of integrity and for that, they must be exposed to multiple ethical-professional problems and also academic problems, developing habits within the classroom so that they can see the problems from a closer perspective and better understand the meaning of their actions. On this point one participant said: (SW 05): “I make them realize how they have to act ethically. I tell them that, instead of reproducing knowledge they need to create new knowledge to guarantee that what they produce does not come from someone else. Teaching that the company must act honestly and not copy the ideas of other companies helps them learn to evaluate the information to know where it comes from”. In Ireland, half of the teachers find ways to connect academic integrity with ethical concerns that could arise in professional practice. One participant described briefly how he discusses ethics in business at his university. He explained that education about ethics includes relate topics of academic integrity with business misconduct to “show students that they can’t cheat in the university and cheat in the business world”. The perception of the professor is that students need to understand the relationship between academic dishonesty and business dishonesty. 4.1.4. Sustainability In summary, many participants in Sweden, and a few in Ireland, perceived ethics in business education as an education that produces citizens with ethical principles and provides a solid basis for informed decision-making and a means of achieving sustainable development. Respondents believed that ethics in education contribute to the creation of sustainability awareness in students minds and influence their present and future actions and reflections. 4.2. Research Question 2: What Could Be Done to Develop Ethics in Business Education? The second research question RQ2 (code 2. Table 3) was: “What might be done to develop ethics in business education?”. Here, the participants expressed their opinions on what they consider necessary to help the students to develop these skills. Two sub-codes emerged: Sub-codes 2.1 (Teach ethics and give examples) and 2.2 (Not my role), coinciding with the professors who thought it was part of their remit and those who did not. On this topic, the differences between countries may be surprising. 4.2.1. Teach Ethics and Integrity and Give Examples 4.2.1. Teach Ethics and Integrity and Give Examples This perception was expressed through the following citation: (IRL 16): “I provide an integral education, so in the module I indicate that they cannot act in an unethical way in the university. I tell them to refer and quote because what they copy today in business schools can be copied tomorrow in the companies. I give them examples of cheating, of plagiarism, and construction of the original knowledge, useful with a more integral approach “. Sustainability 2019, 11, 608 13 of 19 Teachers state that it is important to emphasize these issues, because professional ethics in economics does not merely mean being a good student and knowing the subject, education should be “as broadly based as possible” (SW 02). It is important to talk with students about honesty issues that arise in assignments or exams, establishing relationships between the institution’s honour code and business ethics codes, bad be . . . haviour in the company and bad behaviour at the university. p y y (IT 01): “If you copy an exam what will you do when you have to direct the financial data of a mpany. Will you copy it, too? . . . I have to show you this”. Therefore, they believe that, to avoid these issues, you have to educate from the university. The teachers said that they work with teams, where questions of responsibility and reliability in the group may arise, as well as the balance in the distribution of tasks, and mutual respect and consideration among team members. These issues that are often born in the university in team activities are a stimulus to understanding how students should or should not act tomorrow. If teachers want to counteract attitudes like these, they need to make students understand how their actions can harm different parties and can have negative consequences for themselves and for others. Another interviewee said: (SW 09): “I always try to instil that values are important for us and for society, I tell them that their jobs have to be correct and they cannot violate the rights of others because this will affect their professional future”. Teachers emphasize the importance of training people to reason and know how to think for themselves, and to respect the rules, values and attitudes, without harming the rights of others. 4.2.1. Teach Ethics and Integrity and Give Examples According to these professors, the teaching of ethics in business begins at the university and implies developing an ethical and sustainable project that makes students aware of the need for standards and attitudes that favour coexistence and allow the development not only of individuals but also of society. It seems to be disconcerting that professors in Italy underline the importance to teach ethics and integrity, by giving examples to students, nevertheless this can be explained by the fact that Italy, has a high level of uncertainty avoidance, but low level of power distance (which highlight the ability to approach ethical issues), and is an individualistic country which make it more similar to Ireland and Sweden in term guiding their members towards ethical standards. 5. Discussion and Conclusions The findings allowed us to carry out an in-depth analysis of the teachers’ perceptions of ethics in business education and to know what interventions they consider necessary to be able to promote the development of ethical values in the university and achieve a sustainability education. In order to answer our research questions, a qualitative methodology was used. There were two main reasons for choosing this technique. First, after a detailed review of the literature, no studies were found that analysed in-depth the perceptions of teachers about ethics in education—previous research used surveys and other methodologies of a quantitative nature. Secondly, although teachers have a fundamental role within higher educational institutions in that they can influence student behaviours and be agents of change [71], previous studies have focused more on analysing the perceptions of students on this topic, and in Europe a broad cross-cultural comparison based on Hofstede’s dimension has not been carried out so far. We note that professors’ concepts reveal three categories that are quite similar across all four countries and one category that is different. The professors consider that ethics in business education teaches people to fight against the double standards in organizations that allow them to speak and to behave differently. It is an education that teaches students to be future leaders who act with integrity and according to moral principles, respecting prevailing codes of conduct. Analyzing the data according to Hofstede’s cultural dimension of power distance, uncertainty avoidance, masculinity, and individualism we can argue that some of these dimensions emerged from the data. More specifically, it can be observed that differences and similarities exist for each dimension. First of all, regarding the similarities, as can be noted by these results there is a generalized knowledge in the four countries about what ethics in business education means and its association with morality, the codes of conduct and the integrity. Despite these results, that would appear to indicate that the findings contradict previous research carried out by Hofstede. Namely he found that countries with a high level of power distance, uncertainty avoidance and masculinity may have a lower social and institutional capacity to get involved in ethics and sustainability issues. It is important to highlight that this has some theoretical explanation. The similarity between the four countries, (including Spain and Italy), are related to beliefs and perceptions and not actions. 4.2.2. Not My Role In RQ2, the sub-code 2.2 (Not my role) that was extracted from the interviews of teachers in Spain revealed different conceptions about how ethics in business could be taught in the university. Teachers in Spain stated that in order to raise awareness among students about ethics in business and sustainability issues, there must be a specific course on this subject. Teachers know of the existence of a subject that deals with issues of corporate social responsibility and responsible citizenship. They believe that it is there where it is necessary to delve into key concepts in the field of social responsibility, sustainable development, Human Rights and their connection with economic activity. Almost half of the participants recognized the importance of teaching ethics, but considered that it is not their role to educate students within their subject about ethics, since they believe it is part of a specific and different course. There was a certain resistance on the part of some professors to teaching issues of ethics and values in their class as these are considered issues that do not have to be explained in the university. These professors believe they depend on the social environment and the context where one lives. In this sense, they affirmed that education on ethics and sustainable development cannot be treated by each professor inside the classroom or in the university, since the students have to learn their specific field of study. That is to say, they believe the students of tomorrow have to know how to work in a company, they consider that concentrating on teaching how to act or not to act, what is good or not good is not the teacher’s job. One participant affirmed (SP 11) “My function is to teach my subject, not to form values. That’s what their parents are concerned with, or the sociology subjects”. Sustainability 2019, 11, 608 14 of 19 14 of 19 Others believed that it is a waste of time focusing on these issues rather than devoting time and effort to other activities, such as subject teaching or research. According to the professors, these two issues are the most important in higher education, since this is what determines the position occupied by universities within the national rankings. 4.2.2. Not My Role In this sense, several teachers stressed the need for professors to publish and teach what was established in the curriculum, noting that the current educational system prioritizes having good internationally recognized publications and imparting the established canon, without really being concerned whether or not the student also learns values. Therefore, in Spain a lack of consistency between the professors’ perceptions about what ethics in business education is (and what it includes) and what they think could be done is identified. However, in this sense, the results about what their role is are congruent with Hofstede. 5. Discussion and Conclusions Schepers [72] (based on Hofstede’s) observe, that cultures with high levels of uncertainty avoidance (Italy and Spain) also may know what ethics mean and develop ethic reasoning but not act accordingly. Indeed, differences between Spanish professors’ responses and the other three countries, are related to possible actions and could be determined by the fact that when these cultures have low individualism are less interested in establishing specific rules. This would explain why in Spain, a country which, according to Hosftede [15], has a high level of uncertainty avoidance and at the same time is considered collectivist (unlike in Italy), the professors have knowledge of ethics (like Italy, Sweden and Ireland) but less interest in guide their members toward the appropriate and ethical action (different from Italy Sweden and Ireland). Differences between Spain and Italy can be due to the fact that Italy has a high level of uncertainty avoidance but low level of power distance (which highlight the ability to approach ethical Sustainability 2019, 11, 608 15 of 19 issues) and is an individualistic country which makes it more similar to Ireland and Sweden in term guiding their members towards ethical standards. These results contribute to the literature and also provide confirmation of the findings reported by Zhang, Liang and Sun [73] and by Smith and Hume [43], according to which countries with collectivist cultures are relatively less likely to follow or transmit ethical norms and social values such as honesty and integrity, while cultures with strong individualism seem more committed to bring this debate to the society and to train their citizens. Individualist cultures are more inclined to take corrective measures and create future leaders with strong values of justice, responsibility and respect for human rights and social development [73]. This is why Spanish professors’ have a different perception with respect to Italy, Sweden and Ireland. We found another difference among countries. Many professors, especially in Sweden, believe that ethics in business education also has to do with sustainability. That is, an education that nurtures ethical principles and aims to produce the maximum good for the greatest number of people, which can promote the establishment of standards of conduct, respect and social responsibility to achieve a globalized balanced society. 5. Discussion and Conclusions That said, we could argue that teachers’ perceptions of ethics in business education as an education that fosters sustainability are very much related to the fact that Sweden is among the first European countries concerned with sustainability. In 1996, Sweden passed a law requiring public institutions, such as universities, to contribute to the sustainable development of society [74] (Swedish Environmental Protection Agency). Since the year 2001, all universities in Sweden have to write annual sustainability reports explaining how they have raised awareness among their population of this issue. The universities require their teachers to emphasize the possible effects of business activity for society, to offer continuous knowledge on how human beings and society must manage different environmental problems, thus raising awareness about sustainability policies and ethical practices [75]. So, for teachers in Sweden, ethics in business education is an education that prepares students for the future to become responsible citizens in their practical behaviours. However, the results also showed that in Spain teachers believe that they can do little in this regard since the fundamental values such as honesty, integrity, respect and responsibility are embedded within a society where one is raised and believe that it is not necessary to insist on ethics beyond that specific course. The fact that only in Sweden there is talk of ethics in business education as sustainability education shows how, even though many universities have made efforts for an education towards sustainable development, this has not been fully implemented in all universities and/or disciplines. This is why it is essential that universities commit themselves to develop ethical awareness and achieve social commitment. In this context, as affirmed by Lozano et al. [76] university professors have an essential function, to ensure that ethical and sustainability education is the “Golden Thread” throughout the university system and to educate students to respond to the needs of the present without compromising the capacity of future generations and to strengthen the responsibility for sustainable development within universities. Students continuously learning and emphasizing ethical issues, integrity and sustainability can generate important changes in the ways of valuing the environment. In countries like Spain, it would be necessary to train teachers on the importance of ethics for the development of values among the students and to make them understand that their contribution can be relevant in the creation of future leaders with an understanding of ethics. 6. Recommendations Although an increasing number of studies have stressed the importance of educating towards ethics and sustainability issues, we have observed that most universities tend to respond slowly to this social need. In this sense, teachers are not concerned with creating future leaders with an ethical conscience that implies being sustainable, but rather they focus on teaching the curriculum. Sustainability and ethics are thus still not part of their agenda. Nevertheless, either through public policy or social pressure, some universities (essentially in countries with stronger social commitment) have started to adopt and weave sustainability issues into their curricula. It is evident that universities need to re-evaluate the role that professors play in higher education beyond teaching specific contents. Additionally, attempts must be made to find methods to uniformly implement ethics in business education across the board in business schools. Our findings support the thesis that sustainability should not be left to one specific discipline, but must be fostered and encouraged within each module with the aim of developing future leaders in sustainability. Ideally, universities should enhance the role of teachers in different cultures and create common sources of action. Thus, educating teachers and training them with the aim of implementing new paradigms should be the first step in this journey. Thanks to their role, they could ensure that the ethics and sustainable values of present and future generations will be enhanced. Author Contributions: D.G. carried out the fieldwork, had the idea for the paper and wrote and revised the original and successive drafts. M.d.M.P. supervised the study from inception to design and revised the successive drafts. Funding: This research received no external funding. Funding: This research received no external funding. Acknowledgments: I would like to acknowledge the Mobility centre at URV for the mobility funding provid Conflicts of Interest: The authors declare no conflicts of interest. Conflicts of Interest: The authors declare no conflicts of interest. 5. Discussion and Conclusions In an increasingly globalized world where companies move in a complex environment, and especially for faculties of business administrations that create future entrepreneurs and business managers, teachers can be agents of change and promote ethics. Teachers can help students understand the realities of the world and respect them by participating in the realization of a fairer and more equitable world. A broader collaboration that nurtures the participation of all faculty members could identify ways to support the ethical development of students and achieve sustainability education. Sustainability 2019, 11, 608 16 of 19 16 of 19 Therefore, in this paper we focused on the concept of ethics in business education. The findings show that the concept is not unitary and the differences can be alienated throughout four dimensions: Morality (good or bad), codes of conduct and rules, integrity and honesty and sustainability. After carrying out a comparative the study with professors from four different countries, we found that these differences about the concept are not only due to individual characteristics, but also to cultural contexts. 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https://openalex.org/W2123347589
https://tspace.library.utoronto.ca/bitstream/1807/83606/1/12889_2013_Article_6375.pdf
English
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Incidence rates of sickness absence related to mental disorders: a systematic literature review
BMC public health
2,014
cc-by
10,013
* Correspondence: carolyn.dewa@camh.ca 1Centre for Research on Employment and Workplace Health, Centre for Addiction and Mental Health, 33 Russell Street, Toronto M5S 2S1, Canada 3Department of Psychiatry, University of Toronto, 250 College Street, Toronto M5T 1R8, Canada Full list of author information is available at the end of the article RESEARCH ARTICLE Open Access Dewa et al. BMC Public Health 2014, 14:205 http://www.biomedcentral.com/1471-2458/14/205 Carolyn S Dewa1,3*, Desmond Loong1, Sarah Bonato2 and Hiske Hees4,5 Carolyn S Dewa1,3*, Desmond Loong1, Sarah Bonato2 and Hiske Hees4,5 Abstract Background: Over the past decade, growing attention has been given to the mental health of workers. One way to examine the mental health of workers is to look at the incidence rates of mental illness-related sickness absence. There is a scarcity of literature in which the incidence rates of mental illness-related sickness absence among different countries have been considered together. The purpose of this systematic literature review is to address the question: Are there similarities and differences in the incidence rates of mental disorder-related sickness absence among and within OECD identified Social Democratic, Liberal and Latin American country categories? In this paper, we seek to identify differences and similarities in the literature rather than to explain them. With this review, we lay the groundwork for and point to areas for future research as well as to raise questions regarding reasons for the differences and similarities. Methods: A systematic literature search of the following databases were performed: Medline Current, Medline In-process, PsycINFO, Econlit and Web of Science. The search period covered 2002–2013. The systematic literature search focused on working adults between 18–65 years old who had not retired and who had mental and/or substance abuse disorders. Intervention studies were excluded. The search focused on medically certified sickness absences. Results: A total of 3,818 unique citations were identified. Of these, 10 studies met the inclusion/exclusion criteria; six were from Social Democratic countries. Their quality ranged from good to excellent. There was variation in the incidence rates reported by the studies from the Social Democratic, Liberal and Latin American countries in this review. Conclusions: The results of this systematic review suggest that this is an emerging area of inquiry that needs to continue to grow. Priority areas to support growth include cross jurisdictional collaboration and development of a typology characterizing the benefit generosity and work integration policies of sickness absence schemes. Finally, the literature should be updated to reflect changes in sickness absence benefit schemes over time. Keywords: Sickness absence, Mental disorders, Incidence Similar endorsements have been made in Australia [2], the United States [3] and Canada [4]. Background Global focus on worker mental health © 2014 Dewa et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Global focus on worker mental health The impetus for greater attention to worker mental health has also been spurred by a growing awareness of the impact of mental disorders on the workplace. Indeed, an expanding body of literature indicates that mental ill- ness takes its workplace toll in the form of work absences and decreased productivity (e.g., [5-7]). Because of the length of their absences [8-12] and their rates of recur- rence [10,13-15], sickness absences related to mental ill- ness are one of the most costly types of sickness absences. Over the past decade, increasing attention has turned to the mental health of workers and its effects on the workplace. For example, European Ministers of Health have advocated that employers include mental health programs as part of occupational health and safety [1]. Dewa et al. BMC Public Health 2014, 14:205 http://www.biomedcentral.com/1471-2458/14/205 Page 2 of 14 Dewa et al. BMC Public Health 2014, 14:205 http://www.biomedcentral.com/1471-2458/14/205 Thus, there is an interest in promoting worker mental health. work integration policies. Countries in this category are the United Kingdom, the United States and Canada. Corporatist countries are characterized being relatively moderate – they were not as generous as the Social Democratic countries but not as conservative as the Liberal countries with respect to benefits and work inte- gration policies. These countries include Austria, Belgium, and France. Importance of incidence rates of mental illness-related sickness absence One way to examine the mental health of workers is to look at the incidence rates of mental illness-related sick- ness absence. That is, the better the mental health status of workers, the lower the incidence rates of mental illness- related sickness absence. But, because there are differences in the way various countries approach the mental health of their working populations [16], it could be useful to consider incidence rates by jurisdictions. Jurisdictions with higher rates might be places where further exploration could take place to identify the types of approaches to avoid. In contrast, those with lower rates may be places where further studies could be conducted to learn about effective practices. Because they are emerging welfare states, the Latin American countries generally are treated as a unique cluster [21,22]. Gap in the literature There is a scarcity of literature in which the incidence rates of mental illness-related sickness absence among different countries have been considered together. Part of this gap in the literature may reflect the challenge intro- duced by the heterogeneity with which countries approach sickness absence. The OECD classification system offers a way to describe systemic similarities and differences among countries. In turn, this information can be used as a first step toward studying effective systemic practices. The purpose of this systematic literature review is to take this first step. We address the question: Are there simi- larities and differences in the incidence rates of mental disorder-related sickness absence among and within OECD categories? In this paper, we seek to identify dif- ferences and similarities rather than to explain them. With this review, we lay the groundwork for and point to areas for future research. In doing so, we also raise questions regarding reasons for the differences and similarities. Considering country variations One of the challenges of examining the rates of sickness absence incidence among countries is related to the het- erogeneity of country system factors that affect workers. Examples of these system factors include country work integration policies such as employer sickness absence obligations, employment rehabilitation programs and work incentives. Another group of factors is related to country compensation policies such as the population covered, dis- ability benefit eligibility and criteria. Recognizing the heterogeneity among countries, the Organization for Economic Co-operation and Develop- ment (OECD) developed a classification system to be used to understand the similarity among countries with respect to their work integration and worker compensation pol- icies. The classification builds on work from the political economy literature that was developed to compare social policies across diverse jurisdictions (e.g., [17-19]). The classification focuses on the types of public policies (e.g., work integration schemes) that would affect work-related outcomes (e.g., employment rates) [20]. The classification system facilitates discussion without becoming entrenched in the complexities of individual systems [19]. Methods For the purposes of this systematic review, five electronic databases were searched. They included: (1) Medline Current (an index of journal articles in biomedical re- search and clinical sciences), (2) Medline In-process (an index of journal articles in biomedical research and clinical sciences that are awaiting indexing into Medline Current), (3) PsycINFO (an index of journal articles, books, chapters, and dissertations in psychology, social sciences, behavioral sciences, and health sciences), (4) Econlit (an index of journal articles, books, working papers and dissertations in Economics) and (5) Web of Science (an index of journal articles, editorially selected books and conference proceed- ings in life sciences and biomedical research). A search strategy was developed and executed for each database with the help of a professional health science librarian (SB). Medline Current, Medline In-process and PsycINFO were searched using the OVID platform. Econlit and Web of Science were searched using the ProQuest and Thomson Reuters search interface, respectively. The search was completed between February 2013 and The OECD [16] examined the disability policies of 15 OECD countries. Disability policies were evaluated based on the generosity of their compensation and the extent of their work integration policies. The OECD [16] catego- rized countries into three main groups: (1) Social Demo- cratic, (2) Liberal and (3) Corporatist. Social Democratic countries were characterized as being relatively the most generous and having the most exten- sive work integration policies. These countries include Finland, Denmark, the Netherlands, and Norway. In con- trast, the Liberal countries were characterized as being relatively the least generous and with the least extensive Page 3 of 14 Page 3 of 14 Dewa et al. BMC Public Health 2014, 14:205 http://www.biomedcentral.com/1471-2458/14/205 Dewa et al. BMC Public Health 2014, 14:205 http://www.biomedcentral.com/1471-2458/14/205 March 2013 and was limited to English language jour- nals published between 2002 and 2013. The complete search strategy used for each database can be found in Appendix 1. Eligibility criteria h 2. The data source is well described. 3. The study sample is representative of the target population. The systematic literature search focused on working adults between 18–65 years old who had not retired and who had mental and/or substance abuse disorders. Inter- vention studies were excluded. The search focused on medically certified sickness absences that included sick leave, short-term disability leave, long-term disability leave or sickness absence. For the purposes of this review, sick- ness absence was defined as a work absence requiring a medical certification. These income replacement or disabil- ity benefits (i.e., short-term or long-term work disability) could be either publicly or privately sponsored. In terms of cause of disability, we focused our search on “no cause” disability leaves. That is, the worker did not need to prove that the disability was caused by work. 4. Mental disorders are included and reported. 5. The system of diagnosis/classification is described. 6. The criteria for sickness absence is reported (i.e., pre-sickness absence days to qualify for sickness absence). 7. The denominator is clearly reported. 8. The numerator is clearly reported. 9. Uncertainty of estimates is reported. 10. The stated research objective is met. One point was awarded for each met criterion for a maximum score of 10. Scores between 1 and 4 were regarded as ‘fair/weak’ quality and scores between 5 and 8 were ‘good’. Scores of 9 and 10 were regarded as ‘excellent’ quality. All search results were screened by title, followed by abstract and full-text review for relevant articles. The reference lists of the articles that made it to the full-text review stage were also hand-searched. The screening process was completed independently by two reviewers, CSD and DL, using the following eligibility criteria: Quality assessment Articles that passed the three-stage screening process were assessed for quality using the following criteria: 1. The study population is well described. Description of inclusion and exclusion The electronic literature search resulted in the identifi- cation of 3,818 unique citations (Figure 1). From these, 24 entries that were commentaries were excluded. Based on the title review, 3,524 citations were excluded. Based on the abstract review, another 160 citations were ex- cluded; this left 110 articles for full-text review. After the full-text review, 10 articles remained. Reasons for article exclusion included: (1) did not have information about medically certified sickness absence related to mental disorders (n = 33), (2) were based on select populations (n = 13), (3) used pre-2000 data (n = 3), (4) did not report incidence rates from medically certified sickness absence related to mental disorders (n = 49) and (5) the study population did not consist of adults eligible for sickness absence (e.g., the study population included people who were not employed) (n = 2). 1. The study reported on medically certified sickness absences due to mental illness and/or addiction problems. 2. The study reported the incidence of medically certified sickness absences due to mental illness and/ or addiction problems. 3. The study analyzed data collected in the year 2000 or later. 4. The study sample was not from a select population (i.e. clinical trial, clinical populations). The year 2002 was used as the starting point for inclu- sion because the 1990s were a period of global change in employment policies [23]. Thus, we focused on the last decade because during this time, there were relatively fewer policy changes related to workers. Because pre-2000 data were collected under systems that existed before the policy changes of the 1990s, studies that used pre- 2000 data were also excluded. The 10 included studies were conducted in countries that clustered into three country types: (1) Social Demo- cratic (n = 6), (2) Liberal (n = 1) and (3) Latin America (n = 3). The Social Democratic category included studies from Norway, Finland and the Netherlands. The study in the Liberal category was from Canada. Finally, all the Latin American studies were from Brazil. Discussions were held in instances where there were disagreements until consensus was reached. The inter- rater reliability which corrected for chance agreement was calculated for CSD and DL to be 0.93. Review arti- cles and commentaries were excluded when possible during the screening process. Consensus regarding the inclusion of the final articles was reached among CSD, DL and HH. Quality assessment h l The quality assessment rated three of the 10 studies as excellent and the remaining seven as good (Additional file 1: Table S1, Additional file 2). The identified Dewa et al. BMC Public Health 2014, 14:205 http://www.biomedcentral.com/1471-2458/14/205 Page 4 of 14 Abstracts retrieved (n = 270) Unique citations identified through database and hand search (n = 3818) Commentaries Excluded (n = 24) Total studies screened (n = 3794) Excluded based on title (n = 3524) Excluded based on abstract (n = 160) Full-text articles retrieved (n = 110) Excluded based on full-text (n=100) Studies assessed for quality (n = 10) Excluded based on quality (n = 0) Excellent (n =3) Good (n = 7) Fair/weak (n = 0) Identification Screening Studies included (n = 10) Quality assessment Figure 1 Flowchart of literature search results and inclusions/exclusions. Unique citations identified through database and hand search (n = 3818) Excluded based on abstract (n = 160) Abstracts retrieved (n = 270) Full-text articles retrieved (n = 110) Excluded based on full-text (n=100) Studies assessed for quality (n = 10) Quality assessment Studies included (n = 10) Excluded based on quality (n = 0) Figure 1 Flowchart of literature search results and inclusions/exclusions. data were either from their sickness absence insurer, work- place or healthcare provider. limitations of these studies include: the study sample was not representative of the target population (8 studies), uncertainty of the incidence rate estimate was not re- ported (4 studies) and the stated research objective was not met (3 studies). With the exception of one of the studies, which used the International Classification of Primary Care (ICPC), the studies used the International Classification of Diseases 10th edition (ICD-10). However, Virtanen et al.’s [24] study also reported the ICD-10 equivalents to the ICPC. Overview of the studies There was variability among the included studies with respect to the primary diagnoses of the sickness absence cases that were included in the analyses. However, there were also similarities; all studies included absences re- lated to depressive, anxiety and stress-related disorders. Table 1 contains the descriptions of the included studies. All of the included studies used administrative data from either an insurer or healthcare group practice. As a result, all of the studies represented identifiable complete popula- tions of people at risk of having a sickness absence and Table 1 Description of individual studies Author(s) Country Study population Data source Year(s) of data Diagnostic classification system used Absence days to qualify for sickness absence benefit Social democratic Virtanen et al. [24] FI Participants from the Finnish Public Sector Study covering employees in 10 towns and 21 public hospitals in Finland; who were not on long-term sick leave or disability pension at the time of the survey; and who were employed for at least 6 months during the study between 1997-2005 Administrative data from the National Health Insurance, employer records and national health register records and the Finnish Public Sector Study 1997-2005 International Classification of Diseases,10th edition (ICD-10) Long-term sickness absence = sickness absence of ≥90 days Roelen et al. [28] NL Employees of firms who were clients of an occupational health services provider from 2001-2007 Administrative sickness absence data from ArboNed 2001-2007 ICD-10 Sickness absence: absence of ≥28 sick days requiring a medical certificate from an occupational physician Koopmans et al. [26] NL Dutch Post and Telecommunication employees from 2001-2007 Administrative sickness absence data from ArboNed 2001-2007 ICD-10 Sick leaves of > 3 weeks require a medical certificate from an occupational physician Roelen et al. [27] NL Dutch Post and Telecommunication employees from 2001-2007 Administrative sickness absence data from ArboNed 2001-2007 ICD-10 Sick leaves of > 3 weeks require a medical certificate from an occupational physician Roelen et al. [29] NL Employees covered in a sickness absence benefit program from 2001-2010 Administrative sickness absence data 2001-2010 ICD-10 Sickness absence = absence of > 3 weeks requiring a medical certificate from an occupational physician Hensing et al. Overview of the studies [25] NO People who were 16–66 years in 1994, 1996, 1998 and 2000 who were compulsory members of the Sickness Benefit Scheme Administrative data from the Norwegian National Sickness Administration 1994, 1996, 1998, 2000 International Classification of Primary Care (ICPC) Medical certification is required for sick leave > 4 days Liberal Dewa et al. [11] CA (Ontario) Employees from a large resource sector company from 2003-2006 Administrative sickness absence data 2003-2006 ICD-10 Sickness absence = sickness absence of > 5 continuous work days requiring a medical certificate Latin America Barbosa-Branco et al. [32] BR All employees registered in private sector jobs in 2008 Administrative data from health service provider 2008 ICD-10 Sickness absence = ≥15 consecutive days absent requiring a medical certificate Reis et al. [30] BR Workers from a university hospital who were employed from 2000-2007 Administrative data from health service provider 2000-2007 ICD-10 Not described Barbosa-Branco et al. [31] BR All employees registered in private sector jobs in 2008 National Benefits System and National Social Information Database 2008 ICD-10 Sickness absence = ≥15 consecutive days absent requiring a medical certificate Table 1 Description of individual studies Dewa et al. BMC Public Health 2014, 14:205 http://www.biomedcentral.com/1471-2458/14/205 Dewa et al. BMC Public Health 2014, 14:205 http://www.biomedcentral.com/1471-2458/14/205 Page 6 of 14 Thus, there appeared to be consistency among the stud- ies with regard to a core set of mental disorders. 31.5/1,000 among women. Using a similar dataset, Roelen et al. [27] observed an incidence density of 27.7/1,000 worker-years for mental and behavioral disorders. There was variability in the number of absence days needed to qualify for sickness absence benefits. The number of qualifying days used for the studies ranged from 3 days to 90 days. It should be noted, that while the days required to qualify for benefits in Finland is 9 days, due to limited availability of the data, the Finnish study [24] examined the incidence of sickness absences that were >90 days. Based on data from an occupational health service provider, Roelen and colleagues [28] calculated the 12- month incidence rates for sickness absences related to CMD from 2001–2007. During that time period, it ap- peared that 12-month incidence rates related to CMD ranged from a high of 27/1,000 employees in 2003 and 2004 to 20/1,000 employees in 2007. In a separate study, Roelen et al. Numerators: measures of sickness absence rates In general, the studies used two types of incidence mea- sures. The first type of measure reported the incidence of workers with sickness absence. That is, either only the first episode or the worker who had an episode was counted. Liberal countries Based on data from one organization with a province-wide employee base, Dewa et al. [11] reported a rate of 21/1,000 worker-years for sickness absences related to mental disorders. When stratified by sex, the incidence rate for men was 17/1,000 worker-years and 36/1,000 worker- years for women. In contrast, the second type of measure reported the incidence of sickness absences. It counted the number of episodes occurring in a defined time period. Thus, if a person had more than one sickness absence during the time period of interest, s/he was counted as many times as there was a discrete sickness absence. Social democratic countries Using year 2000 administrative data from the national sickness administration, Hensing et al. [25] observed that the age-adjusted cumulative incidence of men with sickness absence ranged from 0.9/1,000 workers for psychosis-related absences to 13/1,000 for depression- related absences (Table 2). In contrast, for women, the cumulative incidence rates ranged from 1/1,000 for psychosis-related absences to 30/1,000 for depression- related absences. Overview of the studies [29] estimated that 12-month inci- dence rates related to mental and behavioral disorders ranged from 21.1/1,000 employees in 2001 to 17.7/1,000 employees in 2010. Cohorts: measures of sickness absence rates All of the Latin American studies in this review were from Brazil. Using 2000–2007 data from one university hospital, Reis and colleagues [30] reported an incidence density of 0.33/100 worker-months or approximately 39.6/1,000 worker-years. Based on 2008 national data of private sector companies, Barbosa-Branco et al. [31,32] observed an incidence rate of 45.1/10,000 or 4.5/1,000 workers. For specific mental disorders, incidence rates varied by primary disorder from 15.4/10,000 workers (1.5/1,000) for sickness absences related to depression and 2.8/10,000 (0.3/1,000) workers for reaction to severe stress. Among the included studies, two types of cohorts were used. One was a 1-year cohort. It included people who were at risk of a sickness absence during a 12-month period. The other was a dynamic cohort for which mul- tiple years of data were used such that the denominator was calculated with the number of workers at risk of sickness absence in terms of either worker-years or worker-months. Discussion The quality ratings of included studies ranged from good to excellent. There was variation in the incidence rates reported by the studies from the Social Democratic, Liberal and Latin American countries in this review. For the studies conducted in the Social Democratic countries, the incidence rates of sickness absences related to mental disorders ranged between 19 and 28/1,000 workers or 1,000 worker-years. In contrast, the incidence rate re- ported by the study from the Liberal country was 21/1,000 workers. In addition, there was variation in the rates reported by the studies from the Latin America and ranged from 2 - 40/1,000 workers. Virtanen et al. [24] used linked data from 1997–2005 and reported the cumulative incidence of long-term sickness absence that ranged from 2/1,000 for absences related to schizophrenia and schizotypal and delusional disorders to 19/1,000 for absences related to depression. Virtanen et al. [24] used linked data from 1997–2005 and reported the cumulative incidence of long-term sickness absence that ranged from 2/1,000 for absences related to schizophrenia and schizotypal and delusional disorders to 19/1,000 for absences related to depression. Using data from 2001–2007 from one organization with a nation-wide employee base, Koopmans et al. [26] reported an incidence density of 21.8/1,000 worker-years for common mental disorders (CMD) among men and Using data from 2001–2007 from one organization with a nation-wide employee base, Koopmans et al. [26] reported an incidence density of 21.8/1,000 worker-years for common mental disorders (CMD) among men and The differences reported raise a number of questions. Are they a reflection of the differences in system struc- tures? For instance, among countries in which there is relatively more government involvement (i.e., Social Table 2 Results of individual studies Author(s) Country Mental disorders Measure Denominator Numerator Reported incidence Social democratic Virtanen et al. Discussion [24] FI Mental and behavioral disorders including: depressive disorders, mania and bipolar affective disorder, anxiety disorders (phobias, panic disorder, obsessive compulsive disorder and generalized anxiety disorder), reaction to severe stress and adjustment disorders, personality disorder, schizophrenia, schizotypal and delusional disorders and mental and behavioral disorders due to psychoactive substance use (ICD-10 Chapter F) Study participants followed for an average of 6.3 years n = 141,917 Depressive disorder = 2,679 Cumulative incidence of disability benefit receipt: Depressive disorders = 1.9% Cumulative incidence Mania and bipolar affective disorder =150 Mania and bipolar affective disorder = 0.1% Anxiety disorder = 314 Anxiety disorder = 0.2% Reaction to severe stress and adjustment disorders = 275 Reaction to severe stress and adjustment disorders = 0.2% Adult personality and behaviour disorders = 54 Adult personality and behaviour disorders = 0.04% Schizophrenia and schizotypal and delusional disorder = 283 Schizophrenia and schizotypal and delusional disorder = 0.2% Mental and behavioural disorders owing to psychoactive substance use = 62 Mental and behavioural disorders owing to psychoactive substance use = 0.04% Roelen et al. [28] NL Common mental disorders (CMD) included distress (ICD-10 R45), other stress-related disorders (ICD-10 F43), depressive disorders (ICD-10 F32) and anxiety disorders (ICD-10 F40 and F41) Total Employees: Number of episodes: 12-month incidence of sickness absence for CMD by year/100 employees (95% CI): Dynamic cohort study 12-month incidence of total certified sickness absence = number of medically certified sickness absence episodes/number of employees covered 2001 = 956,623 2001 = 21,140 2001 = 2.2 (2.2, 2.2) 2002 = 962,235 2002 = 22,803 2002 = 2.4 (2.3, 2.4) 2003 = 937,030 2003 = 24,917 2003 = 2.7 (2.6, 2.7) 2004 = 1,037,149 2004 = 27,533 2004 = 2.7 (2.6, 2.7) 2005 = 961,890 2005 = 22,682 2005 = 2.4 (2.3, 2.4) 2006 = 970,390 2006 = 20,013 2006 = 2.1 (2, 2.1) 2007 = 921,741 2007 = 18,513 2007 = 2 (2, 2) Koopmans et al. Koopmans et al. [26] NL Common mental disorders (CMD) from medical certification: stress-related (distress and adjustment disorders) (ICD-10 R45, F43) and psychiatric (mild to moderate depressive and anxiety disorders) (ICD10 F32.0, F32.1, F40.0, F40.1, F40.2, F41.0, F41.1, F41.2, F41.3) Virtanen et al. [24] FI Mental and behavioral disorders including: depressive disorders, mania and bipolar affective disorder, anxiety disorders (phobias, panic disorder, obsessive compulsive disorder and generalized anxiety disorder), reaction to severe stress and adjustment disorders, personality disorder, schizophrenia, schizotypal and delusional disorders and mental and behavioral disorders due to psychoactive substance use (ICD-10 Chapter F) Discussion [26] NL Common mental disorders (CMD) from medical certification: stress-related (distress and adjustment disorders) (ICD-10 R45, F43) and psychiatric (mild to moderate depressive and anxiety disorders) (ICD10 F32.0, F32.1, F40.0, F40.1, F40.2, F41.0, F41.1, F41.2, F41.3) Dynamic cohort study Number of employees = 137,172 From 2001–2007, CMD densities/1,000 worker-years (95% CI): Index episode = one episode during research period Worker-years = 363,461 Men: Men: Incidence density of index episodes = # of employees with a first episode of sickness absence due to CMDs between 2001 and Stress = 4,704 Stress = 19.7 (19.1, 20.2) Psychiatric = 723 (2.8, 3.2) Psychiatric = 3.0 (2.8, 3.2) Dewa et al. BMC Public Health 2014, 14:205 Page 7 of 14 http://www.biomedcentral.com/1471-2458/14/205 Denominator Numerator Reported incidence Table 2 Results of individual studies (Continued) Dewa et al. BMC Public Health 2014, 14:205 http://www.biomedcentral.com/1471-2458/14/205 ( ) 2007/worker-years of the total population at risk Total CMD = 34,603 Total CMD = 21.8 (21.2, 22.4) Women: Women: Stress = 3,298 Stress = 27.8 (26.8, 28.7) Psychiatric = 612 Psychiatric = 5.2 (4.7, 5.6) Total CMD = 18,026 Total CMD = 31.5 (30.5, 32.5) Roelen et al. [27] NL Mental and behavioral disorders from medical certification (ICD-10 F00-F99) Dynamic cohort Number of employees = 137,172 Mental and behavioural disorders = 7,197 From 2001–2007, incidence density/1,000 worker-years Mental and behavioural disorders (95% CI): Incidence density = incident episodes of sickness absence/worker-years at risk Worker-years = 363,461 Incidence density = 27.7 (27.0, 28.4) Roelen et al. [29] NL Mental and behavioral disorders from medical certification: emotional disturbance (ICD-10 R45), depressive disorders (ICD-10 F32), anxiety disorders (ICD-10 F40-41) and stress-related disorders (ICD-10 F43) Incidence/year 2001 = 956,623 Not described Incidence of sickness absence by year/1,000 employees (95% CI): 2001 = 21.1 (20.8, 21.4) 2002 = 962,235 2002 = 22.5 (22.3, 22.8) 2003 = 937,030 2003 = 25.3 (25.0, 25.6) 2004 = 1,037,149 2004 = 25.5 (25.2, 25.8) 2005 = 961,890 2005 = 22.9 (22.6, 23.2) 2006 = 970,390 2006 = 20.0 (19.7, 20.3) 2007 = 913,266 2007 = 20.1 (19.8, 20.4) 2008 = 924,300 2008 = 19.4 (19.1, 19.7) 2009 = 1,033,072 2009 = 16.9 (16.6, 17.2) 2010 = 1,006,861 2010 = 17.7 (17.4, 18.0) Hensing et al. Discussion [25] NO Included: Psychoses (ICD-10 F20-31, F35-39), anxiety (ICD-10 F40-F43), neurotic conditions (ICD-10 F44-48, F99), depression (ICD-10 F32-F34), personality disorders (ICD-10 F60-69), alcohol/drug abuse (ICD-10 F10-F19) Cumulative incidence = # of individuals with ≥1 sickness absence episode initiated in each year studied/# of individuals entitled to sickness benefits during that year Denominator: Not described Age-adjusted cumulative incidence of sickness absence in 2000 (95% CI): Men: n = 1,219,338 Women: n = 1,063,423 Men: Psychoses = 0.09% (0.09, 0.09) Anxiety disorders = 0.20% (0.19, 0.20) Neurotic conditions = 0.54% (0.54, 0.54) Depression = 1.31% (1.29, 1.33) Personality disorders = 0.01% (0.01, 0.01) Excluded: Dementia, organic psychoses, mental retardation and child and adolescent psychiatry ewa et al. BMC Public Health 2014, 14:205 Page 8 of tp://www.biomedcentral.com/1471-2458/14/205 Total CMD = 34,603 Total CMD = 21.8 (21.2, 22.4) 2007/worker-years of the total population at risk Roelen et al. [27] NL Mental and behavioral disorders from medical certification (ICD-10 F00-F99) Roelen et al. [29] NL Mental and behavioral disorders from medical certification: emotional disturbance (ICD-10 R45), depressive disorders (ICD-10 F32), anxiety disorders (ICD-10 F40-41) and stress-related disorders (ICD-10 F43) Hensing et al. [25] NO Included: Psychoses (ICD-10 F20-31, F35-39), anxiety (ICD-10 F40-F43), neurotic conditions (ICD-10 F44-48, F99), depression (ICD-10 F32-F34), personality disorders (ICD-10 F60-69), alcohol/drug abuse (ICD-10 F10-F19) Excluded: Dementia, organic psychoses, mental retardation and child and adolescent psychiatry Table 2 Results of individual studies (Continued) Dewa et al. BMC Public Health 2014, 14:205 http://www.biomedcentral.com/1471-2458/14/205 Dewa et al. BMC Public Health 2014, 14:205 http://www.biomedcentral.com/1471-2458/14/205 Alcohol and drug disorders = 0.09% (0.09, 0.09) Women: Psychoses = 0.10% (0.10, 0.10) Anxiety disorders = 0.35% (0.34, 0.35) Neurotic conditions = 1.11% (1.09, 1.13) Depression = 3.01% (3.00, 3.04) Personality disorders = 0.01% (0.01, 0.02) Alcohol and drug disorders = 0.02% (0.02, 0.03) wa et al. BMC Public Health 2014, 14:205 p://www.biomedcentral.com/1471-2458/14/205 Alcohol and drug disorders = 0.02% (0.02, 0.03) Liberal Dewa et al. Discussion [11] CA (Ontario) Schizophrenia, mood disorders, stress-related disorders and mental and behavioral disorders due to psychoactive substance use (ICD-10 F00-F99 and Z502, Z503, Z561-566, Z630-Z639, Z729, Z733, Z738, Z864 and Z915) Incidence = Number of sickness absence episodes/worker-years at risk n = 12,407 employees Total = 698 Incidence of disability/100 worker-years (95% CI): n = 33,028.79 worker-years Men = 449 Mental disorders: Women = 249 Total = 2.1 (2.0, 2.3) Men = 1.7 (1.6, 1.9) Women = 3.6 (3.2, 4.1) Latin America Barbosa- Branco et al. [32] BR Disorders in the ICD-10 Mental and Behavioral Disorders Chapter 5 Case = a newly granted sickness absence claim n = 32,590,239 Not described Age and sex standardized rates of sickness absences for mental and behavioral disorders/10,000 workers = 45.1 Cases that were within 60 days of each other for the same diagnosis were considered to constitute one case Incidence = number of sickness benefit claims due to mental disorders/average number of workers at risk Reis et al. [30] BR Mental and behavioral disorders (ICD-10 F00-F99) Incidence density = number of new sickness absence/total worker-time at risk for the first sickness absence n = 1,542 workers n = 324 Mental and behavioral disorders: Incidence density/100 worker-months = 0.33 Barbosa- Branco et al. [31] BR Disorders in the ICD-10 Mental and Behavioral Disorders Chapter 5 Case = a newly granted sickness absence claim n = 32,590,239 Prevalence of sickness absence claims/10,000 workers: Cases that were within 60 days of each other for the same diagnosis were considered to constitute one case Any mental disorder = 147,105 Any mental disorder = 45.1 Depressive episode = 50,289 Depressive episode = 15.4 Table 2 Results of individual studies (Continued) Dewa et al. Discussion BMC Public Health 2014, 14:205 http://www.biomedcentral.com/1471-2458/14/205 esults of individual studies (Continued) Other anxiety disorder = 19,508 Other anxiety disorder = 6.0 Incidence = number of sickness benefit claims due to mental disorders/average number of workers at risk Recurrent depressive episode = 14,524 Recurrent depressive episode = 4.5 Multiple drug use = 11,224 Multiple drug use = 3.4 Bipolar affective disorders = 9,504 Bipolar affective disorders = 2.9 Reaction to severe stress = 9,008 Reaction to severe stress = 2.8 Use of alcohol = 8,545 Use of alcohol = 2.6 Schizophrenia = 4,616 Schizophrenia = 1.4 Use of cocaine = 3,468 Use of cocaine = 1.1 Unspecified nonorganic psychosis = 2,950 Unspecified nonorganic psychosis = 0.91 Phobic anxiety disorders = 2,023 Phobic anxiety disorders = 0.6 Unspecified nonorganic psychosis = 1,794 Unspecified nonorganic psychosis = 0.6 ewa et al. BMC Public Health 2014, 14:205 ttp://www.biomedcentral.com/1471-2458/14/205 Incidence = number of sickness benefit claims due to mental disorders/average number of workers at risk Page 11 of 14 Page 11 of 14 Dewa et al. BMC Public Health 2014, 14:205 http://www.biomedcentral.com/1471-2458/14/205 Democratic), the range in the incidence rates is rela- tively small. The Liberal country group consisted of one country. Yet, the Liberal countries have the least gov- ernment involvement in benefit and work integration schemes. If that is the case, sickness absence rates are based on definitions of work disability and benefit quali- fication criteria that depend on private schemes that could be as varied as they are numerous. Such variation in schemes could in turn impact the variation in rates. To further pursue this line of inquiry and to understand variations within country types, it will be useful if a typ- ology similar to the OECD’s [16] were developed especially in countries where there is less government involvement. In addition to describing countries, such a typology could characterize sickness absence benefit schemes. should be noted that the majority of sickness absences related to mental disorders are attributable to depres- sion, anxiety and stress-related disorders [34,35]. This suggests that inclusion of these disorders would capture a large proportion of the sickness absences related to mental disorders. A limitation of the studies was the variation in the years they captured. Although all studies used post-2000 data, there could have been changes within systems that could have affected incidence rates. Strengths and limitations of the search strategy g gy While five databases were searched, it is possible that an article could have been missed if it did not appear in any of the databases. However, that possibility is small given the broad scope of each of the databases. Another limi- tation is that the search was limited to English-language journals. Thus, it did not identify research that was not published in English. However, it should be noted that despite the language constraint, the included studies came from Europe, North America and Latin America. This suggests that at least some of the researchers from countries in which English is not a first language are publishing in English-language journals. Strengths and limitations related to interpreting the literature There were a number of strengths of the current body of literature reviewed. First, all of the studies represented identifiable complete populations of people at risk of having a sickness absence. At the same time, it is im- portant to note that there was variation in the breadth of the populations covered from entire countries to sin- gle organizations. Thus, it will be important for future work to examine whether the rates reported hold for larger populations and for different populations within the same country. Another strength was that all of the studies used stan- dardized diagnostic classification systems. All included depressive and anxiety disorders as well as stress-related disorders. However, there was variability in the other type of disorders considered. This could have made some rates higher than others. At the same time, it Discussion For example, in the Netherlands, extensive legislative changes occurred be- tween 2000 and 2013 which affected rates [23,36]. In fact, the changes are reflected in the rates reported by Roelen et al. [29]. Similarly, changes could have been implemented in other countries such that rates could vary depending on year. Another question that arises is why there was such variation among the Latin America studies given they were all from Brazil? Was it because there are significant differences in the mental health among workers? Or are there significant differences in the benefit schemes (i.e., qualification criteria)? Here, a typology characterizing the benefit generosity (i.e., the population covered, dis- ability benefit eligibility and criteria) and work integration schemes (i.e., employer obligations for sickness absence, employment rehabilitation programs and work incen- tives) of individual plans could assist in answering these questions. Another limitation was variability in the absence days cut-offs used. A low number of qualifying days could have made the rates higher compared to benefit schemes with a greater number of qualifying days. At the same time, all schemes required medical certification and an assessment of work ability. To the extent that symptoms manifest over several weeks, it may be that workers seek a sickness absence at similar phases of their mental dis- order. If the acute phase at which they apply for a sick- ness absence leave is similar, the actual variation may be minimized. On the other hand, if there is variation in when workers seek medical certificates, incidence rates may be higher in studies where medical certification takes place in an earlier phase of sickness absence. However, the results of Roelen et al.’s [29] study did not seem to support the latter hypothesis. But, this suggests another area for future inquiry – when do workers apply for sickness absence? The results also indicate that among the studies that report incidence rates by sex, there is a trend toward a higher incidence rate among women than men. This corroborates findings from Hensing and Wahlstrom’s [33] systematic review of risk factors associated with sickness absence. They found evidence suggesting that women have a higher risk of having an absence related to mental disorders. Future directions Both the causes of and the effective return to work strat- egies for sickness absences related to mental disorders are multifactorial and complex [37,38] and extend be- yond the scope of this paper. Indeed, the results of this Dewa et al. BMC Public Health 2014, 14:205 http://www.biomedcentral.com/1471-2458/14/205 Page 12 of 14 (substance$ adj3 depend$).mp. OR (drug$ adj3 abus$). mp. OR (drug$ adj3 depend$).mp. OR addiction$.mp.] AND [exp Absenteeism/OR exp Sick Leave/OR exp Return to Work/OR exp Personnel Turnover/OR Social Welfare/OR Public Assistance/OR exp Insurance Disability/ OR exp Insurance Benefits/OR exp Salaries/OR exp Fringe Benefits/OR exp Social Security/OR exp Retirement/OR (sick$ adj3 day$).mp. OR (illness$ adj3 leave$).mp. OR (disabilit$ adj3 leave$).mp. OR (short term disabilit$). mp. OR (long term disabilit$).mp. OR (work$ adj3 ab- sence$).mp. OR (return$ to work$).mp. OR (work$ adj3 turnover$).mp. OR (employ$ adj3 turnover$).mp. OR (disabilit$ benefit$).mp. OR (employ$ benefit$).mp. OR (work$ benefit$).mp. OR (sick$ benefit$).mp. OR (inca- pacit$ benefit$).mp. OR (social$ welfar$).mp. OR (pub- lic$ assistanc$).mp. OR (insurance$ disabilit$).mp. OR (insurance$ benefit$).mp. OR (old$ age$ assistanc$). mp. OR (social$ securit$).mp. OR retire$.mp.] AND [sn.fs. OR ep.fs. OR preval$.mp. OR incid$.mp. OR stat- istic$.mp. OR exp Epidemiologic Methods/]. (substance$ adj3 depend$).mp. OR (drug$ adj3 abus$). mp. OR (drug$ adj3 depend$).mp. OR addiction$.mp.] AND [exp Absenteeism/OR exp Sick Leave/OR exp Return to Work/OR exp Personnel Turnover/OR Social Welfare/OR Public Assistance/OR exp Insurance Disability/ OR exp Insurance Benefits/OR exp Salaries/OR exp Fringe Benefits/OR exp Social Security/OR exp Retirement/OR (sick$ adj3 day$).mp. OR (illness$ adj3 leave$).mp. OR (disabilit$ adj3 leave$).mp. OR (short term disabilit$). mp. OR (long term disabilit$).mp. OR (work$ adj3 ab- sence$).mp. OR (return$ to work$).mp. OR (work$ adj3 turnover$).mp. OR (employ$ adj3 turnover$).mp. OR (disabilit$ benefit$).mp. OR (employ$ benefit$).mp. OR (work$ benefit$).mp. OR (sick$ benefit$).mp. OR (inca- pacit$ benefit$).mp. OR (social$ welfar$).mp. OR (pub- lic$ assistanc$).mp. OR (insurance$ disabilit$).mp. OR (insurance$ benefit$).mp. OR (old$ age$ assistanc$). mp. OR (social$ securit$).mp. OR retire$.mp.] AND [sn.fs. OR ep.fs. OR preval$.mp. OR incid$.mp. OR stat- istic$.mp. OR exp Epidemiologic Methods/]. paper raise more questions than they answer. Why are there differences among country types? Do these differ- ences truly exist? Or, are they anomalies of the data used? What role does the sickness absence benefit structure play in the incidence rates? What is the appropriate benchmark for sickness absence related to mental disorders? Conclusions The results of this systematic review suggest that this is an emerging area of inquiry that needs to continue to grow. This review identified only 10 studies that were published in the last 10 years; four of them came from a single country. As this literature continues to expand and if countries are to learn from one another, cross juris- dictional collaboration should be pursued and supported. Perhaps, it could begin among countries categorized in the same OECD category. In addition, as benefit schemes respond to economic circumstances, it will be important that this literature be updated to reflect these changes. Finally, to facilitate a meaningful international dialogue regarding sickness absence, the development of a typ- ology characterizing the sickness absence benefit gener- osity and work integration policies of sickness absence schemes should be a research priority. Database: Medline In-process Database: Medline In-process Search Terms: [exp Mental Disorders/OR exp Mentally Ill Persons/OR (mental adj3 disorder$).mp. OR (mental$ adj3 ill$).mp. OR (psychiatric$ adj3 disorder$).mp. OR (psychiatric$ adj3 ill$).mp. OR exp Substance-Related Disorders/OR exp “Diagnosis, Dual (Psychiatry)”/OR (con- current$ adj3 disorder$).mp. OR (dual$ adj3 diag$).mp. OR (alcohol$ adj3 abus$).mp. OR (alcohol$ adj3 depend $).mp. OR (substance$ adj3 abus$).mp. OR (substance$ adj3 depend$).mp. OR (drug$ adj3 abus$).mp. OR (drug $ adj3 depend$).mp. OR addiction$.mp.] AND [exp Ab- senteeism/OR exp Sick Leave/OR exp Return to Work/ OR exp Personnel Turnover/OR Social Welfare/OR Public Assistance/OR exp Insurance Disability/OR exp Insurance Benefits/OR exp Salaries/OR exp Fringe Benefits/OR exp Social Security/OR exp Retirement/ OR (sick$ adj3 day$).mp. OR (illness$ adj3 leave$).mp. OR (disabilit$ adj3 leave$).mp. OR (short term disabilit$). mp. OR (long term disabilit$).mp. OR (work$ adj3 ab- sence$).mp. OR (return$ to work$).mp. OR (work$ adj3 turnover$).mp. OR (employ$ adj3 turnover$).mp. OR (dis- abilit$ benefit$).mp. OR (employ$ benefit$).mp. OR (work $ benefit$).mp. OR (sick$ benefit$).mp. OR (incapacit$ benefit$).mp. OR (social$ welfar$).mp. OR (public$ assis- tanc$).mp. OR (insurance$ disabilit$).mp. OR (insurance$ benefit$).mp. OR (old$ age$ assistanc$).mp. OR (social$ securit$).mp. OR retire$.mp.] AND [sn.fs. OR ep.fs. OR preval$.mp. OR incid$.mp. OR statistic$.mp. OR exp Epidemiologic Methods/]. Future directions Cooperation and data sharing among countries as well as between database holders and researchers could help to increase the understanding regarding the similarities and differences of incidence rates. Access to the data necessary to calculate incidence rates often presents a challenge [39,40]. Rather than relying on primary data collection, these types of studies rely on administrative data. This means that the researchers are often not in- volved in the dataset design. As a result, the calculation of incidence estimates is often influenced by the data limitations. In the future, it would be useful if data warehouses were created where data necessary for this type of research were accessible. It would also help to advance the field if the database managers and researchers were able to work together to design databases that meet administrative and research needs. This would help to promote understanding of incidence rates for a broader range of workers and increase interpretability of the inter- national literature. Appendix 1: Search strategy Database: Medline Current OR exp Insurance/OR exp Salaries/OR exp em- ployee benefits/OR exp Social Security/OR exp Retire- ment/OR (sick$ adj3 day$).mp. OR (illness$ adj3 leave $).mp. OR (disabilit$ adj3 leave$).mp. OR (short term disabilit$).mp. OR (long term disabilit$).mp. OR (work $ adj3 absence$).mp. OR (return$ to work$).mp. OR (work$ adj3 turnover$).mp. OR (employ$ adj3 turn- over$).mp. OR (disabilit$ benefit$).mp. OR (employ$ benefit$).mp. OR (work$ benefit$).mp. OR (sick$ benefit $).mp. OR (incapacit$ benefit$).mp. OR (social$ welfar$). mp. OR (public$ assistanc$).mp. OR (insurance$ disabilit $).mp. OR (insurance$ benefit$).mp. OR (old$ age$ assistanc$).mp. OR (social$ securit$).mp. OR retire$. mp.] AND [preval$.mp. OR incid$.mp. OR statistic$. mp. OR exp Epidemiology/OR ext Data collection/OR epidemiolog$.mp. OR (data collection$).mp. OR survey $.mp. OR questionnair$.mp.]. incapacit* benefit* OR social* welfar* OR public* assis- tanc* OR insurance* disabilit* OR insurance* benefit* OR old* age* assistanc* OR social securit* OR retire*] AND [preval* OR incid* OR statistic* OR epidemiolog* OR data collection* OR survey* OR questionnair*]. Authors’ contributions CSD led the conception, design, data acquisition, analysis and interpretation of the data. DL collaborated on the design, data acquisition and analysis. SB collaborated on the design and data acquisition. HH collaborated on the analysis and interpretation of the data. All authors read and approved the final manuscript. Acknowledgements The authors gratefully appreciate the helpful comments and suggestions offered by Drs. Corné Roelen, Sandra Helena van Oostrom and Marianna Virtanen. Dr. Dewa gratefully acknowledges the support provided by her CIHR/PHAC Applied Public Health Chair. Any views expressed or errors are the sole responsibility of the authors and do not reflect the views of the funder. References 1. World Health Organization: Mental Health Action Plan for Europe. Facing the Challenges, Building Solutions. Copenhagen: World Health Organization; 2005. 2. The Australian Human Rights Commission: Workers with Mental Illness: a Practical Guide for Managers, Volume 2011. Sydney: The Australian Human Rights Commission; 2010. 3. Achieving the Promise: Transforming Mental Health Care in America. [http://www.nami.org/Content/NavigationMenu/Inform_Yourself/ About_Public_Policy/New_Freedom_Commission/Default1169.htm] 4. The Standing Senate Committee on Social Affairs, Science and Technology: Out of the Shadows at Last Transforming Mental Health, Mental Illness and Addiction Services in Canada. Ottawa: The Senate; 2006. 5. Lim KL, Jacobs P, Ohinmaa A, Schopflocher D, Dewa CS: A new population-based measure of the economic burden of mental illness in Canada. Chronic Dis Can 2008, 28(3):92–98. 1. World Health Organization: Mental Health Action Plan for Europe. Facing the Challenges, Building Solutions. Copenhagen: World Health Organization; 2005. Abbreviations CMD: Common mental disorders; GP: General practitioner; ICD-10: International Classification of Diseases 10th edition; ICPC: International Classification of Primary Care; OECD: Organization for Economic Co-operation and Development. Appendix 1: Search strategy Database: Medline Current Search Terms: [exp Mental Disorders/OR exp Mentally Ill Persons/OR (mental adj3 disorder$).mp. OR (mental$ adj3 ill$).mp. OR (psychiatric$ adj3 disorder$).mp. OR (psychiatric$ adj3 ill$).mp. OR exp Substance-Related Disorders/OR exp “Diagnosis, Dual (Psychiatry)”/OR (concurrent$ adj3 disorder$).mp. OR (dual$ adj3 diag$). mp. OR (alcohol$ adj3 abus$).mp. OR (alcohol$ adj3 depend$).mp. OR (substance$ adj3 abus$).mp. OR Database: PsycINFO Search Terms: [exp Mental Disorders/OR exp Psychiatric patients/OR (mental adj3 disorder$).mp. OR (mental$ Dewa et al. BMC Public Health 2014, 14:205 http://www.biomedcentral.com/1471-2458/14/205 Page 13 of 14 adj3 ill$).mp. OR (psychiatric$ adj3 disorder$).mp. OR (psychiatric$ adj3 ill$).mp. OR exp Drug Abuse/OR exp Drug Addiction/OR exp Drug Dependency/OR exp Alco- hol Abuse/OR exp Addiction/ OR exp Dual Diagnosis/OR (concurrent$ adj3 disorder$).mp. OR (dual$ adj3 diag$). mp. OR (alcohol$ adj3 abus$).mp. OR (alcohol$ adj3 depend$).mp. OR 321$.cc.[psychological disorders class code] OR 3233.cc.[Substance abuse & addic class code] OR (substance$ adj3 depend$).mp. OR (drug$ adj3 abus $).mp. OR (drug$ adj3 depend$).mp. OR addiction$. mp.] AND [exp Employee Absenteeism/OR (absenteeism $).mp. OR exp Employee Leave Benefits/OR exp Reem- ployment/OR exp Employee Turnover/OR (social welfar $).mp. OR exp Insurance/OR exp Salaries/OR exp em- ployee benefits/OR exp Social Security/OR exp Retire- ment/OR (sick$ adj3 day$).mp. OR (illness$ adj3 leave $).mp. OR (disabilit$ adj3 leave$).mp. OR (short term disabilit$).mp. OR (long term disabilit$).mp. OR (work $ adj3 absence$).mp. OR (return$ to work$).mp. OR (work$ adj3 turnover$).mp. OR (employ$ adj3 turn- over$).mp. OR (disabilit$ benefit$).mp. OR (employ$ benefit$).mp. OR (work$ benefit$).mp. OR (sick$ benefit $).mp. OR (incapacit$ benefit$).mp. OR (social$ welfar$). mp. OR (public$ assistanc$).mp. OR (insurance$ disabilit $).mp. OR (insurance$ benefit$).mp. OR (old$ age$ assistanc$).mp. OR (social$ securit$).mp. OR retire$. mp.] AND [preval$.mp. OR incid$.mp. OR statistic$. mp. OR exp Epidemiology/OR ext Data collection/OR epidemiolog$.mp. OR (data collection$).mp. OR survey $.mp. OR questionnair$.mp.]. adj3 ill$).mp. OR (psychiatric$ adj3 disorder$).mp. OR (psychiatric$ adj3 ill$).mp. OR exp Drug Abuse/OR exp Drug Addiction/OR exp Drug Dependency/OR exp Alco- hol Abuse/OR exp Addiction/ OR exp Dual Diagnosis/OR (concurrent$ adj3 disorder$).mp. OR (dual$ adj3 diag$). mp. OR (alcohol$ adj3 abus$).mp. OR (alcohol$ adj3 depend$).mp. OR 321$.cc.[psychological disorders class code] OR 3233.cc.[Substance abuse & addic class code] OR (substance$ adj3 depend$).mp. OR (drug$ adj3 abus $).mp. OR (drug$ adj3 depend$).mp. OR addiction$. mp.] AND [exp Employee Absenteeism/OR (absenteeism $).mp. OR exp Employee Leave Benefits/OR exp Reem- ployment/OR exp Employee Turnover/OR (social welfar $).mp. Additional files Additional file 1: Table S1. Quality Assessment Checklist. The additional file contains the quality checklist criteria used to determine the quality of papers being analyzed for the systematic literature review and the scores for each article. Author details 1 Search Terms: [mental disorder* OR mental disorder* OR mental ill* OR psychiatric* OR concurrent* disorder* OR dual* diag* OR alcohol* OR substance* abus* OR sub- stance* depend* OR drug* abus* OR drug* depend* OR addic*] AND [absent* OR sick* OR ill* OR disabilit* leav* OR short term disabilit* OR long term disabilit* OR work* OR absence* OR return* to work* OR work* turnover* OR employ* OR benefit* OR welfar* OR pub- lic* assistanc* OR insurance* OR old* age* assistanc* OR social securit* OR retire*]. 1Centre for Research on Employment and Workplace Health, Centre for Addiction and Mental Health, 33 Russell Street, Toronto M5S 2S1, Canada. 2Library Services, Centre for Addiction and Mental Health, 33 Russell Street, Toronto M5S 2S1, Canada. 3Department of Psychiatry, University of Toronto, 250 College Street, Toronto M5T 1R8, Canada. 4Department of Mood Disorders, Pro Persona, Wagnerlaan 2, 6815 AG Arnhem, The Netherlands. 5Department of Psychiatry, Academic Medical Center, University of Amsterdam, Meibergdreef 5, Room PA1-156, 1105 AZ Amsterdam, The Netherlands. 1Centre for Research on Employment and Workplace Health, Centre for Addiction and Mental Health, 33 Russell Street, Toronto M5S 2S1, Canada. 2Library Services, Centre for Addiction and Mental Health, 33 Russell Street, Toronto M5S 2S1, Canada. 3Department of Psychiatry, University of Toronto, 250 College Street, Toronto M5T 1R8, Canada. 4Department of Mood Disorders, Pro Persona, Wagnerlaan 2, 6815 AG Arnhem, The Netherlands. 5 5Department of Psychiatry, Academic Medical Center, University of Amsterdam, Meibergdreef 5, Room PA1-156, 1105 AZ Amsterdam, The Netherlands. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 27. 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Lim KL, Jacobs P, Ohinmaa A, Schopflocher D, Dewa CS: A new population-based measure of the economic burden of mental illness in Canada. Chronic Dis Can 2008, 28(3):92–98. Database: Web of Science Roelen CA, van Rhenen W, Koopmans PC, Bultmann U, Groothoff JW, van der Klink JJ: Sickness absence due to mental health disorders–a societal perspective. Occup Med (Oxford) 2012, 62(5):379–381. 30. Reis RJ, Utzet M, La Rocca PF, Nedel FB, Martin M, Navarro A: Previous sick leaves as predictor of subsequent ones. Int Arch Occup Environ Health 2011, 84(5):491–499. 30. Reis RJ, Utzet M, La Rocca PF, Nedel FB, Martin M, Navarro A: Previous sick leaves as predictor of subsequent ones. Int Arch Occup Environ Health 2011, 84(5):491–499.
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Who Should Act? Distancing and Vulnerability in Technology Practitioners' Accounts of Ethical Responsibility
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ACM Reference Format: ACM Reference Format: Kristina Popova, Clàudia Figueras, Kristina Höök, and Airi Lampinen. 2024. Who Should Act? Distancing and Vulnerability in Technology Practitioners’ Accounts of Ethical Responsibility. Proc. ACM Hum.-Comput. Interact. 8, CSCW1, Article 157 (April 2024), 27 pages. https://doi.org/10.1145/3637434 Kristina Popova, Clàudia Figueras, Kristina Höök, and Airi Lampinen. 2024. Who Should Act? Distancing and Vulnerability in Technology Practitioners’ Accounts of Ethical Responsibility. Proc. ACM Hum.-Comput. Interact. 8, CSCW1, Article 157 (April 2024), 27 pages. https://doi.org/10.1145/3637434 Who Should Act? Distancing and Vulnerability in Technology Practitioners’ Accounts of Ethical Responsibility CLÀUDIA FIGUERAS, Department of Computer and Systems Sciences, Stockholm University, Sweden KRISTINA HÖÖK, Media Technology and Interaction Design, KTH Royal Institute of Technology, Sweden AIRI LAMPINEN, Department of Computer and Systems Sciences, Stockholm University, Sweden Attending to emotion can shed light on why recognizing an ethical issue and taking responsibility for it can be so demanding. To examine emotions related to taking or not taking responsibility for ethical action, we conducted a semi-structured interview study with 23 individuals working in interaction design and developing AI systems in Scandinavian countries. Through a thematic analysis of how participants attribute ethical responsibility, we identify three ethical stances, that is, discursive approaches to answering the question ‘who should act’: an individualised I-stance (“the responsibility is mine”), a collective we-stance (“the responsibility is ours”), and a distanced they-stance (“the responsibility is someone else’s”). Further, we introduce the concepts of distancing and vulnerability to analyze the emotion work that these three ethical stances place on technology practitioners in situations of low- and high-scale technology development, where they have more or less control over the outcomes of their work. We show how the we- and they-stances let technology practitioners distance themselves from the results of their activity, while the I-stance makes them more vulnerable to emotional and material risks. By illustrating the emotional dimensions involved in recognizing ethical issues and embracing responsibility, our study contributes to the field of Ethics in Practice. We argue that emotions play a pivotal role in technology practitioners’ decision-making process, influencing their choices to either take action or refrain from doing so. CCS Concepts: • Human-centered computing →Empirical studies in interaction design. Additional Key Words and Phrases: ethics, emotion, ethical stance, vulnerability, distancing, responsibility, ethics in practice 1 INTRODUCTION Scholarly conversations about ethics have traditionally excluded emotion. Instead, moral philoso- phers have worked on defining the rational ground for ethical behaviour. This approach has been challenged by ethics of care, where theorists have demonstrated the situated nature of ethics and the role that feelings play in performing ethical action [72]. In the phenomenology of ethics, Varela Authors’ addresses: Kristina Popova, kpopova@kth.se, Media Technology and Interaction Design, KTH Royal Institute of Technology, Stockholm, Sweden, ; Clàudia Figueras, claudia@dsv.su.se, Department of Computer and Systems Sciences, Stockholm University, Stockholm, Sweden; Kristina Höök, khook@kth.se, Media Technology and Interaction Design, KTH Royal Institute of Technology, Stockholm, Sweden; Airi Lampinen, airi@dsv.su.se, Department of Computer and Systems Sciences, Stockholm University, Stockholm, Sweden. This work is licensed under a Creative Commons Attribution International 4.0 License. © 2024 Copyright held by the owner/author(s). ACM 2573-0142/2024/4-ART157 https://doi.org/10.1145/3637434 Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. This work is licensed under a Creative Commons Attribution International 4.0 License. This work is licensed under a Creative Commons Attribution International 4.0 License. © 2024 Copyright held by the owner/author(s). ACM 2573-0142/2024/4-ART157 https://doi.org/10.1145/3637434 Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 157:2 Kristina Popova, Clàudia Figueras, Kristina Höök, and Airi Lampinen challenges the understanding of ethics as a matter of rational judgment, demonstrating the role of embodied and, therefore, felt ethical reasoning [103]. Similarly, in the field of technology pro- duction, ethics has long been approached through rules and guidelines as attempts to avoid the biases of individual judgements that draw upon emotions and gut feelings. Recently, though, the role of emotion has been re-centered in approaches such as experience-centered design [4, 39]. Publications that examine the ethical concerns of technology practitioners empirically have also demonstrated the importance of emotions in both recognizing an ethical issue and acting upon it [99, 107]. We continue this exploration by looking at the connection between discourses regarding ethical responsibility and the feelings they generate among technology practitioners. We argue that attending to emotion can shed light on why taking ethical responsibility can be so demanding for technology practitioners. This is why feelings need to be considered in discussions of ethics, alongside principles like fairness and transparency. g p p p y Building on previous work that points at the importance of emotion in recognizing ethical concerns [107], we ask: What emotion work [4] does ethical responsibility place on technology practitioners? By ‘emotion work,’ we understand the management and navigation of emotional processes in work settings [49, 51]. Emotion work needs to be done when we, for example, handle emotions that conflict with the feeling rules of the workplace [49], that is, the established ways of demonstrating emotions. Such work is common when it comes to advancing ethical designs [4, 50, 95] or dealing with ethical contention in situations of value conflict [112]. We follow the tradition of studies of Ethics in Practice [13, 19, 42, 64, 65, 79, 91] by approaching the attribution of ethical responsibility from technology practitioners’ perspective. We present an empirical study based on two independently collected sets of semi-structured interviews with altogether 23 practitioners in various sectors, from interaction designers working in academia to AI practitioners working for government agencies. Here, we focus on the affective dimension, that is, the emotions and feelings produced by different stances. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. This work is licensed under a Creative Commons Attribution International 4.0 License. We relate our exploration to studies of ethics in action in design and HCI literature [33, 48, 95] together with the studies focused on the emotion work of design researchers [4, 25, 50, 55, 82]. Our analysis is structured into three parts: First, through thematic analysis of the interview data, we identify three ethical stances – I, We, They – which indicate different approaches to attributing ethical responsibility. The I-stance ascribes responsibility directly to an individual, granting them a possibility to own the outcomes of an action but also putting them into a vulnerable position. The we-stance ascribes responsibility to a collective without specifying the role of an individual’s action. The they-stance externalizes ethical responsibility, attributing it to a group or entity that the technology practitioner is not a part of. The stances should be understood as discourses: they are systematic ways of creating an ethical subject rather than individual psychological dispositions. Second, we analyze the differences between the three stances through the paired concepts of distancing and vulnerability. Each stance presumes a different degree of emotional engagement and personal involvement in the outcomes of the practitioner’s work – engagement that makes a practitioner vulnerable to the risks related to both action and inaction. This can include emotional and material consequences. Third, we analyze the work that the three stances do in different types of environments and what consequences they may have for practitioners and the development of technology. We distinguish between low-scale and high-scale settings, that is, small vertically integrated design projects and technology development processes that rely on complex interdependencies of services and often go across organizations. We make an empirical contribution by articulating three ethical stances, generated from our interviews with a diverse set of technology practitioners. Further, we introduce the concepts of distancing and vulnerability to analyze the emotion work that the ethical stances place on technology practitioners in situations of low- and high-scale technology development. Noting that 157:3 Who Should Act? none of the stances is unproblematic, we articulate their emotional consequences: We argue that both over-individualizing ethical responsibility for the outcomes of complex coordinated activities and distancing oneself from acting ethically can become problematic in terms of the emotional distress and moral burden they place on technology practitioners. 2 BACKGROUND Attributing responsibility for ethical action is not straightforward, especially not when it comes to the development of complex technical systems with multiple organizations and collaborators involved [19, 42, 44, 65, 91]. Even in situations where there is consensus on what it means to act ethically, the question of who should act often remains unresolved: an individual reflective designer, an organization following legislative and ethical guidelines, trained ethicists, social movements, or citizens? We draw inspiration from Social Movements Studies that analyze conditions for successful collective action for social change. According to the frame theory of social movements [9], providing a coherent narrative explaining what is wrong, what should be done to make things better, and who should act to implement the necessary changes is a key success factor. If social initiatives fail to articulate a motivating and comprehensive answer to the question of who should act, reasons for participation become unclear [9]. In order to demonstrate the parallels between the work that social movements do to motivate their supporters and the work done to promote ethical development of technology, we attend to three dimensions of scholarly debates regarding ethics in technology design and development: (1) introducing new values; (2) translating new values into practice, and; (3) attributing responsibility for taking ethical action. We demonstrate that the studies of ethics in design and AI do similar work by formulating problems, outlining solutions, and attributing responsibility. We centre our attention on the last dimension, attributing responsibility, by studying answers to the question who should act. Finally, we provide a review of prior empirical studies of ethics in practice and emotion work in relation to ethical work practice – the line of literature that most closely resonates with our research. This work is licensed under a Creative Commons Attribution International 4.0 License. Here, we connect with scholars of Ethics in Practice [83, 92, 110] who argue for a more systemic approach to ethics that avoids placing too much pressure on individual technology practitioners. We conclude by discussing how we might draw upon resources from first-person design methods [52–54, 96] and research strategies from Science and Technology Studies [23, 28, 41, 61, 101] to shape conversations on ethics in a way that avoids over-individualizing ethical responsibility and alienating practitioners from the outcomes of their work. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 2.2 How to implement new agendas? Translating values into practice There have been numerous attempts at making values impactful and actionable for designers through creating toolkits and methodologies for ethics in design. For example, Value-Sensitive Design [34, 35] was developed to make the values of both designers and stakeholders part of the design process. A lot of attention has been paid to describing how to design ethically: how to visualise values, reconcile conflicting values, or identify stakeholders. After the introduction of the initial framework, researchers have applied Value-Sensitive Design to different areas of practice [5, 36, 38], evaluated the outcomes [37], and developed scalable toolkits [110] intended for different technological domains, including AI [111]. Within AI Ethics, the principle-based approach has been criticised for claiming universal applica- bility [45] and for not providing mechanisms to enact the principles [67]. The critique has pointed to the lack of guidance on navigating value tensions in practice [31, 45, 65] and framing ethics as a matter of expert oversight, where ethics is approached solely as a matter of technical or legal expertise rather than political discussion [45]. Answering the call for more actionable mechanisms for AI ethics (transitioning to an ethical how instead of the what [70]), technical teams have built AI ethics toolkits for implementing values in AI systems [60]. Most frequently, such toolkits concern fairness, bias mitigation, anti-discrimination, privacy, explainability, or accountability [46]. Many of the resulting toolkits are aimed at individual practitioners [110], which has led to criticism that the toolkits ignore the potential for collective action [110], as well as the role of organizational factors in ethical decision-making [84]. Another line of criticism relates to the claims that these tools are also limited in their attention to structural inequality [10, 26, 68] and the need for greater participation of marginalized, impacted communities, and the Global South, at large [26, 58, 81, 86]. 2.1 Which values? Introducing new ethical frameworksi The first dimension to consider is which values are promoted — or even seen as relevant enough to take into account. A key line of work to consider is Design Ethics, by which we mean efforts to make ethics tangible and applicable to the process of technology development (with approaches including but not limited to Value-Sensitive Design [34], Values at Play [32], Reflective Design [90], and Critical Design [6]); guiding for design [22, 63]; and visible in the details of the design process [33, 43, 91, 95]. Within Design Ethics, the work of introducing new values is most noticeable in the articulation of new agendas, such as ethics of care [24], feminist Human-Computer Interaction (HCI) [7], autonomous design [29], or design justice [22]. For example, in their article Designing for existential crisis, Light and Shklovski [63] suggest a new set of values and directions: designing for dignity instead of fairness, compassion instead of formalized rules, and re-imagining responsibility rather than empowering. They also urge readers to abandon other values and ways of thinking typical for HCI, such as separating between a designer and a user, designing for ease, and excluding non-human actors. This is an attempt to set up a new ethical agenda. Once such an agenda has been accepted or it has gained sufficient support so that it can be advanced, the emphasis can shift 157:4 Kristina Popova, Clàudia Figueras, Kristina Höök, and Airi Lampinen to how values can be translated into practice. This entails a practice-oriented approach to how values come to matter within design processes and technology deployment. The question of which values are given priority is also central in the AI Ethics literature1. We understand AI Ethics as a subfield of digital ethics that addresses political, social, and psychological implications of AI, including machine learning, big data analytics, and blockchain technologies [59], and seeks to mitigate or pre-empt harms that AI systems can cause [2, 16, 74]. AI Ethics advocates for developing AI that is trustworthy [1], ethical [75], or transparent, explainable, and accountable [106]. There is a lot of work within AI Ethics that sets out high-level ethical principles, such as privacy, fairness, and transparency [56] or responsibility and trustworthiness [1, 27, 75], to create guidelines for AI development teams. ur attention to AI, here, relates to the fact that half of our interviewees work in the domain of AI development. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 2.4 Empirical studies of ethics in practice Research on ethics in practice studies how ethics is done as an integral part of the design process [33, 48, 82, 95]. For example, Shilton conducted an ethnography to analyze how software developers balance functionality and privacy requirements in designing new technology [91]. Together with Shilton, Boyd developed the concept of ethical sensitivity to distinguish between three analytical steps of implementing ethics in practice: noticing an ethical problem, understanding the situation, and taking a decision [13]. Within Participatory Design, Spiel showed how everyday work with participants, in their case children with autism, is an ongoing, processual, ethical matter [94, 95]. g g A recent line of studies has focused on the emotion work of design researchers who work in close proximity with their research participants, drawing attention to the traditionally hidden processes of managing emotions in research work [4]. Subsequent studies have drawn explicit attention to the relation between emotion work and ethics in design research [39, 50, 82], breaking the artificial dichotomy between felt experiences and rational behaviour. Considering the technology industry, Su and colleagues [99] conducted a study of critical affects among workers. Using the concept of emotion habitus, they analyzed how technology practitioners react to public criticisms of their field, removing the usual separation between ethics and emotions. Similarly, Widder and colleagues [107] demonstrated empirically the high psychological cost of raising ethical concerns within companies that develop AI products. Their work points to the importance of focusing the discussion on the distribution of power to solve ethical issues rather than just noticing them.i Scaling up and out from the specifics of individual participants in research settings, there is a growing number of empirical studies examining how ethics is enacted both in academia [105] and industry [19, 42, 64, 79, 109, 110]. One of the critical conclusions from this line of research is that organizational practices matter. While hardly a surprise to CSCW scholars, it is an observation with the potential to shape how we approach ethical responsibility in the organizational and collaborative settings where technology development takes place. Even in situations where an individual designer adheres to high moral standards, it is not guaranteed that deceptive design will be avoided [18, 19, 43, 110]. Learning how to navigate ethical complexities in organizations, then, is as important as adhering to the right set of values [19]. 2.3 Who should act? Attributing responsibility for ethical action For us, the central question for ethics in technology design and development is who should act? Calls for designers to shoulder more moral responsibility are common both in scholarly discussion [57, 64, 69, 71] and popular design literature [69, 76]. This has led to efforts to make ethics part of design education, training reflexivity, and bringing new ethical perspectives into education [30]. For example, Sabie and Parikh [85] report on their experience of teaching students to engage with the communities they are designing for with care and engaging with values beyond productivity. In a recent systematic review of ethical studies in design, attributing responsibility for ethical action to a designer is shown to be the most common approach in Design Ethics [73]. There is a similar tendency in AI Ethics to place the responsibility on individuals, which has taken the form Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. Who Should Act? 157:5 of providing toolkits for developers [65, 66, 83] or stating the importance of ethical education to engineers and computer science students [13, 30]. Attributing ethical responsibility for technology development is complicated by the fact that responsibility is often distributed. For instance, since AI systems involve many actors working on production, data collection, and data mining, to name just a few, the distribution of responsibility is complex and related to the problem of many hands [102]. Coeckelbergh [21] points out that collective responsibility may mean that either a collective agent, such as an organization, is held responsible [77, 84, 88]) or that responsibility is distributed over a group of individual agents, each one bearing responsibility independently. In any case, individual responsibility may be considered less relevant in the context of digital technologies [87] because of the complex relationships among diverse actors, spaces and organizations. This resonates well with empirical studies focused on ethical complexity in organizations that demonstrate how organizational dynamics can prevent the fulfilment of individual good intentions [19, 65, 83, 92, 109, 110]. Not all long-term consequences can be accessed by an individual designer or taken into account within a complex organizational context [42]. Because of this, many studies conclude with recommendations to develop organizational procedures for implementing ethical design rather than placing the responsibility with the individual [42, 85], thus shifting the focus from the individual to organizational practice. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 2We do not consider interaction design as separated from AI in terms of the technologies practitioners work on/with: some of our Interaction Design participants used AI-based technology in their projects. Yet, we see these as two separate communities with different vocabularies and ways of reasoning. 3.1 Positionality and conceptual approach to ethics We are a team working in academic institutions in Northern Europe, with diverse positions (back- ground details anonymised for review). Importantly for this paper, we all work in close proximity to Interaction Design and the AI community. Our approach to ethical responsibility is contextualised by our own experiences of working predominantly in small teams but in overlaps with industry practitioners. Our diverse disciplinary backgrounds and our commitment to interdisciplinary re- search projects, from the social sciences to AI/ML, have made us more accepting of a diversity of approaches to ethics and differing understandings of what one’s professional responsibilities can be seen to entail. Our approach to ethics is grounded in feminist theories of care [24, 40, 100], the phenomenological approach to ethics [103] and a growing body of work that challenges dichotomies between emotional and rational ethical reasoning as developed with the concept of felt ethics [39]. We presume the multiplicity of ethical frameworks and the existence of competing ethical agendas. We assume that technology is not neutral but situated in political and social contexts [108]. Values are exercised in all parts of design and deployment of technology: from interaction within a design team to the deployment of technology and its application by users. 3 RESEARCH MATERIALS AND METHODOLOGICAL APPROACH We present an empirical study of how technology practitioners approach the question of who should act? Before presenting our materials and methods, we reflect on our positionality and explain the theoretical assumptions about ethics that guided our research. 2.4 Empirical studies of ethics in practice This practice-based view of ethics in organizations provides footholds for approaching ethical reasoning as an empirical matter and a part 157:6 Kristina Popova, Clàudia Figueras, Kristina Höök, and Airi Lampinen of work practice where individuals need to reconcile their own assessment over what constitutes ethically justified action with the rules and structures constraining them. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 3.2 Data Collection 3.2.1 Data set 1. Interviews with interaction designers based in academia. The first data set consists of 10 semi-structured interviews that the first author conducted with academic interaction design researchers. Details on the interviewees’ backgrounds are provided in Table 1. The interviews lasted one hour on average. They were centred around details of the participants’ design work and the aspects of their work that they perceived as ethically challenging. The interview protocol included open-ended questions such as “Have you ever encountered any ethical issues in your projects” and “Have you encountered any ethical issues during planning/implementing/presenting your project”. Major parts of each interview were centred around follow-up questions prompted by participants’ initial answers, engaging with the details that participants viewed as ethically challenging. 3.2.2 Data set 2. Interviews with AI practitioners in the public sector. The second author conducted 10 semi-structured interviews with AI practitioners. The interviewees mostly worked with de- veloping AI systems for government agencies. The agencies were responsible, among others, for the distribution of social benefits, taxation, and management of natural resources. The interviews lasted 40 minutes on average. The data was collected as part of a research project on the enactment of ethical principles by AI practitioners working in public organizations. The interviews began with open-ended questions, such as “Are you trying to make AI systems ‘ethical’? If so, how do you do it?”, “What do you understand by ‘ethical AI’?” and “What does it involve in practice to develop ‘ethical AI’?”. Afterwards, the EU Ethics Guidelines for Trustworthy AI [1] were used as a prompt to engage with the interviewees’ perceptions of ethical values in everyday practice. Examples of questions asked in this second part of the interview include “Did you establish mechanisms to ensure fairness/transparency/accountability in your AI systems?” or “Did you assess whether there could be persons or groups who might be disproportionately affected by the negative implications of developing/deploying such systems?” 3.2.3 Complementary data collection. The interviews were initially conducted as two separate projects. Our motivation to combine the data-sets stemmed from the significant overlaps in how participants approached responsibility for taking ethical action. After a preliminary round of analysis of differences and overlaps between the two data sets, we conducted three additional interviews with AI practitioners outside of government agencies and four follow-up interviews with interviewees of Data set 2. 3.2 Data Collection The empirical and conceptual findings of our paper were developed based on two distinctive sets of interviews with interaction designers and developers of AI2. Some of our participants are based in research institutions, while others work in the public sector, with a few working in private businesses or switching between academia, governmental agencies, and private companies. More details about the interviewees can be found in Table 1. All participants, except one HCI practitioner based in North America (the quote from this interview is marked in the footnote), worked in Scandinavian countries at the time of the interviews. The initial participants among interaction design practitioners were recruited from the authors’ social networks. The initial participants among AI practitioners were selected through purposive sampling, recruiting among those who worked in the Swedish public sector and had at least ten years of experience. The rest of the participants in both data sets were recruited through snowball sampling. All participants were given information about the study and an opportunity to ask questions, before signing an informed consent form. We emphasized that participants could skip any questions they preferred not to answer, and that they were free to withdraw their participation at any time. As the present research does not fall within the purview of ethical review in the country where the authors work, we have attended to ethical research practice through ongoing conversation among the author team and our broader community. In preparing the findings for presentation, we have either chosen not to include or have gotten additional approval for including details that 157:7 Who Should Act? Table 1. Summary of interview participant demographics. Table 1. Summary of interview participant demographics. Gender Female (9), Male (14), Non-binary (0). Background/education Engineering (14), design (5), economy/business (2), other (2). Job title/Role Academia (12): Associate professor (2), postdoctoral researcher (3), PhD student (3), research engineer (4). Public sector (9): senior researcher (3), product owner (1), senior advisor (1), IT strategist (1), research director (1), project leader (1). Private company (3): product owner (1), research director (1), developer (1). Years of experience 1-5 years (1), 5-10 years (11), 10-20 years (3), > 20 years (8). are sensitive or might disclose an interviewee’s identity or the organization they work for. Out of concern for anonymity, the description of the interviewees’ professional roles is not uniform across the text, yet sufficient to understand their positions in the respective organizational hierarchies. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 3.2 Data Collection The follow-up interviews were aimed at targeting the interviewees’ personal attitudes towards ethics. The interviewees were asked for examples of work situations where they could not act in line with their views (in situations of ethical distress [17]) and prompted to describe how they behaved and reasoned in such situations. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. Kristina Popova, Clàudia Figueras, Kristina Höök, and Airi Lampinen 157:8 3.4 Limitations We acknowledge that our research materials have certain limitations, some inherent to all interview research. First, we cannot, nor are we trying to, provide a comparison between different ‘groups’ because (1) participants were not selected to represent a particular community or organizational setting and (2) the questions asked in the two sets of interviews differed, even though we intention- ally established similarities through complementary data collection. Second, there are important limitations to what can be established from interview accounts without observing participants’ activities in situ or studying the organizational practices and structures that shape their work set- tings. While a more extensive organizational study would be worthwhile, it is beyond the scope of this paper. Limiting ourselves to the analysis of interview data, we focus on practitioners’ accounts of ethical responsibility rather than their practices. 4 WHO SHOULD ACT? THREE ETHICAL STANCES We now turn to the three ethical stances we synthesised through our analysis: The I-stance is an individual approach focused on personal action. The we-stance attributes action to the collective, leaving the role of an individual unclear. The they-stance outsources ethics, positioning ethics as someone’s else responsibility. The stances should be understood as discourses that underpin answers to the question of who should act rather than as individual dispositions. Taking one of the discursive stances is not bounded to an individual: referring to different situations, one person can shift between all three stances. The stances are related to but not solely defined by the use of I, we- or they- pronouns. Most importantly, they differ in emphasising different actors as the subject of action. In this section, we present each stance as an analytically distinct discourse. In section 5, we will discuss the relationships between the three stances, the emotion work that comes with them, and how they play out in different contexts. 3.3 Analytical Processi The first and second authors had initially analyzed the data separately through thematic analysis [14]. After discussing the findings we had generated in this way, we combined the two data sets and performed a new, collaborative thematic analysis [20], encompassing the research materials as a whole. First, we analyzed three interviews together to agree on how to approach coding the materials. After that, we coded two interviews independently and synchronized the codes. The rest of the data was coded separately, with regular discussions about the themes we were generating and further directions for analysis. We performed inductive back-and-forth analysis to refine the codes, establish common patterns, and probe our hypotheses. Eventually, in collaboration with the third and fourth authors, we centred our analysis around different discourses underpinning the interviewees’ reasoning around attributing ethical responsibility. The three ethical stances described in Section 4, as well as the elaboration of their connections, the emotion work they entail, and the work that they perform for practitioners in low- and high- scale environments, were generated from the interview data as a result of this thematic analysis process. While our research materials are not suited for advancing a systematic comparison between practitioners working in different domains, in presenting the findings, we indicate which data set each quote is drawn from so as not to obscure emerging distinctions between accounts from different types of practitioners. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 4.1 I-stance But in this case [the project discussed in the interview] it was how do you engage other people in your work and create a space where everyone feels comfortable about sharing their opinions through words, or through movements, you know, like a place where you can create trust.” (Interaction design practitioner, early- career academic, female) “There are national ethical reviews, guidelines, but it’s more like the daily practice of how you’re doing research. I have examples of ethical ways of working with myself, my own body. But in this case [the project discussed in the interview] it was how do you engage other people in your work and create a space where everyone feels comfortable about sharing their opinions through words, or through movements, you know, like a place where you can create trust.” (Interaction design practitioner, early- career academic, female) The same interviewee talked about the importance of daily work practices and the ethics of everyday decisions within the design process, such as communicating with teenage research participants. By law, teenagers are not yet fully fledged subjects and do not have to be asked to consent to participate in the study. Instead, their parents should consent on their behalf. Yet, our interviewee chose to communicate with the participants directly, too. What guided their decision to ask the teenagers themselves was a feeling and intuition rather than a formal rule: “They say: it’s their parents, who need to sign it for the minors: if you’re under 18, um, then it’s the parents who sign. Like, it doesn’t matter what the minor thinks, the parent needs to sign it. So I had two documents [informed consents], because I thought that was a bit weird, you know, because these were teenagers, they were almost 18.” (Interaction design practitioner, early-career academic, female) In addition to this instance of a researcher going beyond formal ethical requirements to act in a manner they considered ethical, our data contained multiple further examples when the I-stance was applied in research ethics in ways that the participants deemed successful. However, the personalised take on ethics became demanding and problematic for an individual when touching on structural issues or power imbalances. In such situations, the cost for the individual taking action becomes too high, and operating from an I-stance can have high emotional costs without leading to action. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. 4.1 I-stance The I-stance presumes that a person is an agent of ethical action. Therefore, it attributes high value to personal decisions. This stance places the least possible distance between an individual’s actions and their consequences. It implies a belief that a person can influence relevant outcomes Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. Who Should Act? 157:9 and, hence, can (and should) take the blame or distress related to any potential negative outcomes – or the credit for success. In this section, we consider two types of ethical concerns in turn: first, matters of interpersonal interaction in technology development processes, and second, issues that have to do with the technologies being developed. While these two ethical domains are different, we highlight that they intertwine in the development of technology since technology ultimately changes relationships between people, even if issues of interpersonal interaction may appear distant in settings complicated by organizational hierarchies. We start presenting the I-stance in the domain of interpersonal interaction where it is represented the best. However, the I-stance can be taken in any domain and at different scales: from interacting with colleagues and research participants ethically to striving to develop a non-biased AI. While practitioners in different settings are faced with different ethical questions, the common denominator here is taking personal responsibility. 4.1.1 I-stance and interpersonal relationships. The I-stance is familiar to researchers as it often needs to be taken when building relationships with research participants [95], creating safe design spaces [4, 11, 25, 82, 98], and steering research projects in a desired direction. These are all settings where individual ethical decisions have a high impact and, thus, the I-stance is at its most powerful. This is illustrated in the following interview excerpt from an interaction design researcher reflecting on the importance of daily work with co-designers of technology. The interviewee highlights the importance of her personal role in setting up trusting relationships with project participants. She stresses that her personal role matters despite the existence of ethical guidelines that outline the direction of action, since guidelines alone are not enough for implementing a project in an ethical way or building interpersonal relationships within a team: “There are national ethical reviews, guidelines, but it’s more like the daily practice of how you’re doing research. I have examples of ethical ways of working with myself, my own body. 4.1 I-stance Publication date: April 2024. 157:10 Kristina Popova, Clàudia Figueras, Kristina Höök, and Airi Lampinen Our second example demonstrates an extreme case of a personal crisis that resulted from a situation of ethical distress. The interviewee felt obliged to act but, in the end, did not act because of the high personal cost that taking action would have entailed. In the quote, the participant refers to an internship at a company where they spent parts of their time as a doctoral student. The interviewee strongly disapproved of the company’s work practices. This made working for the company an ethical problem and left the participant balancing the intention to act with the fear of negative consequences: “Should I say something to anyone? And then I realised that the guy who has a company, “Should I say something to anyone? And then I realised that the guy who has a company, he is pretty powerful figure [...] and he has a lot of connections, and I didn’t want to start moving things there. I mean, it wasn’t my job in that case. I stayed there for three months and my whole self was destroyed, because I returned back and I started feeling that I should have said something and I didn’t.” (HCI practitioner, early-career academic, female) Experiencing emotional struggle, in their case, led not to an action but to a personal crisis related to the feelings of losing one’s agency. This kind of strong emotional response – although typically in a less damaging form – was common for interviewees who took personal responsibility for the consequences of their actions while finding themselves in situations where they perceived acting to have too high personal costs. 4.1.2 I-stance in the development of complex technology. Operating from the I-stance can be challenging when multiple parties are involved. In our data, this was especially relevant in accounts related to AI development. AI systems exist in complex environments compared to the contexts where designers and users meet face-to-face. The complexity stems from the fact that many participants are involved in the development of AI, from data labelling and developing mathematical models to applying AI-based technology in practical settings. Nonetheless, the process of AI development, too, features situations where individuals operate from the I-stance. 4.1 I-stance The following example describes a situation when an interviewee, who occupies a senior position in his organization, was planning to install a facial recognition system in a shared, open-plan office. The interviewee, who led the project, explained that they felt uncomfortable thinking about how his colleagues would react to this facial recognition technology. He explained that his main reason for emotional discomfort was the realisation that computer scientists are sensitive to privacy issues, and it would be difficult to communicate how the system would not store any data. It is interesting how the interviewee makes a distinction between the ‘ethical’ and the ‘emotional’. To define the problem as ethical, they refer to their feeling and other people’s feelings rather than a rule, but this makes them initially doubt whether the issue is, indeed, ethical: “I don’t know... ethical? It was just also just... emotional. I mean, how, how do people feel about having some system taking their picture and processing it and, and how do you communicate that: no, it’s okay because we don’t store the pictures. (...) it has the nature of some kind of surveillance, right? No, but it feels like it’s: so you have a camera and whenever I pass by that, you will record that. (...) That particular demo machine was just outside my office, and I wanted it to be in play. And there was definitely an ethical problem to force that upon [others], because [...] There are slightly more people within computer science who are more sensitive about such issues, who are very careful about computer security and integrity and all of that.” (AI practitioner, senior academic, male) “I don’t know... ethical? It was just also just... emotional. I mean, how, how do people feel about having some system taking their picture and processing it and, and how do you communicate that: no, it’s okay because we don’t store the pictures. (...) it has the nature of some kind of surveillance, right? No, but it feels like it’s: so you have a camera and whenever I pass by that, you will record that. (...) That particular demo machine was just outside my office, and I wanted it to be in play. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 4.1 I-stance And there was definitely an ethical problem to force that upon [others], because [...] There are slightly more people within computer science who are more sensitive about such issues, who are very careful about computer security and integrity and all of that.” (AI practitioner, senior academic, male) Another example comes from a researcher who worked on developing machine learning algorithms in a commercial company. Given that the company had other specialisations than developing AI, 157:11 Who Should Act? the interviewee’s team had to explain their work to other teams. Reporting the results of ML models can sometimes be challenging [104] because the statistical results of the model evaluation can be easily misinterpreted. The interviewee found himself in a situation where the results reported by their team were misinterpreted by others (the 90% accuracy rate model was treated as a sign of success, which it actually was not). At that time, the team was facing criticism from management, so the presentation was a high-stakes event. The interviewee described feeling conflicted because he wanted neither to mislead colleagues nor to ruin relationships within the team: “If we know that people can misunderstand that 90%, we’re responsible to make sure that, you know, they don’t hype it too much or disregarded too. We are responsible for that. So even if the team is in a tough spot and people are putting pressure on it, I don’t think it’s like really ethical to go and say but we got 90%. (...) I wasn’t sure, but in the end I was like ‘okay let’s do this’ [say that 90% was not a success], but it was risky. I actually went and spoke with the team, but it didn’t go well for me. Exactly what I expected happened that people were thinking ’this person wants to act all like ethical and sabotage the team’ and it was kind of dismissed.” (AI practitioner, research engineer in a private company, male) Taking the I-stance and accepting personal responsibility can be problematic when action cannot be taken or the costs of taking action seem too high. What is more important, the outcomes of taking the I-stance in situations of developing complex technology were not necessarily beneficial when individuals did not have power to act upon their will. 4.2 We-stance The distinction became especially noticeable when interviewees who were asked directly about their individual opinions defaulted to collective ‘we’ answers: “-In case you would experience a tension between, for example, you and your projects or your organization, what do you think you would rely on for solving that problem? For example, would you rely on your inner feeling or go to maybe some documents and guidelines and procedures or talk with other colleagues or with experts?i - So, how would I go about fixing that? That’s something we do pretty much every day when we run a company [...].” (AI practitioner, research director in a company, male) - So, how would I go about fixing that? That’s something we do pretty much every day when we run a company [...].” (AI practitioner, research director in a company, male) Referring to a company as the actor holding responsibility in front of another collective — the ‘customer base’, users, or stakeholders – is one example of operating from the we-stance. This is also a common discourse in AI Ethics, which often considers groups and organizations as the relevant units of action [88]. These two quotes, from different participants, provide further examples: “I think we also might have an educational obligation as a company. We should actually “I think we also might have an educational obligation as a company. We should actually inform, in some sense, our customer base what it means to buy a data-driven product.” (AI practitioner, research director in a private company, male) “I think we also might have an educational obligation as a company. We should actually inform, in some sense, our customer base what it means to buy a data-driven product.” g g p y y inform, in some sense, our customer base what it means to buy a data-driven product.” (AI practitioner, research director in a private company, male) “[Interviewer]: So, how is transparency applied for example in the systems or in the methods? [Respondent]: Again and I would say that’s by education because it’s like a two front war if you want. 4.2 We-stance The we-stance considers a collective – an organization, a company, a governmental agency – as a unit of ethical action. The individual is considered a part of this collective subject rather than an independent actor. When technology is produced in large organizations with a strict distribution of labor, taking an I-stance towards ethical action may not be feasible. In such cases, taking the we-stance – where the organization as a whole is seen as the unit of action that is responsible for acting ethically – may seem like the default choice for the individual practitioner. The we-stance corresponds to a sense of belonging to a group, as illustrated by the next interviewee who shares the collective responsibility of sustaining the reputation of civil servants: “In [the country] we have a very strong type of culture within the administration. For many, many years people have had a huge trust to it. It has been open and we have built that in many hundred years I would say. So, we need to maintain that within the digital age.” (AI practitioner, senior advisor in governmental sector, male) The we-stance is not specific to government officials and AI developers. Interaction designers in industry settings have also been documented to frame their responsibility vis-à-vis the organization in this way, as demonstrated by Lindberg and colleagues [64, p. 5]. The main distinction between the two stances is that the role of individual responsibility is not emphasized in the we-stance, and not positioned in conflict with a collective. Individuals taking the we-stance consider themselves part of a collective but do not necessarily see a direct connection between themselves and any necessary ethical action. In the interviews, the collective take on ethics could often be noted through the use of the personal pronoun ‘we’ instead of ‘I’, along with expressions referring to the group, such as ‘in our team/organization’ or ‘as a company’. The use of the pronouns ‘I’ or ‘We’ is not the only indication we rely on in categorizing interview accounts as instances of a particular stance. Rather, in recognizing the we-stance, the emphasis is on the absence of any specification of an individual’s personal role. In contrast, in identifying the I-stance, the key is whether interviewees describe Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 157:12 Kristina Popova, Clàudia Figueras, Kristina Höök, and Airi Lampinen themselves as responsible. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 4.2 We-stance Because we have to educate our clients and stakeholders at the same time as we are trying to make systems for them.” (AI practitioner, research director in a private company, male) The we-stance does not emphasize an individual’s role in taking action, so it does not provoke internal conflicts between personal intentions and external circumstances. In that sense, to the individual, operating from the we-stance is a safer choice, as it can help them to avoid both the risks related to taking action (losing one’s job or ruining workplace relationships) and the emotional burden of non-action. Because the we-stance distances the individual from ethical action, interview accounts that use the we-stance are less emotionally charged than those given from the I-stance. Most of the examples of ethically challenging situations discussed within the we-stance include, for instance, educating users – a significantly less emotionally challenging activity than the situations described with the I-stance. User education is considered a step towards developing ethical AI because it increases users’ awareness of potential issues related to data-driven products. This educational approach transfers some part of ethical responsibility to the product’s users, implying that they should be aware of potential negative consequences resulting from its use and, further, should stop using products that lead to excessive negative outcomes. Overall, when interviewees speak from the we-stance, their accounts involve less emotional expression in words and intonations, both in what is said and how it is said. The lack of emotion may be specific to our data set, where collectives were mostly formal workplace collectives or units of bureaucratic organisations rather than self-organised groups. In our data, strong emotional responses were noted when the participant personally witnessed negative consequences of technology. In the following case, which we consider transitional between the I- and we-stances, the interviewee was working in a team that used a machine learning algorithm for facial recognition – originally developed for other purposes – to build their technology. As it turned out, the algorithm, developed by a third party, did not recognize participants with dark skin during the testing stage. The bias was discovered at the latest stages of the process when the team finished the artifact and started to use it as part of their work. Despite the discovery, it was decided to continue the project – a decision that resulted in the interviewee’s feeling of moral discomfort. Proc. ACM Hum.-Comput. Interact., Vol. 4.2 We-stance 8, No. CSCW1, Article 157. Publication date: April 2024. 157:13 Who Should Act? “The device was finished, the vehicles finished, it took months to create and someday you have this [discovering the bias] we cannot start the project over again. The dis- cussion was like ‘we have to be careful (...) One month ago we were doing another thing with [...] and two students came to see how they work, because I was going to give them the cameras and everything. I didn’t remember that one of them was black. I forgot about that. I was showing them how to use it. [...] And then he’s like ‘can I try, can I try it?’ And it doesn’t work. And I was like ‘Oh, I don’t know.’ We were very ashamed.” (academic researcher) “The device was finished, the vehicles finished, it took months to create and someday you have this [discovering the bias] we cannot start the project over again. The dis- cussion was like ‘we have to be careful (...) One month ago we were doing another thing with [...] and two students came to see how they work, because I was going to give them the cameras and everything. I didn’t remember that one of them was black. I forgot about that. I was showing them how to use it. [...] And then he’s like ‘can I try, can I try it?’ And it doesn’t work. And I was like ‘Oh, I don’t know.’ We were very ashamed.” (academic researcher) In this case, the problem of bias was not abstract but related to personal face-to-face encounters with users from an excluded group. The interviewee described the feeling of shame they experienced when showing the device to the user. The way the participant talked about bias also visibly expressed discomfort. However, they do not separate themselves from the team as a collective subject and do not attribute the responsibility for developing biased technology to themselves personally. They talk about shame as a shared emotion and attribute the responsibility for the problem to the team as a whole, who attempted to remedy the situation by contacting the algorithm developer, but did not discontinue the project because the cost – in terms of money and invested resources – appeared too high. In general, the we-stance puts fewer demands on personal judgement compared to the I-stance. 4.2 We-stance In the realms of formal institutions, it considers ethical decision-making a result of following the established set of organizational procedures rather than the ethical judgements of a reflective practitioner. In commercial and bureaucratic organizations, it implies the need to translate ethics into formalised language of organizations: into guidelines, norms, procedures, and metrics. When there are no guidelines at play, and ethics ‘is not measured,’ ethical issues become intangible – as noted both by other researchers [64] and our interviewees: “In all of this, to also keep track of something that is quite elusive as ethics, I think you need to actually bring it down to a measurement so it’s easy to see. Preferably in a dashboard or some spreadsheet or whatever that people could look at. Perhaps this might sound crude, but put a price tag on it.” (AI practitioner, research director in a private company, male) Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 4.3 They-stance But we don’t take responsibility for the applications that are directed out to the citizens. That’s up to the government agencies, and that’s their responsibility and their ownership of those techniques.” (AI practitioner, senior researcher in governmental sector, male) “I mean our work in this project is more about the core technologies and we also support the government agencies when they build their in-house applications. But we don’t take responsibility for the applications that are directed out to the citizens. That’s up to the government agencies, and that’s their responsibility and their ownership of those techniques.” (AI practitioner, senior researcher in governmental sector, male) This logic could be applied to any service that does not directly interact with end-users. The interviewee in the following quote applies a similar technique when he talks about a service developed for the needs of the governmental agency, claiming that the service is not subject to considerations regarding transparency because it is a time-saving intermediary device. Yet again, this discourse implies that the tasks of ‘mere optimisation’ are free of ethical considerations – a type of reasoning that imagines technology as neutral and that has long been challenged by Social Studies of Science [12, 41, 47, 108]. While ethical considerations may arise, the responsibility for identifying and addressing them is assigned to a third party who interacts with end-users. This leads to overlooking the potential responsibility of both the system itself and, more importantly for our discussion, the developers of the system: “So they do email classifications, so when people ask questions by email they try to classify which topic the email is about and then they route that to the correct administrative person. And I don’t really think that transparency enters into that process at all because it’s just a sort of a filtering step. We don’t actually make any automatic decisions based on the system. It’s only like routing the messages to the correct person, so it simply just saves time for the administrative part of [a government agency].” (AI practitioner, senior researcher in governmental sector, male) “So they do email classifications, so when people ask questions by email they try to classify which topic the email is about and then they route that to the correct administrative person. And I don’t really think that transparency enters into that process at all because it’s just a sort of a filtering step. 4.3 They-stance The last ethical stance we discuss is the they-stance. The they-stance externalises responsibility for taking ethical action. Here, ethics was not claimed to be unimportant, but participants acknowledged it as someone else’s responsibility, be it a particular group of developers, ethicists or social scientists, users of the resulting system, or another group that the technology practitioners did not belong to. Below, we report the most common rhetorical techniques that served to distance our participants from ethical responsibility (whether as an intentional move to avoid responsibility or not).i As a first technique, participants distinguished between the ‘ethical’ and the ‘technical.’ The quote below refers to a work domain where the end-users are other engineers. The interviewee draws on a distinction between ‘ethical’, which has to do with people, and ‘technical,’ which has to do with interaction between machines and, thus, is not taken to imply any ethical considerations. This removes the possibility of recognizing an ethical concern in ‘purely’ engineering matters: “Many of the applications we address within [our organization], they have to do with looking at images of cracks in composite plastics and assessing them in different ways. There aren’t that many ethical concerns in that. Or maybe if you help me, I might find [the concerns], but often not. Many applications are very technical, or they have to do with process automation, industrial process automation, take the readings of 157:14 Kristina Popova, Clàudia Figueras, Kristina Höök, and Airi Lampinen various sensors and do something with these. These ethical concerns, typically, of course, come when you have to do with people in one way or another.” (AI practitioner, senior researcher in governmental sector, male) Another technique implies limiting the domain of ethics to end-user interaction. Such considerations were expressed by an interviewee who developed an AI-based system for government agencies. Here, the developed system is not solely a machine-to-machine application – and it will be used by other people who are not engineers – but it is not explicitly designed for the end-user (i.e., citizens). As expressed in the quote, the presence of an intermediary – an administration – implies that ethical considerations should be the concern of the client, not the developer: “I mean our work in this project is more about the core technologies and we also support the government agencies when they build their in-house applications. 4.3 They-stance Therefore, direct involvement or action is considered optional by those who do not work within those realms. 4.3 They-stance We don’t actually make any automatic decisions based on the system. It’s only like routing the messages to the correct person, so it simply just saves time for the administrative part of [a government agency].” (AI practitioner, senior researcher in governmental sector, male) Finally, our last example represents a slightly different technique where an ethical problem is recognized, but it is attributed to a particular domain unrelated to the area of the participant’s job. Working in certain domains is acknowledged to be ethically problematic (such as recommender systems or automation of work), but the position is taken that ethical concerns can be avoided simply by not engaging with these sensitive AI applications: “- have you ever encountered any ethical issues, during any of these projects or any other project? “- have you ever encountered any ethical issues, during any of these projects or any other project? - Ethically? Like how, for example? I mean, if I was doing something like for automation factories or like self-driving cars maybe I would have ... I don’t think I will do it. It depends on the... cause like when you work in this kind of stuff, there is always like the question ‘who are you working for?’. Especially in AI. I have friends that went to do internships at Amazon. They don’t have a problem with that, but I will not do it.” (AI practitioner in a research institution, male) - Ethically? Like how, for example? I mean, if I was doing something like for automation factories or like self-driving cars maybe I would have ... I don’t think I will do it. It depends on the... cause like when you work in this kind of stuff, there is always like the question ‘who are you working for?’. Especially in AI. I have friends that went to do internships at Amazon. They don’t have a problem with that, but I will not do it.” (AI practitioner in a research institution, male) Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 157:15 Who Should Act? Within this approach, ethics is recognized as relevant only to particular domains, specific AI techniques, or applications thereof. Therefore, direct involvement or action is considered optional by those who do not work within those realms. Within this approach, ethics is recognized as relevant only to particular domains, specific AI techniques, or applications thereof. 5 EMOTION WORK AND THE THREE ETHICAL STANCES In the previous section, we considered three ethical stances – discourses that attribute responsibility for ethical action to an individual, an organizational entity, or someone else. The stances are not fixed individual dispositions: the same person may shift between different stances even within a single conversation as they produce an account of their actions and experiences. While the stances intertwine, they are analytically distinguishable and perform different work in attributing responsibility to specific actors. The most crucial distinction is between the I-stance, which places ethical responsibility on an individual, nudging them to act, and the they-stance, which help individuals avoid feeling obligated to act through techniques that transfer responsibility to another actor. With the help of the concepts of distancing and vulnerability, we now consider the relations between the three stances and the emotion work that operating from each can place on technology practitioners. We also analyze the work that the stances perform for technology practitioners operating in different settings, making a distinction between low- and high-scale environments. 5.1 Vulnerability and distancing Vulnerability in HCI is commonly considered to be a characteristic of a group affected by technology or social structures, but, here, we apply the concept to technology practitioners who may end up in vulnerable positions while (and because of) trying to act ethically. By vulnerability, we refer to the risks related to direct personal engagement in the outcomes of one’s action [82]. Being vulnerable, then, implies carrying on emotion work [4, 49] as a part of technology design and development. Vulnerability implies both emotional burdens and material consequences, such as the risk of losing one’s job or entering into a conflict with a powerful figure, an action that can lead to multiple negative consequences. The I-stance is prone to evoke vulnerability in situations of asymmetrical power relationships. As we illustrated in the Section 4.1, taking action in such situations can mean, for example, risking one’s job, whereas not acting can cause emotional distress. The interviewees who had to act against their ethical views reported feelings of losing one’s agency, accompanied by emotional discomfort of varying intensity, including feelings of guilt, sadness, and shame (as illustrated in the quote in section 4.1.2 where an academic researcher expresses having felt really ashamed). In the most extreme case, the same interviewee who felt that her ‘whole self was destroyed’ (see 4.1.1) due to not taking action, discussed feeling guilty because of her role in enabling the status quo: “They [the company] will manage to get more funding only because I was there. Of course, if I wasn’t there, they would have done it themselves, it wasn’t something... it was a manual work. But still I helped. [...] I felt trapped and I could, I explain that I felt trapped. I couldn’t speak, I was scared and so on. [...] And at the moment I was like, I was crying.” (HCI practitioner, early-career academic, female). “They [the company] will manage to get more funding only because I was there. Of course, if I wasn’t there, they would have done it themselves, it wasn’t something... it was a manual work. But still I helped. [...] I felt trapped and I could, I explain that I felt trapped. I couldn’t speak, I was scared and so on. [...] And at the moment I was like, I was crying.” (HCI practitioner, early-career academic, female). 5.1 Vulnerability and distancing The techniques that we encountered in the interviews included limiting the sphere of the ‘ethical’: setting up a dichotomy between ‘ethical’ and ‘technical’ issues, excluding everything that does not have to do with ‘people’ (end users) as well as excluding service intermediary technologies from the domain of ethical concern, or assuming that only specific technologies belong to ‘ethically sensitive’ domains. Following this logic, the need to consider ethics is acknowledged only in certain situations Within the they-stance, distancing is achieved by externalizing ethics to a third party. The techniques that we encountered in the interviews included limiting the sphere of the ‘ethical’: setting up a dichotomy between ‘ethical’ and ‘technical’ issues, excluding everything that does not have to do with ‘people’ (end users) as well as excluding service intermediary technologies from the domain of ethical concern, or assuming that only specific technologies belong to ‘ethically sensitive’ domains. Following this logic, the need to consider ethics is acknowledged only in certain situations – and commonly, these do not include the domains of the interviewee’s employment. Within such a discourse, the need to take ethical action is either not recognized at all or it is attributed to a third party. In our interviews, this third party was usually a client who interacted directly with end-users, but it could also be the end-users themselves, a person responsible for ethics in the company, or a particular algorithm [89]. The ultimate example of distancing is what Seaver has labelled as ‘decorrelational ethics’ [89], a move to fulfill the dream of creating a technology that avoids value conflict altogether, thus eliminating the need for conversations about ethics. – and commonly, these do not include the domains of the interviewee’s employment. Within such a discourse, the need to take ethical action is either not recognized at all or it is attributed to a third party. In our interviews, this third party was usually a client who interacted directly with end-users, but it could also be the end-users themselves, a person responsible for ethics in the company, or a particular algorithm [89]. The ultimate example of distancing is what Seaver has labelled as ‘decorrelational ethics’ [89], a move to fulfill the dream of creating a technology that avoids value conflict altogether, thus eliminating the need for conversations about ethics. 5.1 Vulnerability and distancing Another example of feeling sad and frustrated comes from a researcher who, because of institutional pressure, had to step away from a research project she wanted to engage in: “You know, I was really sad that I was told to not do it because I was really excited about it and I felt, you know, this was a research question driving me with curiosity, and I was told to not do it. So, I was like, okay, what should I do then? And then I felt Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 157:16 Kristina Popova, Clàudia Figueras, Kristina Höök, and Airi Lampinen like, then I did turn on quite a different track to do something else. You can definitely imagine frustration” (Interaction design practitioner, early-career academic, female). like, then I did turn on quite a different track to do something else. You can definitely imagine frustration” (Interaction design practitioner, early-career academic, female). In these cases, the interviewees were economically and emotionally vulnerable: while recognizing ethical concerns, they also recognized that they would face negative consequences for both acting and not acting – either in the form of losing their employment or in terms of experiencing the loss of self. Ethical responsibility in such cases was not a question of distributing duties within an organization, but something that was felt – sometimes intensely – as a somatic experience of discomfort.i In contrast, the we- and they-stances evoke a significant degree of emotional distancing, which is achieved by situating subjectivity outside of the individual. The we-stance considers the collective – an organization, a company, or a community of AI developers – to be the relevant subject of ethical action without specifying a personal role for the individual practitioner. Within the we-stance, distancing can be additionally achieved through rules and formal procedures that reduce the need for personal judgement to a minimum. These distancing mechanisms move ethical responsibility away from an individual by turning ethical decision-making from a felt phenomenon to an organizational one. Within the they-stance, distancing is achieved by externalizing ethics to a third party. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 5.2 Low and high-scale technology design and productionf So all of these are very, very important problems to solve from a science and engineering perspective, but from where I am coming from – small town, surrounded by very rural villages – their problems were not addressed in my education. So it didn’t reflect. Like I was learning, I was really working hard on my education, but that wasn’t like, that didn’t feel very directly useful for the community I belong to... [...] that was really a shock for me! I was in my first year, I was so upset that my politics failed.” (HCI practitioner, early-stage academic, female3). In contrast, the they-stance was usually taken when the implications of the technology could not be directly observed or felt or when the practitioners clearly lacked resources to address the issue. When the implications became visible for the practitioner, such as in the cases of observing the effects of a racially biased algorithm (see 4.2) or setting up a system that could threaten colleagues’ sense of privacy (see page 10), interviewees often switched to describe situations from the I-stance. Shilton has reported similar findings from her fieldwork, noting that software engineers could more easily relate to privacy and other ethical issues after testing the system that they were designing [91]. In situations of high-scale technology development, actors are separated from each other by the power hierarchy within an organization, and most of them do not have much control over the course of technology development. Any case of developing AI/ML-based technology would be a high-scale technology development, given the amount of participants involved at each stage of the project, from data collection, labelling and storage, to algorithm deployment. The I-stance becomes problematic in such environments, since the high personal costs of operating from it do not necessarily pay off in terms of meaningful impact. Accepting responsibility will not necessarily lead to action if multiple other actors are involved, the action feels too risky, or mechanisms for action are unclear. The overly individualistic approach to owning ethics [66] has a clear risk of becoming too demanding, overwhelming, and ultimately not actionable if the sense of responsibility is not paired with possibilities to make a change. 3The participant was working in North America during the interview. 5.2 Low and high-scale technology design and productionf The stances perform different work in low- and high-scale situations of technology design, where they can either help practitioners see the connections between their actions and outcomes or become discouraging by placing unrealistic expectations on an individual who lacks power to act upon them. By low-scale, we refer to situations with a limited number of participants, where practitioners have more control and power over the development of technology. Following Brown and colleagues, we presume that these situations usually occur in the context of vertically integrated designs [15] that do not depend on broad ecologies of technologies that are interdependent (such as the Google suite of services). Instead, they are intended for a known context, where practitioners can control the use of the system, and they are meant to be used only by a limited number of people. These are often the kinds of situations where practitioners can directly interact with the intended users of the technology.i Unsurprisingly, the I-stance seems to fit best in situations of low-scale design work, where an individual has power to act according to their values. In our interviews, taking a proactive I-stance in controlled circumstances – for example, when interacting with research participants who were also the intended users of the technology in question – allowed practitioners to achieve desired outcomes and experience a sense of fulfilment from work well done: establishing a trusting atmosphere Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 157:17 Who Should Act? withing a project, treating co-design participants with care and respecting their autonomy (see the accounts from an interaction design practitioner in 4.1.1 and 4.1.1). Such interactions were often related to a context where the consequences of one’s activity were directly visible and the researcher had sufficient resources to act. Yet the successful examples of individual interventions were not common beyond the scope of small research projects. Despite good intentions to serve communities, technology practitioners were often discouraged by their lack of capabilities to induce change. The following quote illustrates the shock this can entail: “I did my undergrad in electrical engineering, where I learned how to do machine learning, how to design circuitry and devices, technology so that they work faster. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 5.2 Low and high-scale technology design and productionf Given the difficulty of implementing individual values in relation to complex AI technologies, operating from distanced we- and they-stances is a safer and more reasonable (from the individual point of view) choice for developers employed by large organizations. Distance creates safety by removing the uncomfortable and potentially paralyzing burden of inescapable uncertainty (‘Am I acting ethically?’). Further, they- and we-stances often translate ethics into an organizational phenomenon: a matter of guidelines, rules, and bureaucratic matters. While we certainly do not advocate for relying solely on emotion in managing ethical issues, the opposite, too, can be problematic. When ethics is approached only through organizational lenses – quantified and measured, not felt – it becomes detached from lived experience and it can no longer be experienced as a field where personal action matters. This alone is problematic as it can lead to feelings of losing one’s agency and sense of control. Distanced stances reduce feelings of personal involvement and meaningfulness, creating 157:18 Kristina Popova, Clàudia Figueras, Kristina Höök, and Airi Lampinen detachment that can be damaging for a person [49]. Considering ethics solely through organizational lenses, then, overshadows the lived experience of people affected by technology, along with those who are developing algorithmic systems. Given the contextual nature of ethical action that cannot be adequately formalized through a system of rules [40, 100, 103], this is problematic for efforts to develop technology ethically. Strict reliance on organizational understandings of ethics poses the risk of ignoring broader questions, such as asking whether the technology should be built in the first place [97], since considering such questions is unlikely to be part of organizational guidelines and, thus, a matter of no one’s responsibility. 6 DISCUSSION: BETWEEN VULNERABILITY AND DISTANCING Our analysis follows the tradition of studying ethics in practice: approaching ethics on the tech- nology production site as a matter of concrete technical decisions [91], situated in organizational contexts [13, 42, 64]. With a few exceptions [31, 64, 95], prior studies have focused on the work of industry practitioners based in the US. We broaden the state of the art by interviewing technology practitioners who are located in Scandinavia and by including participants who work in the public sector and in academia. Crucially, our research furthers the exploration of the emotional aspect of ethical responsibility, started by researchers in Interaction Design [4, 50, 82] and scholars of Ethics in Practice who have analyzed how recognizing ethical concerns affects technology practitioners’ feelings [99, 107]. We contribute to the study of ethics in practice by focusing on the emotion work related to recognizing an ethical issue and accepting (or refusing) the responsibility to act on it. While the importance of developing ethical sensitivity – the ability to recognize ethical issues – has been studied and documented thoroughly in prior work [13, 64, 91], we focus on the next step: taking responsibility for ethical action. We argue that the emotional aspect of attributing responsibility is central to practitioners’ subsequent choices to act or to refrain from taking action. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 6.1 Taking ethical responsibility involves bearing an emotional burdenif Our findings describe how the three stances place a different degree of responsibility on the individual practitioner, either liberating them from any personal need to act or putting them on the spot. While taking the actionable I-stance may seem empowering in that it positions the individual with high responsibility and high personal involvement, it also makes the individual vulnerable. Taking more distanced we- and they-stances can, in contrast, help individuals to avoid emotional discomfort, at least in the short term. With the we-stance, the interviewees were able to speak about ‘we’ or ‘us’, defining themselves as part of a collective subject while avoiding a direct personal need to act. Within the distanced they-stance, we have noted three separate distancing techniques, which our interviewees deployed to frame ethics as a matter of someone else’s responsibility: (1) distinguishing between the ethical and the technical; (2) limiting the domain of ethics to interactions with end-users; and (3) marking only certain areas of technology application as ethically problematic and, thus, as requiring ethical reflection. These distancing techniques also explain why participants come to not feel the need to carry ethical responsibility in their workplaces. We highlight that the very fact of noticing an ethical issue and acknowledging one’s personal role in acting puts emotional burden on the individual. Due to the high emotional costs of responsibility, it is reasonable that accounts from the I-stance were not that common among our participants. Here, our findings align with the research of Widder and colleagues [107] that reports on emotional distresses and sensations of anxiety, depression, and isolation related to raising ethical concerns in tech companies by workers who had no actual power to address such issues. Similarly, when taking the I-stance, most of our interviewees ended up feeling distressed: sad, ashamed, frustrated, or left with a feeling of ‘losing oneself.’ In our data, positive examples of taking ethical action came Who Should Act? 157:19 from the area of research ethics, especially from interactions with participants in participatory design and co-design projects where the interviewees had the power and resources to act in line with their ethical sensitivities and to work on establishing trusting and respectful relationships.i Taking the I-stance could certainly be beneficial for the development of more ethical technology, yet given the high cost of acting upon ethical issues, it is only reasonable that our interviewees deployed a variety of distancing techniques. 6.1 Taking ethical responsibility involves bearing an emotional burdenif Given the similarities between our findings and the findings from Widder and colleagues [107], we can say that individuals located in different institutional and regional settings – yet in structurally similar circumstances where they lacked the power to address their ethical concerns – were experiencing similar feelings of distress, anxiety, or disappointment. This structural shaping of how emotions come to matter for ethical (in)action is a central part of why emotions should not be dismissed from conversations about ethics as individual and volatile matters. Both we- and they-stances allow interviewees to distance themselves from ethical responsibility, hence largely avoiding negative emotional consequences related to even recognizing an ethical issue. The distanced stances relieve practitioners from discomforting feelings of powerlessness, responsibility, and internal conflict related to the discovery of injustice. But there is a danger in both taking the I-stance and not taking it. The danger of being too vulnerable relates to the possibility of experiencing an emotional crisis and/or facing material consequences for acting. The danger of distancing, on a psychological level, manifests in the form of alienation and detachment – something that Hochschild noted in workers who had to engage in emotion work in the service industry [49]. At scale, the danger of distancing lies in that it may enable broader negative outcomes, such as the production of harmful technology. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 6.2 Steps toward making ethics actionable While we make an analytic distinction between situations of low- and high-scale technology development, there is no opposition between the scales: all high-scale projects demand personal involvement. Large AI systems are built by workers who pay attention to seemingly ‘small’ things, such as cleaning data sets [80, 89]; ethical guidelines are driven by enthusiasts fighting their way through organizational structures [65, 84]; the implementation of otherwise abstract principles is made possible by very concrete people [78, 109]. The I-stance has to be taken in order to make high-scale settings work, but taking the I-stance is likely to have serious negative consequences for individuals with limited power. That said, we join claims to refrain from framing ethical matters as matters of individual decision-making and responsibility [8, 87]. Our suggestions align with prior calls to develop more supporting structures for practitioners who take responsibility for acting ethically, both in low-scale projects and within big tech companies [65, 83]. In what follows, we discuss three strategies to make ethics actionable: normalizing a degree of vulnerability and discomfort, developing organizational structures that support individual ethical action, and developing hands-on approaches to demonstrate the limits of individual action at the technology production site. 6.2.1 Normalizing vulnerability and discomfort. Taking an actionable stance is needed to implement change. Therefore, our first conclusion is that we have to normalize the vulnerability of the I- stance along with a degree of discomfort that follows from recognizing ethical issues. Inspiration for becoming more vulnerable and shouldering more personal responsibility can be taken from first person perspectives in interaction design, such as soma design [52, 53], that strive to reduce the distance between the creator and their work, and encourage approaching one’s inherent vulnerability not as a problem that needs to be hidden but as a ground for connection and a reason to care for each other [82]. Approaching design work from a vulnerable standpoint means accepting Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 157:20 Kristina Popova, Clàudia Figueras, Kristina Höök, and Airi Lampinen the value of our felt experiences, discomfort, and failures [55] and respecting gut feelings – the not-yet-fully formalized discomforts and sensations of unease that often point at problems which were not yet articulated. 6.2 Steps toward making ethics actionable Such an approach to social interaction on the technology production site implies building new grounds for collective action, based on ideas of the inherent relationality (rather than a supposed autonomy) of human beings. This aligns closely with ethics of care [24, 100]. ( pp y) g g y [ ] Normalizing vulnerability means accepting that a degree of discomfort will always be part of an ethical practice of technology production, especially in high-scale settings. Further, even within low-scale projects, an individual’s control is limited. For example, design materials carry their own histories [23] which can introduce traces of colonialism and oppression even into the purportedly most ethical and justice-oriented designs. Accepting and articulating the discomfort of not having total control over the process while still feeling called upon to bear responsibility for its outcomes can be the first necessary step in changing the status quo. 6.2.2 Developing structures to support ethical action. Besides normalizing a certain degree of vulnerability and the discomfort that comes with it, we need to develop structures to support ethical action. Otherwise, it is very likely that those who are in the least powerful positions will end up being the most vulnerable and bearing the biggest cost for taking responsibility. Vulnerability is not a meaningful answer when there is no safety and equality, or when urges to be vulnerable come from those in positions of power and security towards those who are located lower in a social hierarchy. Liboiron and colleagues provide an example of a situation where the request for being vulnerable evokes nothing but anger: “I was giving a talk about CLEAR’s feminist science at a university reputed for its progressive politics. It hosted one of the most alienating Q&As I’ve ever been invited to (except for that time in that philosophy department). One question stood out. A woman asked me to share my failures in the lab. I asked what she meant. She said, “I want you to be more vulnerable.” I obliged her. Later, I was furious. Furious for the question—asking me to be more vulnerable, standing at the front of the room as a small, Native woman whom academia and dominant science are built to erase, trying to work where there are few roadmaps. I was furious no allies in the room stepped in to intervene, despite their rhetoric of allyship. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 6.2 Steps toward making ethics actionable But mostly I was furious at myself for obliging instead of burning that house down.” [62, p. 147]. Attempts to label ethical decision-making on the technology production site as solely an individual responsibility are dangerous for those who have little protection within organizational hierarchies. Vulnerability has to be equalized with the degree of power. When the outcomes of an action are beyond one’s control or when one lacks the ability to make the necessary sacrifices, distancing becomes a matter of safety. While protocols, guidelines, and formal procedures are imperfect, at the lack of better means, they may serve to ensure the safety of individuals in structurally vulnerable positions within their organizations. However, solely relying on protocols is insufficient. It is crucial to have supporting structures that provide checks and balances, such as technology workers’ associations. 6.2.3 Reckoning with the limits of individual agency. The conversation about ethics of technology is too often framed with an excessive focus on the individual as the main actor of change. This is in stark contrast to the distributed nature of technology development. To create a more realistic perspective on the value of individual efforts that are not supported by associations and other forms of political power, we suggest bringing forms of reasoning from Science and Technology Studies (STS) closer to tech practitioners, that is, making the consequences and limitations of individual action more readily felt. Attending to the histories of design goals and materials, including data, Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. Who Should Act? 157:21 can stimulate us to recognize the limits within which we can induce change and help us respect the unexpected consequences our work may have. We see an opportunity here to bring ideas that are almost taken for granted within STS, such as the agency of technological artifacts and politically motivated decisions that can underlie their design [108], closer to design and engineering practice. In practice, this can mean developing historical sensibility [93] – demonstrating the historical, consecutive nature of technological development – through hands-on work with design practition- ers. One successful example of this type of interdisciplinary translation is the implosion exercise that Dumit developed based on Haraway’s scholarship [28]. 6.2 Steps toward making ethics actionable In this exercise, participants are asked to pick up a simple artifact, like a pen, and answer a number of questions to trace its connection to the world outside of the classroom/workshop. The questions can be very concrete: “How was it produced and who is involved in its production?”, “What materials are involved in its production and mainte- nance? Where have these materials come from?” [28]. An example of using the implosion exercise to uncover the connections between cultural discourses, media, and intellectual lineages in emotion detection can be found in recent work by Arnelid, Harrison and Johnson [3]. Another example is Joler and Crawford’s [23] critical map of the Amazon Echo device which uncovers the supply chain of materials, planetary resources, data, and human labor behind the device, highlighting injustices and exploitation at each stage of the process. What we suggest as a next step is experimenting with making the complexity and relationality of technology production as tangible and felt as possible to highlight the limitations of individual agency in relation to the complex histories of materials. We encourage CSCW scholars to work on developing hands-on ways to introduce historicism into the technology production site and to have conversations about ethics, not only analytically but also in a way that appeals to our felt sense as technology practitioners. This is not an easy task since high-scale technology by its nature produces distance between its users and creators and renders the underlying complexity of its production invisible. Yet, we believe such an intervention is needed to bridge the gap between our emotional understanding of ethics and the distributed nature of technology production. This can help us not only to avoid placing unrealistic hopes on the potential of an individual reflexive practitioner but also to recognize opportunities to make change collectively. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. 7 CONCLUSION We have considered emotions related to recognizing ethical issues on the technology production site and attributing responsibility for acting on them. While the scholarly conversation about ethics has traditionally focused on searching for a rational ground for ethical action, we argue for the need to attend also to the emotional component of ethics as it helps explain why taking responsibility and even recognizing an ethical issue can be so problematic. Based on an interview study with technology practitioners from academia, technology industry, and the public sector, we have identified three ethical stances – I, we, and they – that technology practitioners use when providing accounts for taking or not taking ethical responsibility. Further, we have introduced the concepts of vulnerability and distancing to analyze the differences between these stances and the emotion work that they entail. Our analysis demonstrates that recognizing an ethical issue and taking an actionable I-stance towards it places a heavy burden on practitioners, especially when they do not have the power to act upon their ethical sensitivities. Cultivating ethical sensitivity, then, is important but not sufficient to enable productive ways of acting. We argue for the importance of building new organizational practices and safe conditions for taking personal responsibility for ethical action. We invite researchers to draw upon first person perspectives in design and STS approaches to acknowledge the limitations of individual control over technology development and to recognize opportunities to make change collectively. 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Interact. 5, CSCW2, Article 355 (oct 2021), 28 pages. https://doi.org/10.1145/3479499 [110] Richmond Y. Wong, Michael A. Madaio, and Nick Merrill. 2023. Seeing Like a Toolkit: How Toolkits Envision the Work of AI Ethics. Proc. ACM Hum.-Comput. Interact. 7, CSCW1, Article 145 (apr 2023), 27 pages. https: //doi.org/10.1145/3579621 [111] Haiyi Zhu, Bowen Yu, Aaron Halfaker, and Loren Terveen. 2018. Value-Sensitive Algorithm Design: Method, Case Study, and Lessons. Proceedings of the ACM on Human-Computer Interaction 2, CSCW (Nov. Proc. ACM Hum.-Comput. Interact., Vol. 8, No. CSCW1, Article 157. Publication date: April 2024. REFERENCES 2018), 1–23. https://doi.org/10.1145/3274463 g [112] Jarrett Zigon. 2007. Moral breakdown and the ethical demand: A theoretical framework for an anthropology of moralities. Anthropological Theory 7, 2 (2007), 131–150. https://doi.org/10.1177/1463499607077295 Received January 2023; revised July 2023; accepted November 2023
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Thermodynamic Evaluation of the Intermediate Liquid Compounds (ILC) from Biomass Fast Pyrolysis
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IOP Conference Series: Earth and Environmental Science IOP Conference Series: Earth and Environmental Science PAPER • OPEN ACCESS Thermodynamic Evaluation of the Intermediate Liquid Compounds (ILC) from Biomass Fast Pyrolysis To cite this article: Guiyu Xiao et al 2021 IOP Conf. Ser.: Earth Environ. Sci. 651 022001 View the article online for updates and enhancements. This content was downloaded from IP address 194.167.201.151 on 12/02/2021 at 12:01 s content was downloaded from IP address 194.167.201.15 This content was downloaded from IP address 194.167.201.151 on 12/02/2021 at 12:01 3rd International Conference on Green Energy and Sustainable Development IOP Conf. Series: Earth and Environmental Science 651 (2021) 022001 IOP Publishing doi:10.1088/1755-1315/651/2/022001 3rd International Conference on Green Energy and Sustainable Development IOP C f S i E th d E i t l S i 651 (2021) 022001 d i 1 doi:10.1088/1755-1315/651/2/0 *Corresponding author e-mail: marion.carrier@mines-albi.fr Abstract. Biomass fast pyrolysis process is a technology that converts renewable solids into a dense liquid. This study aims to apprehend the thermodynamic behaviour of the Intermediate Liquid Compounds (ILCs) observed during the biomass fast pyrolysis. The system studied was a closed system (20 mL) with air and a mixture solution of five components (Acetic acid (AA), hydroxyacetone (HX), phenol; furfural (FF) and methanol) at 90°C and under atmospheric pressure. The flash calculation was conducted at a given temperature and pressure. The vapor-liquid equilibrium compositions were determined combining equation of state and activity coefficient models, the Soave- Redlich-Kwong (SRK) equation of state coupled with Modified Huron-Vidal (MHV2) mixing rules incorporating the UNIversal Functionnal Activity Coefficient (UNIFAC) model. Theoretical calculations of vapor-liquid equilibrium compositions were experimentally validated by using a Head-Space GC-MS system. A quantitative agreement between simulated and measured concentrations in the liquid phase was achieved with this combined state-predictive model of SRK-MHV2-UNIFAC model; thus, confirming that it accounts well for the nonidealities. Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 Guiyu Xiao 1, 3, Marion Carrier 2, *, Jean-Jacques Letourneau 2, Yani Zhang 1, 3, Yi Wang 1, 3 Guiyu Xiao 1, 3, Marion Carrier 2, *, Jean-Jacques Letourneau 2, Yani Zhang 1, 3, Yi Wang 1, 3 1 China-EU Institute for Clean and Renewable Energy, Huazhong University of Science and Technology, Wuhan, 430074, China 2 RAPSODEE, CNRS UMR 5203, Université de Toulouse, IMT Mines Albi, Campus Jarlard, 81013 Albi CT Cedex 09, France 3 State Key Laboratory of Coal Combustion, School of Energy and Power Engineering, Huazhong University of Science and Technology, Wuhan, 430074, China Yi Wang 1 China-EU Institute for Clean and Renewable Energy, Huazhong University of Science and Technology, Wuhan, 430074, China 2 RAPSODEE, CNRS UMR 5203, Université de Toulouse, IMT Mines Albi, Campus Jarlard, 81013 Albi CT Cedex 09, France 3 State Key Laboratory of Coal Combustion, School of Energy and Power Engineering, Huazhong University of Science and Technology, Wuhan, 430074, China *Corresponding author e-mail: marion.carrier@mines-albi.fr Thermodynamic Evaluation of the Intermediate Liquid Compounds (ILC) from Biomass Fast Pyrolysis Guiyu Xiao 1, 3, Marion Carrier 2, *, Jean-Jacques Letourneau 2, Yani Zhang 1, 3, Yi Wang 1, 3 3rd International Conference on Green Energy and Sustainable Development IOP Conf. Series: Earth and Environmental Science 651 (2021) 022001 IOP Publishing doi:10.1088/1755-1315/651/2/022001 3rd International Conference on Green Energy and Sustainable Development IOP Conf. Series: Earth and Environmental Science 651 (2021) 022001 IOP Publishing doi:10.1088/1755-1315/651/2/022001 3rd International Conference on Green Energy and Sustainable Development IOP Conf. Series: Earth and Environmental Science 651 (2021) 022001 IOP Publishing doi:10.1088/1755-1315/651/2/022001 d International Conference on Green Energy and Sustainable Development of ILC is a common stage to all lignocellulosic polymer pyrolysis. Some of the existing research has focused on the formation of ILC during the fast pyrolysis of cellulose. They often called this type of ILC as "active" cellulose or melt cellulose [3-5]. Some essential physical properties of ILC are shown in Table 1. of ILC is a common stage to all lignocellulosic polymer pyrolysis. Some of the existing research has focused on the formation of ILC during the fast pyrolysis of cellulose. They often called this type of ILC as "active" cellulose or melt cellulose [3-5]. Some essential physical properties of ILC are shown in Table 1. The formation of ILC occurs at early stages of fast pyrolysis. As a result, the ILC is considered to have a lower degree of polymerization (DP) and crystallinity [6]. However, the exact structure and chemical composition of ILC is still unknown. Experimental and kinetic studies could be evidently interesting aspect for the study of ILC. However, due to the extremely short life of ILC, it is impossible to obtain ILC samples directly. Therefore, it is an attractive approach to explore the thermodynamic behavior of ILC through model compounds. p Table 1. Main thermophysical characteristics of ILC Table 1. Main thermophysical characteristics of ILC Fraction Formation Temperature (℃) Life Time(s) Liquid Density (kg/m3) Surface Tension (N/m) Viscosity (kg/m*s) Cellulose ILC 300-350 [3] 0.02-0.1[5] 1000 [5] 10-6-10-5 [3] 10-6-10-5 [3] Hemicellulose ILC 200-275 [3] --- --- --- Lignin ILC 200-350 [3] --- --- --- Figure 1 proposed a degradation model for lignocellulose pyrolysis. The objective of this study is to apprehend the thermodynamic behavior of ILC model solutions. Acids, phenols, aldehydes and ketones, furans, and alcohols are the five major chemical families that compose bio-oil. Therefore, five compounds from those five major chemical families will be used to formulate the ILC solution: Acetic acid (AA), hydroxyacetone (HX), phenol, furfural (FF) and methanol. We are interested in predicting the vapor-liquid equilibrium of this model mixture. 3rd International Conference on Green Energy and Sustainable Development IOP Conf. Series: Earth and Environmental Science 651 (2021) 022001 IOP Publishing doi:10.1088/1755-1315/651/2/022001 Simulis thermodynamics software will be used to determine the thermophysical properties and the vapor-liquid equilibrium composition of the mixture. Different models such as Soave-Redlich-Kwong (SRK) equation-of-state combined modified Huron- Vidal (MHV) mixing rule and UNIFAC (SRK-MHV2-UNIFAC) will be tested for simulation. The experimental validation will be done using the Head-Space system coupled with a gas chromatography/mass spectrometry. Figure 1. Proposed degradation model for lignocellulose pyrolysis Figure 1. Proposed degradation model for lignocellulose pyrolysis 1. Introduction d l To respond to global concerns linked to climate changes caused in a large part by the increasing energy demand and fossil fuels use, alternative and renewable energy solutions such as biomass-based conversion technologies have received considerable attention. Conversion of biomass into liquid fuel, namely bio-oil, using a thermochemical process, most specifically the fast pyrolysis, has received great interest [1]. Fast pyrolysis is the rapid thermal decomposition of organic compounds in the absence of oxygen, which can be considered as the superposition of three degradation processes respectively for the three main components that are cellulose, hemicelluloses and lignin [2]. In fact, a melting phenomenon can be observed in the experiment of pyrolyzing biomass, which is called Intermediate Liquid Compound (ILC). The short state-of-the-art on pyrolysis mechanisms suggests that the formation 1 2.2. Modelling approach g pp The system studied is a closed system with air and liquid mixtures under the ambient temperature and atmospheric pressure. Input and output information were collated in an Excel spreadsheet in which was inserted a Simulis Calculator object. The Simulis calculator allows the selection of compounds and models. The initial default temperature was 25°C and pressure was 1 atm. Using pressure and temperature as input values of this system, the pressure was kept at 1 atm and the temperature was determined by the boiling point of liquid phase. The thermodynamic properties such as vaporization rate, density, bubble point, and dew point were determined using TP-Flash Simulis Calculator. The vapor- liquid equilibrium of the system could be predicted by using SRK-MHV2-UNIFAC predictive model at giving temperature and pressure. Then, the concentration of each component in gas and liquid phases could be obtained. 3rd International Conference on Green Energy and Sustainable Development IOP Conf. Series: Earth and Environmental Science 651 (2021) 022001 IOP Publishing doi:10.1088/1755-1315/651/2/022001 3rd International Conference on Green Energy and Sustainable Development IOP Conf. Series: Earth and Environmental Science 651 (2021) 022001 IOP Publishing doi:10.1088/1755-1315/651/2/022001 feedstocks and systems found in the literature [7]. The ILC model mixture was composed of 33.3 wt% acetic acid, 33.3 wt% hydroxyacetone, 13.3 wt% phenol, 13.3 wt% furfural, and 6.8 wt% methanol. feedstocks and systems found in the literature [7]. The ILC model mixture was composed of 33.3 wt% acetic acid, 33.3 wt% hydroxyacetone, 13.3 wt% phenol, 13.3 wt% furfural, and 6.8 wt% methanol. 2.1. Chromatographic methods 2.1. Chromatographic methods The samples were analyzed using a Head-Space system coupled with a GC-MS system (Shimadzu GCMS-TQ8030). The chromatographic conditions were based on Moore et al. [8]. The headspace system (Shimadzu AOC-5000 Plus) is an injection unit of the gas chromatograph using the gas-tight syringe technique. The incubation setting temperature of the Head-Space system should be lower than 180°C, which is a technical parameter of the equipment and represents an operating limitation to reach for pyrolysis conditions. An amount of sample was placed into an incubation oven at a given temperature until the sample reaches the thermodynamic equilibrium. Then, an aliquot of volatile components was taken by the heated syringe and injected into the GC-MS. It is a sensitive technique for testing volatile organic compounds. The column was operated in a constant flow mode, 1.26 mL/min, using Helium (Scientific grade 6.0, Linde France) as the carrier gas with a solvent delay of 1.5 min. The heating program settings were initially maintained at 50 °C for 4 min before to be increased to 250 °C at a heating rate of 10 °C/min, and thereafter held for 5 min. The mass spectrometer was operated in an electron ionization mode at 70 eV. The ion fragments were separated via a quadrupole and the detection range set as 30-600 Da. The selected ion monitoring (SIM) mode was used to quantify the specific analytes. Compounds were identified according to the NIST Standard Reference database (NIST SRD 69). The incubation temperature needs to be lower than the bubble point of the solution to ensure that the air pressure in the glass vial of 20 mL (Restek) does not change during the incubation process. This precaution prevents damage to the syringe caused by high air pressure in the glass vial. A thermodynamic calculation using Simulis Thermodynamics® was performed to determine the bubble point of each mixed solution to select the incubation temperature. The temperature of the syringe should be higher than that of the incubator to prevent condensation on the surface of the syringe when taking samples. The direct injection mode was used for analysing liquids and the Headspace injection mode was used for gas analysis. Duplicate measurements were made to determine the reproducibility. The methanol was not quantified as its retention time was lower than that of the solvent. 2. Materials and Methods Standard solutions for calibrations and model intermediate compounds were prepared adding primary standards together: Acetic acid (AA, 99.9%, Sigma-Aldrich), hydroxyacetone (HX, 95%, Alfa Aesar), phenol (99.9%, Sigma-Aldrich), furfural (99%, Acros Organics), methanol (99.9%, Sigma-Aldrich). Acetone (99.9%, Suprasolv®) and fluoranthene (98%, Sigma-Aldrich) were used respectively as solvent and internal standard for preparing the standard solutions. The acetone was selected as solvent because it is a polar solution, in which the model pure compounds were miscible. The selection of main compounds was made according to the chemical composition of bio-oils produced from a wide range of 2 3.2. Optimization of chromatographic conditions p f g p By using the internal standard method for calibration to determine the concentration of analytes in unknow sample, the GC conditions were optimized to avoid the electronic saturation of the MS detector. To do this, different split ratios were used to control the detector response. When the response was too weak the Selective Ion Monitoring (SIM) mode was used to detect the specific analytes. The selection and tracking of specific mass fragments can increase the detector sensitivity relative to full scan mode. The main parameter to be optimized is the split ratio according to the analyte’s concentration. It was found that there is a non-linear correlation coefficient between different split ratios when analysing the same analyte. The relative Split Ratio Factor (A/B) (equation 1) was used to describe this phenomenon. Relative Split Ratio Factor ቀ A Bቁ= RSRF( A B) = [X]A [X]B (1) (1) Where [X]A is the calculated concentration of X for a split ratio A; [X]B is the calculated concentration of X for a split ratio B. Where [X]A is the calculated concentration of X for a split ratio A; [X]B is the calculated concentration of X for a split ratio B. By using this factor that describes the effect of split ratio value on concentration, it is possible to relate two measured concentrations for a same component determined at different split ratios. Those relationships depend on the factor used to draw the calibration curves: when using Concentration (X- axis) and Area ratio (Y-axis), the RSRF (50/10) is equal to 0.95±0.00008 and the RSRF (200/10) corresponds to 0.385±0.005; when using Concentration (X-axis) and Area of Analyte (Y-axis), the RSRF (200/10) is 18.9±2.18. Table 2 shows all the equations of calibration curve used to determine the analytes concentration in the ILC model mixture. It can be seen, all the regression coefficients are above 0.99, indicating that the calibration curve equation fits well the experimental values. le 2. Equations of calibration curves associated with regression coefficient (R2) for ILC mixture according to the chromatographic conditions. Table 2. Equations of calibration curves associated with regression coefficient (R2) for ILC mixtures according to the chromatographic conditions. 3.1. Properties of mixtures The calculated physical properties of the ILC mixture were found to be: 110°C for the bubble point, 151°C for the dew point and 1.048 g/mL for the density. As a result, the incubation temperature of the Head-Space system was set at 90°C, an incubation temperature lower than the bubble point of 110°C. Those experimental conditions allowed the investigation of vapor-liquid equilibrium, which is also a very necessary part of the thermodynamic evaluation process. 3 3rd International Conference on Green Energy and Sustainable Development gy p IOP Conf. Series: Earth and Environmental Science 651 (2021) 022001 g doi:10.1088/1755-1315/651/2/022001 P Conf. Series: Earth and Environmental Science 651 (2021) 022001 doi:10.1088/1755-1315/651/2/02 Figure 2 shows experimental records of the ageing test for the ILC model mixture. The results indicate that ILC model mixture was stable during its preparation and use, and that the substances did not react with each other at 25℃. Indeed, there were no by-products separated by the GC column and detected by the MS detector, which further suggests that no chemical reaction could occur between those model components. Figure 2. Concentrations measured in liquid mixture at 25℃ before heating. Figure 2. Concentrations measured in liquid mixture at 25℃ before heating. 3.2. Optimization of chromatographic conditions Liquid phase Gas phase Injection mode Direct Headspace Concentration range (mg/mL) 70-354 0.0061-0.3550 Product AA HX FF Phenol AA HX FF Phenol Scan or SIM mode Scan Scan Scan Scan Scan Scan Scan Scan Slope 14.74 15.758 126.79 111.43 30,000,000 30,000,000 4000,000 4000,000 y-intercept -0.871 -0.204 -16.69 -15.08 -2000,000 -720998 -598856 -512122 R2 0.9958 0.9976 0.9999 0.9941 0.9968 0.9995 0.9988 0.9926 4 3rd International Conference on Green Energy and Sustainable Development IOP Conf. Series: Earth and Environmental Science 651 (2021) 022001 doi:10.1088/1755-1315/651/2/022001 After optimizing the chromatographic conditions, each component in the ILC model mixture could be detected (Figure 3) and quantified by GC/MS. Figure 3. Chromatogram indicating the separation of each ILC model compound after incubation of 15 min at 90°C. After optimizing the chromatographic conditions, each component in the ILC model mixture could be detected (Figure 3) and quantified by GC/MS. Figure 3. Chromatogram indicating the separation of each ILC model compound after incubation of 15 min at 90°C. 3.3. Vapour-Liquid Equilibrium Analysis of ILC model mixture Figure 4 presents the concentration of components in ILC model mixture after 15 minutes of incubation at 90°C. The vapor-liquid flash was calculated at the 90°C and under atmospheric pressure. The simulated values (L-sim, G-sim) and the measured values (L-exp, G-exp) obtained under the same initial conditions and associated relative deviations were compared. The results indicated that the calculated data were well correlated with the experimental values measured in liquid phase (Figure 4A) for the hydroxyacetone and phenol, the relative deviation (RD) were respectively 7.7% and 5.3%, less than 10%, which was not the case for the acetic acid (RD = 20.4%) and the furfural (RD = 38.3%). With respect to gas phase simulations, only an appropriate relative deviation of 3.6% was confirmed for phenol, while RD of 21.4%, 26.8% and 53.7%, respectively, for furfural, hydroxyacetone and acetic acid were found (Figure 4B). Those large deviations obtained for gaseous concentrations can be explained by the fact that the heated flask containing the liquid mixture was not sealed. As a result, further model investigations and a more appropriate experimental set-up are required to appropriately describe the thermodynamic properties of the liquid and therefore offers a better response to predict quantitative information in both liquid and gaseous phases. Figure 4. 4. Conclusion and Perspectives p Both TP flash thermodynamic model and head-space gas chromatographic methods were developed and used to predict the vapor-liquid equilibrium for the ILC model mixture. The system studied was a closed Air-Mixture solution system at 90°C and under atmospheric pressure. The chemical composition of the ILC model mixture was successfully assessed. The comparative study between simulated and measured values indicates that further investigations are required to confirm the encouraging results provided by the use of SRK-MHV2-UNIFAC model. Meanwhile, a chemical kinetic model should be developed for studying the system under dynamic conditions. Acknowledgments I gratefully acknowledge the support from the French scientific program MOPGA (ANR-18-MPGA- 0013) managed by the National Research Agency and financially supported by the “Investissements d’Avenir" and 'La Région d'Occitanie'. And, my special thanks go to the society ProSim for the academic use of Simulis Thermodynamics. References [1] Liu R, Sarker M, Rahman M M, et al. Multi-scale complexities of solid acid catalysts in the catalytic fast pyrolysis of biomass for bio-oil production-A review [J]. Progress in Energy and Combustion Science. 2020, 80: 100852. [1] Liu R, Sarker M, Rahman M M, et al. Multi-scale complexities of solid acid catalysts in the catalytic fast pyrolysis of biomass for bio-oil production-A review [J]. Progress in Energy and Combustion Science. 2020, 80: 100852. [1] Liu R, Sarker M, Rahman M M, et al. Multi-scale complexities of solid acid catalysts in the catalytic fast pyrolysis of biomass for bio-oil production-A review [J]. Progress in Energy and Combustion Science. 2020, 80: 100852. [2] Yang H, Yan R, Chen H, et al. Characteristics of hemicellulose, cellulose and lignin pyrolysis [J]. Fuel. 2007, 86(12): 1781-1788. [2] Yang H, Yan R, Chen H, et al. Characteristics of hemicellulose, cellulose and lignin pyrolysis [J]. Fuel. 2007, 86(12): 1781-1788. [3] Teixeira A R, Mooney K G, Kruger J S, et al. Aerosol generation by reactive boiling ejection of molten cellulose [J]. Energy & Environmental Science. 2011, 4(10): 4306-4321. [3] Teixeira A R, Mooney K G, Kruger J S, et al. Aerosol generation by reactive boiling ejection of molten cellulose [J]. Energy & Environmental Science. 2011, 4(10): 4306-4321. [ ] gy , ( ) [4] Lédé J. Cellulose pyrolysis kinetics: An historical review on the existence and role of intermediate active cellulose [J]. Journal of Analytical and Applied Pyrolysis. 2012, 94: 17-32. [4] Lédé J. Cellulose pyrolysis kinetics: An historical review on the existence and role of intermediate active cellulose [J]. Journal of Analytical and Applied Pyrolysis. 2012, 94: 17-32. [5] Lédé J, Blanchard F, Boutin O. Radiant flash pyrolysis of cellulose pellets: products and mechanisms involved in transient and steady state conditions [J]. Fuel. 2002, 81(10): 1269- 1279. [5] Lédé J, Blanchard F, Boutin O. Radiant flash pyrolysis of cellulose pellets: products and mechanisms involved in transient and steady state conditions [J]. Fuel. 2002, 81(10): 1269- 1279. [6] Sribala G, Carstensen H, Van Geem K M, et al. Measuring biomass fast pyrolysis kinetics: State of the art [J]. WIREs Energy and Environment. 2019, 8(2): e326. gy ( ) [7] Li M, Zhang M, Yu Y, et al. Ternary System of Pyrolytic Lignin, Mixed Solvent, and Water: Phase Diagram and Implications [J]. Energy & Fuels. 2018, 32(1): 465-474. 3.2. Optimization of chromatographic conditions Concentrations of each component in ILC model mixture: A) in liquid phase and B) in vapour phase after an incubation period of 15 min at 90°C. Figure 4. Concentrations of each component in ILC model mixture: A) in liquid phase and B) in vapour phase after an incubation period of 15 min at 90°C. The overall mass balance is also displayed in Figure 5. It is important to note that experimental concentrations were employed while the total produced liquid and gas volumes were computed. Despite of the large standard deviation found in the case of acetic acid, both chromatographic quantification developments and use of the SRK-MHV2-UNIFAC model appear to be a suitable approach for studying liquid equilibrium. 5 3rd International Conference on Green Energy and Sustainable Development 3rd International Conference on Green Energy and Sustainable Development IOP Conf. Series: Earth and Environmental Science 651 (2021) 022001 IOP Publishing doi:10.1088/1755-1315/651/2/022001 IOP Conf. Series: Earth and Environmental Science 651 (2021) 022001 doi:10.1088/1755-1315/651/2/022001 Figure 5. Mass balance for each analyte before (m0) and after (m) incubation at 90°C. Figure 5. Mass balance for each analyte before (m0) and after (m) incubation at 90°C. References g p gy ( ) [8] Moore A, Park S, Segura C, et al. Fast pyrolysis of lignin-coated radiata pine [J]. Journal of Analytical and Applied Pyrolysis. 2015, 115: 203-213. 6 6
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Multi-objective optimal of surface-groundwater resources in the Dongxiezong irrigation district
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1 Preface Single-objective optimization can offer one and only optimization result[1]. However, for multi-objective optimal allocation of water resources, it is difficult to obtain an optimal solution, and normally, a series of non-inferior solutions are obtained[2]. Sarker and Ray proposed a crowd-based approach for optimization. They solved two types of optimization problems using a multi-objective optimization model based on three different optimization methods[3]. Raju and Kumar established a multi-objective optimization model based on genetic algorithm in order to solve the problem of optimal reservoir scheduling while taking into consideration the crop planting structure[4]. Das and Datta developed a corresponding multi-objective management model group by establishing the relationship between a simulation model and an optimization model using embedding methods so as to use coastal aquifer groundwater more effectively [5]. Peralta and Datta developed a planning model by using embedded methods for regional sustainable income planning. The aim of this model is to maximize the total flow of the pump under the constraints that the pre-specified target water flow is stable and the damage on the existing planting structure is minimized [6]. sorting genetic algorithm (NSGA-II) with that of the sequence genetic algorithm (SGA), it can be seen that NSGA-II model can greatly reduce the computational cost of simulation optimization [7]. Bazargan-Lari et al. proposed a fuzzy multi-objective linear programming model based on the surface-groundwater joint configuration model featuring the conflict resolution method [8]. Rothmanand Mays established a multi-target GA model (MOGA) to assess the performance of sustainable water supply of the system. This model, taking into consideration the balance of the supply and demand of the total and underground, aiming at cost control, aquifer protection and growth, can make decisions on the water distribution of a certain area in a specific year [9]. Artificial neural networks (ANN) are often used to build the tissue structure of the central nervous system[10]. ANN obtains an optimization plan through a learning process similar to that of the human brain. ANN is widely applied in many scientific fields for it creates a good approximation of a complex system through simple structures. Rao et al. established a conceptual regional joint planning model aiming to ensure sufficient water for crops so as to optimize the joint surface-groundwater allocation in the delta region of Eastern India. Rao et al. also proposed that the annealing algorithm can reduce the difficulty brought by the nonlinearity and non-convexity of the model itself. Corresponding author: 1183506777@qq.com Multi-objective optimal of surface-groundwater resources in the Dongxiezong irrigation district Hengyue Yang1,Shaohui Zhang1,Wei Dai1,Yinong Li1,Xin Zeng2 1 State Key Laboratory of Simulation and Regulation of Water Cycle in River Basin, China Institute of Water Resources and Hydropower Research, 20 West Chegongzhuang Rd., Beijing 100038, China. E-mail: 1183506777@qq.com; daiwei@iwhr.com 2Water Recourses and Hydropower Planning and Design General Institute, Beijing 100120, China) Abstract: the water cycle in irrigation districts is extremely complicated under the dual influence of strong human activities and the nature. To establish the multi-water source rational allocation model of irrigation district, this paper first establish a multi-objective function based on economic utility, ecological utility and irrigation performance and improve Hicks optimization method. Then, combine it with chaotic particle swarm optimization algorithm to carry out research on temporal and spatial distribution evolution and optimal allocation of water resources in irrigation districts and collaborative scheduling and regulation of surface-groundwater. The multi-objective rational allocation is an important basis for the efficient use of water resources in irrigation districts and ecological harmony. This paper takes the typical irrigation area of Dongxiezong in Heilongjiang Province as the object for the study of the optimal allocation method of water resources in the irrigation district. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 ses/by/4 0/) , 0 (2018) MATEC Web of Conferences 2 ISWSO 2018 46 2054 , 0 (2018) MATEC Web of Conferences 2 ISWSO 2018 46 2054 https://doi.org/10.1051/matecconf/201824602054 1 Preface The combination of artificial neural network and high-efficiency algorithm of water flow In recent years, Rezapour Tabari and Soltani established a multi-objective optimization model in order to maximize the reliability of the model system while minimizing the cost of water and aquifer backwater utilization. Compare the efficiency of the non-dominant , 0 (2018) MATEC Web of Conferences 2 ISWSO 2018 46 2054 , 0 (2018) MATEC Web of Conferences 246 2054 https://doi.org/10.1051/matecconf/201824602054 movement stimulation can effectively reduce the amount of calculation caused by the water flow model. The results show that ANN solved by the annealing algorithm is effective in the study of model simulation of actual scale [11]. Safavi trained ANN to simulate the surface-groundwater interaction, and established a simulation optimization model by taking the genetic algorithm as the optimization model. In this way, the optimal configuration of surface-groundwater resources under multi-constraint conditions is achieved to meet as many requirements for irrigation as possible. This study shows that the simulation optimization model established by combining ANN with and genetic algorithm is very flexible, and can serve irrigation system management research under different constraints and assumptions [12]. Rao et al. has conducted similar researches and established simulation optimization simulation based on ANN for optimal management of the irrigation system. movement stimulation can effectively reduce the amount of calculation caused by the water flow model. The results show that ANN solved by the annealing algorithm is effective in the study of model simulation of actual scale [11]. Safavi trained ANN to simulate the surface-groundwater interaction, and established a simulation optimization model by taking the genetic algorithm as the optimization model. In this way, the optimal configuration of surface-groundwater resources under multi-constraint conditions is achieved to meet as many requirements for irrigation as possible. This study shows that the simulation optimization model established by combining ANN with and genetic algorithm is very flexible, and can serve irrigation system management research under different constraints and assumptions [12]. Rao et al. has conducted similar researches and established simulation optimization simulation based on ANN for optimal management of the irrigation system. Among all points (A, B, C, D, and E), E is the optimal one, and the decision variable e associated with it is not worse than any other decision variables. 2.2.1 Objective function The optimization objective function in this study is irrigation utility, ecological utility and irrigation performance, which is expressed as: When it comes to optimizing the usage of finite resources, the traditional optimization model based on Parato optimization theory is outperformed by the optimization model based on Hicks optimization theory in terms of global optimality. Therefore, this paper establishes a multi-water source optimal allocation model based on Hicks theory in order to improve the global optimality of the results. ( ) ( ) ( ) log req avg max , , , , , , , , irrigation surfacewater i groundwater i eco y before after Hi i i F U Q Q c R U H H E Z Z Z     ( ) ( ) 0 1 water surfacewateri surfacewater surfacewater i surfacewater j surfacei j p Q Q U Q c Q c R η α λ = = + + +  groundwater water groundwateri i i p Q U R η = ecology = afteri i beforei H U H req req avg req avg 100% 100% i H i Hi Hi H i Z Z Z Z E Z Z Z Z  × ≥  =   × <  　　 　　 （1） ( ) ( ) 0 1 water surfacewateri surfacewater surfacewater i surfacewater j surfacei j p Q Q U Q c Q c R η α λ = = + + +  groundwater water groundwateri i i p Q U R η = ecology = afteri i beforei H U H  surfacewater U = 1 Preface For e in this case, the performance of the indicator can no longer be improved, so solutions such as vector e can be regarded a Hicks optimal solution. p The front end of Hicks optimal solutions Feasible solutions Same as D Better than D Worse than D Same as D The front end of Hicks optimal solutions Feasible solutions Same as D Better than D Worse than D Same as D The front end of Hicks optimal solutions Feasible solutions Same as D Better than D Worse than D Same as D 2.1 Hicks Optimization Theory There is no dominant relationship between Hicks optimal solutions. Improving the performance of one target can cause decline of performance other target. During Hicks improvement, it is acceptable to improve the performance of targets by reducing the performance of a certain target as long as the overall performance of the system is improved. For practical problems in engineering, it is reasonable to reduce the performance of the subsystem to improve the overall performance when the decrease of performance is acceptable in order to obtain the optimal solution of the engineering system. Based on the definitions of Hicks dominance, Hicks optimal solution, Hicks optimal solution set, Hicks frontier, Hicks frontier, and Hicks utility loss obtained from related references and the multi-objective optimization problem described above, Hicks optimization method is improved (see Figure 1). （1） Qsurfacei water is the total amount of surface water (m3) used in area i; Qgroundwateri is the total amount of ground water (m3) used in area i; α is the current value conversion factor, α=r(1+r)m/[(1+r)m-1]. Since the operation and maintenance of the irrigation-drainage dual-use canal system is relatively simpler, the depreciation expense is used as the cost for the convenience of calculation. Assume dual-use canal irrigation systems have the same service life of m years; λ is the annual operating cost coefficient; R is fuel cost, R=γEmnet ，allATzH/102ηpummp efficiency ηwaterTt with γ being water soluble weight (N/m3); Tz is the actual service life of the pump (h); H is the pump design head of delivery (m); η is the pump installed efficiency; E electricity price (yuan / kWh); T is the irrigation cycle (d); t is the time for daily irrigation (h); Hgroundwater after irrigation i is the groundwater level of point i after irrigation (m); Hgroundwater before irrigation i is the groundwater level of point i before irrigation (m); ZHi is the average irrigation depth of 1/2 strip with the lowest average inflation water (mm); Zreqi is the water required for irrigation at point I (mm); Zavg is the average infiltration depth at point i (mm). 3.3 Congestion comparison operator After applying fast non-dominated sorting and congestion calculation, each individual in the group has two attributes: the non-dominated order irank determined After applying fast non-dominated sorting and congestion calculation, each individual in the group has two attributes: the non-dominated order irank determined by the non-dominated sorting and the congestion degree id. The congestion comparison operator based on the above two attributes should meet the following two conditions: There are two objectives for optimization, namely, maximum effectiveness of the irrigation system and optimal irrigation performance. The objective function to be optimized, together with the many constraints, constitutes a multi-objective optimization model for allocating irrigation water in the research area. After that, the model needs to be instantiated according to the actual situation of the research area and the space and attribute data collected and processed in the early stage. The optimized model is solved with the appropriate multi-objective intelligent algorithm so as to obtain the optimal irrigation plan in the research area. by the non-dominated sorting and the congestion degree id. The congestion comparison operator based on the above two attributes should meet the following two conditions: ① If the non-dominated layer of individual i is superior to the non-dominated layer of individual j, that is irank<jrank; ② If they are in the same level, and individual i has a larger congestion distance than individual j, that is irank=jrank and id>jd. When both of the above conditions are met, individual i is superior to individual j. When both of the above conditions are met, individual i is superior to individual j. ① The congestion degree id of each point is set to 0; ① The congestion degree id of each point is set to 0; ② Apply non-dominated sorting to each target, set the congestion of the two individuals on the boundaries as infinite, that is Od=Id=∞; ③ The degree of freedom of the remaining individuals is calculated according to formula (5) ( ) 3 1 1 1 i i d j j j i f f + − = = −  （5） （5） id is the congestion degree of point i; fji+1 is the jth objective function value of point i+1, and fji-1 is the jth objective function value of point i-1. （2） （2） In formula (2), Qmax is the maximum amount of water in the irrigation process (m3), and other parameters have the same meaning as above. θmin is the lower limit of soil moisture content for crops (%), θ0 is the initial water content (%), θm is the volumetric water content of soil increased under the quota of irrigation district (%), and θf is the field water holding ratio (for dry land) or saturated water content (for paddy fields) (%); PAmax is the total irrigation area (mu, 1 mu=666.67 m3); Qg(u) is the actual water flow of the uth main canal in the research area during water delivery (m3/s), Qgmax is the design water flow of the uth main canal (m3/s), R is the number of main canals in the research area (unit: strip); Qp(u) is the actual water flow of the uth main canal in the research area during drainage (m3/s), Qpmax is the increased water flow of the uth main canal (m3/s), R' is the number of main dual-purpose canals for irrigation and drainage in the research area (strip); qg(v) is the actual flow of the vth branch channel in the research area rate during water delivery (m3/s), qgmax is the design flow of the vth branch channel (m3/s), r is the number of branch canals in the research area (strip); qp(v) is the actual flow of the vth branch channel in the research area rate during drainage (m3/s), qpmax is the increased flow of the vth branch channel (m³/s), r' is the number of branch dual-purpose canals for irrigation and drainage in the research area (unit: strip). （4） Each offspring Qt and its parent Pt are sorted according to the total objective function value of the system. The larger the function value, the better. The division of the non-dominated set is similar to that of the traditional NSGA-II. 2.1 Hicks Optimization Theory R=γEmnet ，allATzH/102ηpummp efficiency ηwaterTt with γ being water soluble weight (N/m3); Tz is the actual service life of the pump (h); H is the pump design head of delivery (m); η is the pump installed efficiency; E electricity price (yuan / kWh); T is the irrigation cycle (d); t is the time for daily irrigation (h); Hgroundwater after irrigation i is the groundwater level of point i after irrigation (m); Hgroundwater before irrigation i is the groundwater level of point i before irrigation (m); ZHi is the average irrigation depth of 1/2 strip with the lowest average inflation water (mm); Zreqi is the water required for irrigation at point I (mm); Zavg is the average infiltration depth at point i (mm). Figure 1, Principles of Hicks optimization. Figure 1, Principles of Hicks optimization. 2 https://doi.org/10.1051/matecconf/201824602054 , 0 (2018) MATEC Web of Conferences 2 ISWSO 2018 46 2054 2.2.2 Constraints The constraints are: , , max 1 1 I J i j i j i j PA CN Q = = ≤  min 0 f θ θ θ ≤ ≤ min 0 m f θ θ θ θ ≤ + ≤ , max 1 1 I J i j i j PA PA = = ≤  ( ) max g g Q u Q ≤ u=1,2,...,R ( ) max p p Q u Q ≤ u=1,2,...,R' ( ) max g g q v q ≤ u=1,2,...,r ( ) max p p q v q ≤ u=1,2,...,r' （2） Different weights are assigned to the three sub-goals in the objective function according to different requirements, and the integrated objective function of each water demand node can be expressed as: 1 2 log 3 Mi irrigate eco y F U U E α α α = − − （3） （3） In this formula: α1, α2, and α3 3 are respectively the weights of the irrigation utility function, the ecological utility function, and the irrigation performance function (α1+α2+α3=1). When the three are considered to be equally important, α1=α2=α3; when the utility is considered to be the most important, the risk the second, and the loss the least, α1>α2>α3, and so on. The overall integrated objective function of the pipe network system is expresses as: 1 n M Mi i F F = = （4） 1 n M Mi i F F = = 3.1 Fast non-dominated sorting 3 3 , 0 (2018) MATEC Web of Conferences 2 ISWSO 2018 46 2054 https://doi.org/10.1051/matecconf/201824602054 Figure 2,Canal layout in the research area canal layout. Initial contemporary population Gen=0, which generates initial population P0 and produce offspring Q0 according to crossover and mutation; the number of the individuals is N. The following operations are performed when the evolutionary algebra Gen<Genmax. ① Fast non-dominated sorting. Put the offspring Qt and its parent Pt into the set Rt (the number of individuals is 2N). Perform non-dominated sorting to the elements in Rt according to the evaluation standard of total integrated objective function value of the pipe network system expressed in formula (4-34), thereby form a non-dominated sequence Zi . ② Form an elite population. Put it into the elite population according to the number of layer of Zi and the two conditions in congestion comparison operator until the number of individuals in the elite population reaches the upper limit N. At this moment, the elite population is recorded as a new parent Pt+1. Figure 2,Canal layout in the research area canal layout. ③ Crossover and mutation. A new offspring Qt+1 is generated based on crossover and mutation. Figure 3,Sub-areas in typical irrigation area ④ Gen=Gen+1, enter the next cycle ④ Gen=Gen+1, enter the next cycle 5 Conclusion (a) (a) (b) Three objectives are considered simultaneously during the optimization of water resources in the irrigation area in this study, so it is a multi-objective optimization problem in the field of water resources optimization and scheduling. Therefore, this paper first expounds some concepts and basic theories about multi-objective optimization and displays mathematical expressions accordingly, including the composition of objective functions, decision variable space, constraints, etc. Based on the exploration of multi-objective optimization, this paper defines optimal Hicks and solutions to multi-objective optimization, which is part of the definition of Hicks optimal solution. A multi-objective optimization model with economic utility, ecological utility and irrigation performance as its target is established and applied in the typical area of the main irrigation area of Dongxie. Compared with the existing optimal allocation model of irrigation water resources, the multi-objective optimization model helps to save water by 17%~27% and pull up groundwater level by 2% to 22%. (b) (b) (c) (c) (d) Fi 4 R l f i i i i i i 4.2 Optimization results (a) Figure 5,Comparison of groundwater level before and after optimization Acknowledgments This research is supported by National key R&D Program of China(2017YFC0403201and 2017YFC0403204) This research is supported by National key R&D Program of China(2017YFC0403201and 2017YFC0403204) 4.1 Overview of the research area The main irrigation area of Dongxiezong lies in Youyi County, Heilongjiang Province. It is located in the southeast of Jiamusi City, in the northeast of Shuangyashan City, and on the edge of a large swamp in the Sanjiang Plain. In the typical area, there are a total of 2 branch canals, with a total length of 14.498 km; 18 canals with a total length of 48.735 km (including 7.305 km for 2 single irrigation channels and 41.430 km for 16 irrigation-drainage channels); 174 agricultural canals with a total length of 108.094 km (including 66.769 km for 122 single irrigation canals and 41.324 km for 52 irrigation-drainage canals); the branch travels from north to south with the main tributary ditches, head ditches and field ditches lying vertical with each other. The distance between two nearest head ditches is about 1000 m, while for field ditches, it is about 200m. See Figure 2. Figure 3,Sub-areas in typical irrigation area Figure 3,Sub-areas in typical irrigation area Divide the typical area into 18 sub-areas according to the controlled area of the head ditch, as is shown in Figure 3. g (2) Parameter design of channels in the typical zone The area of controlled drainage is 67,800 mu. The ditches correspond to the channels in the typical area. Set 2 tributary ditches with a length of 16.326 km and 7 branch ditches with a total length of 15.076 km. Set 127 agricultural field ditches under head ditches, with a total length of about 100.488 km. (3) Plant the crop The crop selected in the typical areas is rice. (4) Water source project The main irrigation area of Dongxie is a newly-built one with 6 canal heads (including 3 intake gates and 3 pumping stations). According to irrigation water sources, divided it into three sub-areas, namely, general main canal area of the Yinsong, main canal area of Sanhuanpao, and main canal area of Naoli River. In this research, the typical area is in general main canal area of the Yinsong with one water project and 40m3/s of water intake. 4 , 0 (2018) MATEC Web of Conferences 2 ISWSO 2018 46 2054 https://doi.org/10.1051/matecconf/201824602054 4.2 Optimization results (a) (b) (c) (d) Figure 4,Result of irrigation area optimization Figure 5,Comparison of groundwater level before and after optimization 4.2 Optimization results 4.2 Optimization results Reference (d) 1. Cohon J L, Marks D H. A review and evaluation of multiobjective programing techniques. Water Resources Research, 1975, 11(11):208–220. Figure 4,Result of irrigation area optimization 2. Park C H, Aral M M. Multi-objective optimization of pumping rates and well placement in coastal aquifers. Journal of Hydrology, 2004, 290(1–2):80-99. 2. Park C H, Aral M M. Multi-objective optimization of pumping rates and well placement in coastal aquifers. Journal of Hydrology, 2004, 290(1–2):80-99. 3. Sarker Ruhul, Ray Tapabrata. An improved evolutionary algorithm for solving multi-objective crop planning models. Computers and Electronics in Agriculture, 2009, 68(2):191-199. 3. Sarker Ruhul, Ray Tapabrata. An improved evolutionary algorithm for solving multi-objective crop planning models. Computers and Electronics in Agriculture, 2009, 68(2):191-199. 4. 4.K.Srinivasa Raju, D. Nagesh Kumar. Irrigation Planning using Genetic Algorithms. Water 5 , 0 (2018) MATEC Web of Conferences 2 ISWSO 2018 46 2054 https://doi.org/10.1051/matecconf/201824602054 Resources Management, 2004, 18(2):163-176. Past, Present, and Future. Water Resources Management, 2013, 27(13):4409-4424. Resources Management, 2004, 18(2):163-176. Past, Present, and Future. Water Resources Management, 2013, 27(13):4409-4424. 5. Das A, Datta B. Development of Management Models for Sustainable Use of Coastal Aquifers. Journal of Irrigation & Drainage Engineering, 1999, 125(3):112-121. 10. Rogers L L, Dowla F U. Optimization of Ground Water Remediation Using Artificial Neural Networks. Water Resources Research, 1994, 30(2):457–481. 6. Richard C. Peralta, Bithin Datta. Reconnaissance-Level Alternative Optimal Ground-Water Use Strategies. Journal of Water Resources Planning and Management, 1990, 116(5):676-692. 11. Rao S V N, Bhallamudi S M, Thandaveswara B S, et al. Conjunctive Use of Surface and Groundwater for Coastal and Deltaic Systems. Journal of Water Resources Planning & Management, 2004, 130(3):255-267. 7. Tabari M M R, Soltani J. Multi-Objective Optimal Model for Conjunctive Use Management Using SGAs and NSGA-II Models. Water Resources Management, 2013, 27(1):37-53. 12. Safavi H R, Darzi F, Mariño M A. Simulation-Optimization Modeling of Conjunctive Use of Surface Water and Groundwater. Water Resources Management, 2010, 24(10):1965-1988. 8. Bazarganlari M R, Kerachian R, Mansoori A. A conflict-resolution model for the conjunctive use of surface 13. Rao S V N. Optimal Pumping from Skimming Wells. Journal of Hydrologic Engineering, 2006, 11(5):464-471. 9. Mays L W. Groundwater Resources Sustainability: 6
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A comparative study of eight human auditory models of monaural processing
Acta acustica
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1 Introduction model implementations, there is a chance of applying a model outside its specific signal or parameter range. Thus, studies comparing models’ properties and configurations are important to model users. For example, Saremi et al. [12] compared seven models of cochlear filtering with respect to various parameters describing the nonlinear filtering pro- cess of an active cochlea, and Lopez-Poveda [13] compared eight models of the auditory periphery based on the repro- duction of auditory-nerve properties. Other related studies focus on a specific application (e.g., [10, 11, 14–17]) or pro- vide an introduction to modelling frameworks [18, 19]. Computational auditory models reflect our fundamental knowledge about hearing processes and have been accumu- lated during decades of research (e.g., [1]). Models are used to derive conclusions, reproduce findings, and develop future applications. Usually, models are built in stages that reflect basic parts of the auditory system, such as cochlear filtering, mechanoneural interface, and neural processing, by applying signal-processing methods such as bandpass filtering or envelope processing [2]. Models of monaural pro- cessing often form a basis for binaural models (e.g., [3]) and more complex models of auditory-based multimodal cogni- tion (e.g., [4]). For this reason, combined with the increas- ing popularity of reproducible research [5], it is not surprising that there is an increasing number of auditory models available as software packages (e.g., [6, 7]). In the current study, we compare various monaural auditory models that approximate subcortical neural pro- cessing. For this comparison, we use model configurations that reduce the heterogeneity across model outputs, indi- cating advantages and disadvantages of these configuration choices. These configurations are evaluated using the same set of sound stimuli across models. The selected set of stim- uli illustrates critical model properties that can be used as a guideline for the choice of a specific model. These properties include fast and slow temporal aspects, i.e., temporal fine structure and temporal envelope, that are evaluated for a wide range of presentation levels. However, models must be used with caution because they approximate auditory processes and are designed and evalu- ated under a specific configuration for a specific set of input sounds. While the evaluation conditions are selected to test the main properties of the simulated stages, models may provide different predictions when processing unseen sounds. Combined with the wide and low-threshold availability of We selected a number of auditory models that met two main criteria. A comparative study of eight human auditory models of monaural processing Alejandro Osses Vecchi1,* , Léo Varnet1 , Laurel H. Carney2 , Torsten Dau3 , Ian C. Bruce4 , Sarah Verhulst5 , and Piotr Majdak6 1 Laboratoire des systèmes perceptifs, Département d'études cognitives, École Normale Supérieure, PSL University, CNRS, 75005 Paris, France 2 Departments of Biomedical Engineering and Neuroscience, University of Rochester, Rochester, NY 14642, USA 3 Hearing Systems Section, Department of Health Technology, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark 4 Department of Electrical and Computer Engineering, McMaster University, Hamilton, ON L8S 4K1, Canada 5 Hearing Technology group, WAVES, Department of Information Technology, Ghent University, 9000 Ghent, Belgium 6 Acoustics Research Institute, Austrian Academy of Sciences, 1040 Vienna, Austria Received 4 July 2021, Accepted 28 February 2022 Abstract – A number of auditory models have been developed using diverging approaches, either physiological or perceptual, but they share comparable stages of signal processing, as they are inspired by the same constitutive parts of the auditory system. We compare eight monaural models that are openly accessible in the Auditory Modelling Toolbox. We discuss the considerations required to make the model outputs compara- ble to each other, as well as the results for the following model processing stages or their equivalents: Outer and middle ear, cochlear filter bank, inner hair cell, auditory nerve synapse, cochlear nucleus, and inferior colliculus. The discussion includes a list of recommendations for future applications of auditory models. Acta Acustica 2022, 6, 17  The Author(s), Published by EDP Sciences, 2022 https://doi.org/10.1051/aacus/2022008 Acta Acustica 2022, 6, 17  The Author(s), Published by EDP Sciences, 2022 https://doi.org/10.1051/aacus/2022008 Acta Acustica 2022, 6, 17  The Author(s), Published by EDP Sciences, 2022 https://doi.org/10.1051/aacus/2022008 Acta Acustica 2022, 6, 17  The Author(s), Published by EDP Sciences, 2022 https://doi.org/10.1051/aacus/2022008 Available online at: Topical Issue - Auditory models: from binaural processing to multimodal cognition SCIENTIFIC ARTICLE auditory path beginning with the acoustic input up to *Corresponding author: ale.a.osses@gmail.com This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. *Corresponding author: ale.a.osses@gmail.com This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://crea which permits unrestricted use, distribution, and reproduction in any medium, provided the original w 2 Models Based on our inclusion criteria, some models are excluded from the comparison, e.g., models that have only been evaluated at the level of cochlear filtering, such as models based on Hopf bifurcation [21] and the model of asymmetric resonators with fast-acting compression [22]. Other cochlear models, such as the Gammatone filter bank [23], the dual-resonance nonlinear model [24], the chirp filter bank [25] and the transmission-line model from [26], are included as modules in the selected models. We further excluded models whose structure did not contain one or more of the relevant processing stages that we evaluated in this study (Stages 3–6 in Fig. 2). Two models that entered this category are the power spectrum models, EPSM [27] (no stages 3–5) and GPSM [10, 11] (no stage 5). Lastly, we did not include models focusing on specific psychoacoustic metrics [28–30], despite the fact that such models are often based on comparable auditory stages as those described in this study. We define three model families, classified by their objec- tives [40], which translate into three different levels of detail in simulating the cochlear processing, as schematised in Figure 1. The selected models are listed in Table 1 and are labelled throughout this paper by the last name of the first author and the year of the corresponding publication. This naming system directly reflects the corresponding model functions implemented in AMT 1.1 [8]. [ ] We define the family of biophysical models (Fig. 1a) that use a transmission line consisting of many resonant stages coupled by the cochlear fluid. Biophysical models aim at exploring how the properties of the system emerge from biological-level mechanisms, needing a fine-grained descrip- tion at this level. The biophysical models are represented by verhulst2015 [34] and its extended version, verhulst2018 [35] (model version 1.2 [41, 42]). We further define phenomenological models which primarily predict physiological properties of the system, using a more abstract level of detail than the biophysical models. The phenomenological models considered here rely on dynami- cally adapted bandpass-filtering stages combined with nonlinear mappings (Fig. 1b) and are represented by zilany2014 [32] and its extended version bruce2018 [36], both combined with the same-frequency inhibition- excitation (SFIE) stages for subcortical processing [43]. Fur- ther approximation is given by functional-effective models [44], which target the simulation of behavioural (percep- tual) performance rather than the direct simulation of neural representations. 1 Introduction First, the selected models describe the auditory path beginning with the acoustic input up to This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 2 Figure 1. Model families used in this study. The families are defined by the level of detail in simulating the cochlear processing and are sorted by their complexity from left to right. a) Biophysical models using a nonlinear transmission line that contains resonating stages coupled by the cochlear fluid; b) Phenomenological models using nonlinear filters dynamically controlled by an outer-hair-cell (OHC) model; c) Functional effective models using linear filters, optionally combined with static nonlinear filters. Figure 1. Model families used in this study. The families are defined by the level of detail in simulating the cochlear processing and are sorted by their complexity from left to right. a) Biophysical models using a nonlinear transmission line that contains resonating stages coupled by the cochlear fluid; b) Phenomenological models using nonlinear filters dynamically controlled by an outer-hair-cell (OHC) model; c) Functional effective models using linear filters, optionally combined with static nonlinear filters. subcortical neural stages, in the cochlear nucleus (brain- stem) and the inferior colliculus (midbrain). Consideration of these stages extends previous comparisons of auditory periphery models [12, 13]. Second, the model implementa- tions are publicly available and validated to simulate psy- choacoustic performance and/or physiological properties. We use the implementations available in the Auditory Modelling Toolbox (AMT) [8, 9, 20]. Section 4 is relevant for model users who are interested in a transparent and accurate description of the illustrated model outputs, readers who are only interested in a bigger picture, as it is covered elsewhere (e.g., [1, 2, 13]), may wish to go directly to Sections 5 and 6. 2 Models These models usually approximate the cochlear processing by using static bandpass filtering with an optional nonlinear mapping (Fig. 1c). The linear effective models are represented by dau1997 [31] and osses2021 [39] and the nonlinear effective models are rep- resented by king2019 [37] and relanoiborra2019 [38]. Given that for each model a similar level of approxima- For the sake of simplicity, our analyses are focused on the comparison across models rather than on a comparison with experimental data. Nevertheless, we provide experi- mental references to the simulations that are illustrated throughout this paper. Additionally, to encourage repro- ducible research in auditory modelling, all our paper figures can be retrieved using AMT 1.1, including (raw) waveform representations of intermediate model outputs. The paper is structured as follows: In Section 2 we pro- vide a brief description of the processing stages in the selected auditory models. Their specific configurations are described in Section 3. The model comparison is presented in Section 4 and contains a description of the set of test stimuli as well as a detailed numerical description of the simulation results. Section 5 starts with a list of applications of the evaluated models, including some general considera- tions for the application of auditory models in further mod- elling work. Note that although the detailed analysis in A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 3 Table 1. List of selected models. The model labels used in this study correspond with the model functions in AMT 1.1. and bruce2018 models use a linear middle-ear filter [48, 49] that approximates the forward-pressure measure- ments from [50, 51]. The middle-ear filters in relanoiborra2019 and osses2021 are those designed to represent stapes velocity near the oval window of the cochlea [24, 52]. These models use the same filter implemen- tation, but the filter in osses2021 includes a gain factor to provide a 0-dB amplitude in the frequency range of the passband and a fixed group-delay compensation. study correspond with the model functions in AMT 1.1. Label Reference dau1997 Dau et al. (1997) [31] zilany2014 Zilany et al. (2014) [32] and Carney et al. (2015) [33] verhulst2015 Verhulst et al. (2015) [34] verhulst2018 Verhulst et al. (2018) [35] bruce2018 Bruce et al. (2018) [36] and Carney et al. (2015) [33] king2019 King et al. (2019) [37] relanoiborra2019 Relaño-Iborra et al. 2.3 Cochlear filtering A cochlear filter bank performs a spectral analysis of incoming signals to simulate the mechanical oscillations of the basilar membrane (BM) and organ of Corti at different points along the cochlea. The BM and organ of Corti are thought to have multiple modes of vibration which could drive the transduction currents of the IHCs depending on their amplitudes. The cochlear filtering stages in the evaluated models all aim to describe the filtering properties of the main mode(s) of vibration that explain the frequency- tuning curves of AN fibres. Some of the models include further components aimed at capturing the filtering beha- viour of additional modes of BM and/or organ of Corti vibration to better explain the level-dependent nonlinear filtering of the cochlea. All the included cochlear filtering stages produce a set of N time-domain signals, for N simu- lated characteristic frequencies (CFs). Each cochlear section is assumed to either have relatively sharp frequency tuning (Eq. (1), [53]) or broader tuning (Eq. (2), [54]). For CFs expressed in Hz: 2.1 Outer ear The listener’s head, torso, and pinna filter incoming sounds. The ear-canal resonance further emphasises fre- quencies around 3000 Hz [45]. These effects can be accounted for by filtering the sound with a head-related transfer function (HRTF) (e.g., [46]) or by applying a head- phone-to-tympanic-membrane transfer function, as used in relanoiborra2019 and osses2021. The other six selected models that do not include an outer-ear filter, implicitly assume that either the outer ear does not intro- duce a significant effect in the subsequent sound processing chain, or that the sounds are presented near the tympanic membrane, as is the case for a sound presentation using in-ear earphones. QERB ¼ 12:7  CF =1000 ð Þ0:3; ð1Þ QERB ¼ CF = 24:7  4:37  CF =1000 þ 1 ð Þ ½ : ð2Þ ð1Þ ð2Þ The models verhulst2015 and verhulst2018 use a transmission-line model fitted to otoacoustic estimates of human cochlear filtering [26], whose outputs represent BM velocity [34, 35], which provides the input to a model stage that maps BM velocity to IHC stereociliar deflection. In zilany2014 and bruce2018, the filtering is based on a chirp filter bank [25, 55] tuned to a human cochlea [48, 49, 56], that contains two parallel processing paths of static and outer-hair-cell (OHC) controlled filters (C2 and C1 in [32]), representing BM and organ of Corti motion that drive two separate IHC transduction functions 2 Models (2019) [38] osses2021 Osses and Kohlrausch (2021) [39] Middle-ear filtering not only introduces a bandpass characteristic to the incoming signal (Fig. 3), but also affects the operating range of cochlear compression in models relying on nonlinear cochlear processing, i.e., verhulst2015, verhulst2018, zilany2014, bruce2018, and relanoiborra2019. The passband gains of the middle-ear filters are indicated in Table 2 and range between 66.9 dB (relanoiborra2019) and +24 dB (verhulst2015). In nonlinear models, lower and higher passband gains vary the actual input level to the filter bank, shifting the onset of cochlear compression to higher and lower knee points, respectively. tion has been generally used in the design of subsequent model stages, we use the defined categories to reflect the nature of the entire model. The defined categories are not meant to represent a hard boundary for model classifica- tion. Hence, we do not discard the existence of other more- or less-detailed models than the selected biophysical and effective models, respectively. The selected monaural models share common stages of signal processing, as indicated in the schematic diagrams of Figure 2, with some stages even using the same (digital) implementation. Each model stage mimics, with greater or lesser detail, underlying hearing processes along the ascend- ing auditory pathway. The thick vertical lines in Figure 2 indicate the intermediate model outputs which are the basis for our evaluation. Note that these stages are, conceptually speaking, independent of each other. However, because of nonlinear interactions between them, the processing performed by these stages is not commutative and thus requires a step-by-step approach. 1 The models verhulst2015 and verhulst2018 use an updated version of equation (1): QERB ¼ 11:46  CF =1000 ð Þ0:25 [34, 35]. This equation produces sharp tuning curves but with slightly lower Q-factors compared to those given by equation (1), with values between 6.8 (CF = 125 Hz) and 19.3 (CF = 8000 Hz). 2.2 Middle ear Six of the eight evaluated models include a stage of middle-ear filtering. The transfer functions of the middle- ear filters used in these models are shown in Figure 3. The transfer functions in verhulst2015 and verhulst2018 approximate the middle-ear forward pres- sure gain (“M1” in [47]). The humanised zilany2014 A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 4 Figure 2. Block diagrams of the selected auditory models. Vertical lines: Intermediate model outputs as the basis for the evaluation in the corresponding sections. Blue: Type of hearing impairment that can be accounted for in the corresponding stage (see a brief overview in Sect. 5.1). Figure 2. Block diagrams of the selected auditory models. Vertical lines: Intermediate model outputs as the basis for the evaluation in the corresponding sections. Blue: Type of hearing impairment that can be accounted for in the corresponding stage (see a brief overview in Sect. 5.1). Figure 3. Amplitude spectra of the four middle-ear filters used in six of the evaluated models. The lines were shifted vertically to display their individual maximum at 0 dB. For relanoiborra2019 and osses2021, the grey dashed line shows the combined response of the outer- and middle-ear filters. Literature: Figure 1A from [47] and Figure 3 from [51]. nonlinear (DRNL) filter bank [24]. The cochlear filters of these models are assumed to be tuned according to equation (2). 2.4 Inner hair cell The inner hair cells (IHCs) transform the mechanical BM and organ of Corti oscillations into receptor potentials, subsequently initiating neuronal discharges in the auditory nerve (AN) [58]. In the most simple approach, the IHC processing can be simulated as an envelope extractor that removes phase information for high stimulus frequencies, implemented as a half-wave rectification followed by a lowpass (LP) filter. This approach is used in dau1997, king2019, relanoiborra2019, and osses2021, in which the LP filters have 3-dB cut-off frequencies (fcut-off) between 1000 and 2000 Hz. In zilany2014, bruce2018, and verhulst2015, a nonlinear transformation is applied to the output of the cochlear filter bank, followed by a cascade of LP filters with fcut-off of 3000 Hz (zilany2014 and bruce2018) and 1000 Hz (verhulst2015). The resulting fcut-off of each model ranges between 642 Hz (verhulst2015) and 1000 Hz (dau1997, relanoiborra2019, king2019), as indi- cated in Table 2. In verhulst2018, a more sophisticated IHC model is used [59], that is implemented as a three- channel non-spiking Hodgkin–Huxley type model, with each of the channels representing mechanoelectrical and (fast and slow) potassium-gated processing [35, 59]. Figure 3. Amplitude spectra of the four middle-ear filters used in six of the evaluated models. The lines were shifted vertically to display their individual maximum at 0 dB. For relanoiborra2019 and osses2021, the grey dashed line shows the combined response of the outer- and middle-ear filters. Literature: Figure 1A from [47] and Figure 3 from [51]. [55, 57]. The cochlear filters in these models are assumed to be tuned according to equation (1).1 In dau1997 and osses2021, the linear Gammatone filter bank from [23] is used. King2019 uses the Gamma- tone filter bank from [23] followed by a compressive stage acting above a given knee point. In relanoiborra2019, the cochlear processing is simulated by the dual-resonance 00 dB refer to fi LP filter structu lated IC output us using each of 019 osses20 0.00 474.8 1230.2 1.9 34 0.905 34 0.905 5 1 2000 771 77.2 70 MU 155.9 157.3 0.622 31 0.020 Details of frequency response of the middle-ear filters. Cochlear filter bank: 40 dB and oises of 40 and 100 dB SPL, respectively. IHC: Parameters and frequency response of th modulation filter with the closest BMF to 100 Hz and peak-to-peak amplitude of the sim A) (see Sect. 4.4 for more details). 00 dB refer to fi LP filter structu ulated IC output us using each of 019 osses20 0.00 474.8 1230.2 1.9 34 0.905 34 0.905 5 1 2000 771 77.2 70 MU 155.9 157.3 0.622 31 0.020 2.6 Subcortical neural processing AN firing patterns propagate to higher stages along the auditory pathway, first through the auditory brainstem, then towards more cortical regions [62]. On its way, AN spiking is mapped onto fluctuation patterns by neurons that are sensitive to the amplitude of low-frequency fluctu- ations [63]. This fluctuation sensitivity has been approxi- mated using various approaches. Our analyses focus on model approximations of the modulation processing circuits of the ventral cochlear nucleus (CN) and inferior colliculus (IC) [43], as well as on different modulation-filter-bank vari- ants [27, 31]. As a result, we exclude the analysis of other subcortical structures such as those that play a particular role in the binaural interaction between ears (e.g., the dorsal cochlear nucleus and superior olive) [62, 64]. A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 6 modulation filter centred at a BMF of 82.4 Hz (see Tab. 2) [35, 41]. Further, bruce2018 can be combined with the SFIE model in the UR EAR 2020b toolbox [66]. Note that zilany2014, verhulst2015, and verhulst2018 have used the output of their mean firing rate generator—an output that can be conceptualised as peri-stimulus time histograms (PSTHs) [67]—as an input to the SFIE model. In bruce2018, because of the stochas- tic processes in its spike generator, repeated processing of the same stimulus is recommended to obtain a faithful PSTH that can appropriately account for power-law adap- tation properties (see Sect. 3 in [36]). 2.5 Auditory nerve The transduction from IHC receptor potentials into pat- terns of neural activity can be derived from the interaction between the IHC and AN. Several AN synapse models have been inspired by the three-store diffusion model [58], assum- ing that the release of synaptic material is managed in three storage compartments. For steady-sound inputs, this model predicts a rapid neural firing shortly after the sound onset with a decreasing rate towards a plateau discharge rate, a phenomenon called adaptation (e.g., [60]). The AN synapse models in verhulst2015, verhulst2018, and zilany2014 are based on [58], but zilany2014 further incorporates a power-law adap- tation following the diffusion model from [32]. The synapse model in bruce2018 uses a diffusion model based on [61] to: (1) have limited release sites, and (2) come after the power-law adaptation instead of before it [36]. The outputs of these models simulate the firing of neurons having a specific spontaneous rate of high-, medium-, and/or low- spontaneous rates. The effective models, on the other hand, approximate the subcortical neural processing based on the modula- tion-filter-bank concept [27, 31]. In dau1997, king2019, relanoiborra2019, and osses2021, linear modulation filter banks are used, covering a range of BMFs up to 1000 Hz. In dau1997, twelve modulation filters with a Q-factor of 2 and overlapped at their 3 dB points are used. The same modulation filters are used in relanoiborra2019 and osses2021, but an additional 150-Hz LP filter is applied [27, 68] and the number of filters is limited so that the highest BMF is less than a quarter of the corresponding CF [69]. In king2019, the filter bank is used with a wider tuning (Q = 1, as suggested in [27, 70]), using ten 50%-overlapped filters having a maximum BMF of 120 Hz [37]. The effective models, on the other hand, rely on a more coarse AN simulation, expressed in arbitrary units (a.u.). In king2019, adaptation is simulated by applying a highpass filter with a cut-off frequency of 3 Hz [37]. In dau1997, relanoiborra2019, and osses2021, adaptation is simulated by so-called adaptation loops [44] that introduce a nearly logarithmic compression to stationary input signals and a linear transformation for fast signal fluctuations (Appendix B in [39]). The arbitrary units of these trans- formed outputs are named model units (MUs). 3 Model configuration We evaluated the intermediate model outputs that are indicated by thick vertical black lines in Figure 2. The eval- uation points are located after the cochlear filter bank (Stage 3), the IHC processing stage (Stage 4), the AN synapse stage or equivalent (Stage 5), and after the IC pro- cessing stage or equivalent (Stage 6). Starting with the default parameters of each model, we introduced small adjustments to obtain the most comparable model outputs. All comparisons can be reproduced with the function exp_osses2022 from AMT 1.1 [8]. 2.4 Inner hair cell Performance: Time required to process a 1-s long stim cut-off in this table were measured at the 3 dB points of the amplitude spectrum. Model 4 verhulst2015 verhulst2018 bruce2018 king2019 relanoiborra 24.0 18.0 6.0 – 66.9 601.0 601.1 577.6 – 474.8 2995.3 3993.1 6061.9 – 1230.2 77.9 101.9 44.1 43.9 26.9 59 52 51 34 36 0.505 0.579 0.588 0.904 0.848 12 15 20 33 27 3.046 2.299 1.57 0.925 1.147 1 – 7 1 1 2 – 1 1 2 1000 – 3000 1000 1000 642 – 966 1000 1000 82.4 82.4 85.4 94.0 77.2 45 60 45 65 70 lV lV spikes/s a.u. MU 0.245 0.401 117.6 1.64  104 423.1 0.237 0.407 117.3 1.67  104 408.9 122.9 319.5 163.1 1.86 7.70 401 401 66 31 60 0.306 0.797 2.47 0.060 0.128 A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 3.4 Subcortical neural processing CFn ¼ A0  10axn=1000    A  k; ð3Þ The default configuration of the model stages of subcor- tical processing (Stage 6, Fig. 2) differs in the number of modulation filters (from 1 to 12) and in their tuning across models. In our study, we use only one modulation filter tar- geting a BMF of approximately 80 Hz (see “Theoretical BMF” in Tab. 2) using a Q-factor of approximately 1 for zilany2014, verhulst2015, verhulst2018, bruce2018, and king2019, and a Q-factor of 2 for dau1997, relanoiborra2019, and osses2021. ð3Þ where xn (in mm) can be obtained as x1 + Dx  (n  1), and A = 165.4188 Hz, a = 61.765 1/m, k = 0.85, and A0 = 20682 Hz. Note that when reporting results, we indi- cate the cochlear section number n and its corresponding CFn. The cochlear-filtering parameters of zilany2014 and bruce2018 were those adapted to a human cochlea [48, 49]. Moreover, in order to analyse separately the effects of cochlear filtering and IHC processing in zilany2014, the C1- and C2-path outputs from the chirp filter bank were added and analysed before the IHC nonlinear mapping was applied. This analysis follows a similar rationale as analysing the main output of the DRNL filter bank in relanoiborra2019 (see Fig. 3a from [24]). For the biophysical and phenomenological models, we used the SFIE model [33, 43] using two different configura- tions. The SFIE model [43] integrated in verhulst2015 and verhulst2018 has CN parameters with excitatory and inhibitory time constants of sexc = 0.5 ms and sinh = 2 ms, a delay D = 1 ms, and a strength of inhibition of S = 0.6. The IC stage uses sexc = 0.5 ms, sinh = 2 ms [35], D = 2 ms, and S = 1.5 [43], achieving a BMF of 82.4 Hz. Finally, in king2019, we used a compression factor of 0.3 for all simulated CFs, which is different from the one- channel (on-CF) compression used in [37, 72]. For zilany2014 and bruce2018, the SFIE model is a separate stage [33], implemented as carney2015 in AMT 1.1, where either the mean-rate (zilany2014) or the PSTH outputs (bruce2018) are used as inputs. In our analysis, we only used the output of the band-enhanced IC cell, which corresponds to the SFIE model from [43]. The CN parameters were identical to those for the biophysical models. 3.4 Subcortical neural processing The IC parameters were sexc = 1.11 ms, sinh = 1.67 ms, D = 1.1 ms, and S = 0.9, achieving a BMF of 83.9 Hz [33]. Note the different inhibition strength S between models. In the biophysical models, the IC output is dominated by inhibitory responses (S > 1) whereas in the phenomenological models the IC output is dominated by excitatory responses (S < 1). 3.2 Cochlear filtering The phenomenological and effective models can be set to simulate responses at any CF. However, because of the nature of the transmission-line structure, the selected bio- physical models have a discrete tonotopy that translates into a discrete set of available CFs. The models verhulst2015 and verhulst2018 were set to 401 cochlear sections spaced at Dx = 0.068 mm with tonotopic distances xn ranging between x1 = 3.74 mm and x401 = 30.9 mm, that are related to CFs between CF1 = 12010 Hz and CF401 = 113 Hz, accord- ing to the base-to-apex mapping [71], 3.3 Inner hair cell and auditory nerve Default parameters were used for the IHC and AN stages of the evaluated effective models. However, the biophysical and phenomenological models require the choice of parameters to simulate a population of AN fibres. For each CF we simulated 20 fibres, having either high- (HSR), medium- (MSR), or low-spontaneous rates (LSR), distributed in a 0.6–0.2–0.2 ratio [73, 74], resulting in a 12–4–4 configuration (HSR–MSR–LSR). Note that for verhulst2015 and verhulst2018, this deviates from the standard 13–3–3 configuration [34, 35]. For verhulst2015 and verhulst2018, the spontaneous rates of each fibre type were 68.5, 10, and 1 spikes/s for HSR, MSR, and LSR, respectively, as used in human-tuned simulations [35]. For zilany2014, the spontaneous rates of each fibre type were 100, 4, and 0.1 spikes/s and for bruce2018 were 70, 4, and 0.1 spikes/s for HSR, MSR, and LSR, respectively. We further disabled the random fractional noise generators in zilany2014 and 3.1 Level scaling In king2019, a calibrated knee point (default of 30 dB) is used in its cochlear compression stage (Stage 3). All signal levels are reported as root-mean-square (rms) values refer- enced to 20 lPa, in dB sound pressure level (dB SPL). 3.1 Level scaling The same set of sound stimuli was used as input to all models. The waveform amplitudes were assumed to repre- sent sound pressure expressed in Pascals (Pa). The models zilany2014, verhulst2015, verhulst2018, bruce2018, and relanoiborra2019 use this level convention and did not require further level scaling. The models dau1997, king2019, and osses2021 interpret sound pressures between 1 and 1 Pa as amplitudes in the range ±0.5, thus a factor of 0.5 (attenuation by 6 dB) was applied to the generated stimuli to meet the level convention of these models. For these latter models, which include mostly level-independent stages, such calibration is relevant because the adaptation loops (used in dau1997, osses2021, also extensible to relanoiborra2019) include level-dependent scaling (Eqs. (B1)–(B3) in [39]). The modulation processing in the ventral CN and IC can be simulated using the same-frequency inhibition- excitation (SFIE) model, resulting in a widely tuned modu- lation filter (Q-factor  1) with a best-modulation fre- quency (BMF) depending on the parameters of the model [33, 43]. The SFIE model has already been used in combina- tion with the biophysical and phenomenological models described here. For example, zilany2014 has been com- bined with the SFIE model using between one and three modulation filters (e.g., [65]). Or, verhulst2015 and verhulst2018 have used the SFIE model with one A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 7 bruce2018 [75], and the random spontaneous rates in bruce2018 (“std” from Tab. I in [36] was set to zero). With this configuration, the mean-rate synapse outputs of verhulst2015, verhulst2018, zilany2014, and bruce2018 are deterministic. For this reason, to obtain population responses, we simulated the AN processing of each type of neuron only once and then weighted them by factors of 0.6, 0.2, and 0.2 for HSR, MSR, and LSR fibres, respectively. In contrast, the PSTH outputs that are reported for zilany2014 and bruce2018 are not deterministic, requiring the simulation of each AN fibre for each CF. Therefore, PSTH population responses were obtained by counting the average number of spikes in time windows of 0.5 ms across 100 repetitions of the correspond- ing stimuli. In king2019, a calibrated knee point (default of 30 dB) is used in its cochlear compression stage (Stage 3). All signal levels are reported as root-mean-square (rms) values refer- enced to 20 lPa, in dB sound pressure level (dB SPL). 4 Evaluation In this section we analyse the outputs of the eight selected auditory models in a number of test conditions, whose results are presented in Figures 4–15. We aimed at a comparison across models and thus, for the sake of clarity, we refrained from a direct comparison to ground-truth A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 8 Figure 4. Filter bank responses to pure tones at 500 Hz and 4000 Hz (top two rows) and spectral magnitudes of single-channel responses to white noises (bottom two rows) for sounds of 40-, 70-, or 100-dB SPL (bottom-to-top coloured curves, respectively). In the first two rows, the responses represent excitation patterns at the simulated CFs. The coloured markers indicate the amplitudes at the (off-frequency) CFs one ERBN below (grey) and one ERBN above (yellow or orange) the on-frequency CF (502 or 4013 Hz). These markers are highlighted using the same colours in Figure 5. In the third and fourth rows, the average FFT response of two cochlear- filters (CFs of 502 Hz or 4013 Hz) in response to six 500-ms white noise sections are shown in grey, and the corresponding smoothed responses are shown in colour. This type of responses was used to assess the quality factors of Figure 6. The dashed black lines indicate the corresponding 3-dB filter bandwidths. All responses were shifted vertically by the reference gains given in Table 2 (see the text for details). Literature: Figure 1A from [76], Figure 2C–E from [77], and Figure 2 from [12]. Figure 4. Filter bank responses to pure tones at 500 Hz and 4000 Hz (top two rows) and spectral magnitudes of single-channel responses to white noises (bottom two rows) for sounds of 40-, 70-, or 100-dB SPL (bottom-to-top coloured curves, respectively). In the first two rows, the responses represent excitation patterns at the simulated CFs. The coloured markers indicate the amplitudes at the (off-frequency) CFs one ERBN below (grey) and one ERBN above (yellow or orange) the on-frequency CF (502 or 4013 Hz). These markers are highlighted using the same colours in Figure 5. In the third and fourth rows, the average FFT response of two cochlear- filters (CFs of 502 Hz or 4013 Hz) in response to six 500-ms white noise sections are shown in grey, and the corresponding smoothed responses are shown in colour. 4 Evaluation This type of responses was used to assess the quality factors of Figure 6. The dashed black lines indicate the corresponding 3-dB filter bandwidths. All responses were shifted vertically by the reference gains given in Table 2 (see the text for details). Literature: Figure 1A from [76], Figure 2C–E from [77], and Figure 2 from [12]. 20 and 20000 Hz. All sounds were gated on and off with a 10-ms raised-cosine ramp and had levels of 40, 70, and 100 dB SPL. The obtained responses are shown in Figure 4, which were vertically shifted by the gains indicated in Table 2. These gains were derived for each model using the 1000-Hz pure tone of 100 dB SPL as a reference. The frequency responses were level-dependent for zilany2014, verhulst2015, verhulst2018, king2019,andrelanoiborra2019. Forverhulst2015, verhulst2018, and relanoiborra2019, we further observed a change in the location of their maximum amplitude (green triangles in Fig. 4, fourth row). Although a shift of the responses with increasing the stimulus level is supported by experimental data [76, 78, 80], this argument was later challenged [81] and rather attributed to shallower upper tails in the responses. A final observation is that king2019, zilany2014, verhulst2015 and verhulst2018, showed a certain amount of distortion in the frequency responses of 70- and 100-dB SPL sounds (Fig. 4, second and third rows). For the responses to white noises at CF = 502 Hz, the distortions occur in the upper tail of the cochlear responses as a side effect of the (amplitude) compression stage. This frequency-response distortion is most prominent in king2019 due to its references from physiological data. However, such a com- parison is important and interesting. For this reason, we provide references where similar experimental and/or simu- lation analyses have been presented. These references are indicated as “Literature” in the caption of the corresponding figure. Alternatively, the outputs of the biophysical and phenomenological models may be considered as referential because they have been primarily developed to reflect physiological (human or animal) responses to sounds. This latter assumption always requires a careful consideration, especially when translating findings from animal to human physiology. 2 Note that king2019 was primarily developed to simulate responses to pure tones or narrow-band signals using cochlear channels with CFs that are either on-frequency (one cochlear channel) [110] or spanning ±2 ERBN around the on-frequency CF (five cochlear channels) [37, 72]. The prominent frequency distortions shown in Figure 4e (third row) are thus outside the tested CF ranges for this model. 4.1.1 Compressive growth For zilany2014/bruce2018, the I/O curves were fairly linear in response to 500-Hz tones (top panels) for both on- and off-frequency CFs. For 4000-Hz tones, a prominent compressive behaviour was observed in the on-frequency curves (Fig. 5d) where, additionally, the curve for verhulst2018 turned from a compressive to a lin- ear regime for signal levels above 80 dB. The off-frequency I/O curves obtained for verhulst2018 were similar to those for verhulst2015 but had overall lower and higher amplitudes for the pure tones of 500 Hz (Fig. 5b–c) and 4000 Hz (Fig. 5e–f)), respectively, as a consequence of the differences in their middle-ear filters (see Fig. 3). The tendency to a more linear regime in off-frequency CFs has been shown previously [79]. This is in fact the basis for having compression only applied to the on-frequency channel in king2019 [37, 72]. However, the default compression rate of 0.3 for the on-frequency channel with no compression for off-frequency channels leads to an unrealistic level balance between on- and off-frequency channels. The set of pure tones was further used to assess the curves relating the input stimulus levels with levels at the output of the cochlear filter banks, known as input–output (I/O) curves. For this analysis we included stimulus levels between 0 and 100 dB SPL in steps of 10 dB. The I/O curves were obtained for (1) the on-frequency CF tuned to the frequency of the input stimulus, and (2) the off- frequency responses of cochlear filters tuned to one equiva- lent rectangular bandwidth number (ERBN) [54] below and above the stimulus frequency. The obtained I/O curves are shown in Figure 5 for on- frequency (left panels) and off-frequency simulations (±1 ERBN, middle and right panels). The I/O curves were vertically shifted by the reference gains indicated in Table 2 (as in Fig. 4). As expected for the level-independent Gam- matone filters used in dau1997 and osses2021, the curves were linear in all panels of Figure 5. For the remain- ing models, more compressive behaviour was observed for on-frequency curves (left panels) while more linear curves 4.1 Cochlear filtering Sound processing in the cochlea depends not only on the frequency but also on the level of the input stimulus [78]. The amplitude of the BM vibration displacement increases for higher levels, following an amplitude growth that com- prises linear and compressive regimes [79]. We illustrate this level dependency for a set of pure tones and white noises. The pure tones had frequencies of 500 or 4000 Hz, with a duration of 100 ms. The white noise was generated as a fixed 3-s long waveform with a flat spectrum between A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 9 Figure 5. Input–output (I/O) curves for pure tones at 500 Hz (panels a–c) and 4000 Hz (panels d–f), at six model CFn frequencies (see Eq. (3)). Left (a,d): On-frequency simulations, i.e., output of the cochlear filter with the CF tuned to that of the stimulus frequency. Middle (b,e), right (c,f): Off-frequency simulations, one ERB below and above the on-frequency, respectively. The exact simulated on- and off-frequency CFs are indicated in the title of each panel. The filled markers indicate off-CF amplitudes that are also highlighted in the corresponding frequency responses of Figure 4. All I/O curves were shifted vertically by the reference gains given in Table 2 (see the text for details). Literature: Figures 1–3 from [79] and Figure 3 from [12]. Figure 5. Input–output (I/O) curves for pure tones at 500 Hz (panels a–c) and 4000 Hz (panels d–f), at six model CFn frequencies (see Eq. (3)). Left (a,d): On-frequency simulations, i.e., output of the cochlear filter with the CF tuned to that of the stimulus frequency. Middle (b,e), right (c,f): Off-frequency simulations, one ERB below and above the on-frequency, respectively. The exact simulated on- and off-frequency CFs are indicated in the title of each panel. The filled markers indicate off-CF amplitudes that are also highlighted in the corresponding frequency responses of Figure 4. All I/O curves were shifted vertically by the reference gains given in Table 2 (see the text for details). Literature: Figures 1–3 from [79] and Figure 3 from [12]. broken-stick compression (rise of the amplitudes to the power of 0.3).2 were obtained for off-frequency CFs (middle and right panels), except for relanoiborra2019 and king2019, that had on- and off-frequency compression. 4.1.2 Frequency selectivity: Filter tuning The frequency selectivity of each filter bank was com- puted in response to the described frozen noise waveform, presented at 40, 70, and 100 dB SPL. The estimates of frequency selectivity were obtained from FFT responses averaged across 500-ms non-overlapped analysis windows, A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 10 Figure 6. Filter tuning expressed as quality factors Q for noises of 40, 70, and 100 dB SPL (panels a–c), and Q-factor difference obtained from the results of 40- and 100-dB noises (panel d). Literature: Figure 4 from [53] and Figure 4B from [12]. within one ERB are similar to Q3 dB values. The results for 40-dB noises show that the frequency selectivity follows either the analytical tuning of equation (1) (zilany2014, bruce2018, verhust2015, and verhulst2018) or the tuning of equation (2) (dau1997, relanoiborra2019, king2019, and osses2021). When looking at the results for higher levels (Fig. 6b–c), no change in tuning was observed for dau1997 and osses2021, as expected for linear models. For the nonlinear models, the results for 70-dB noises in Figure 6b showed overall lower Q factors, but with only a small change for king2019 and relanoiborra2019. The results for 100-dB noises in Figure 6c showed a further lowering of Q factors in the biophysical and phenomeno- logical models, reaching values as low as Q  2 in verhulst2015, lower Q factors for frequencies up to about 4000 Hz in relanoiborra2019, and virtually unaffected Q factors in king2019. A closer inspection to the outputs of king2019 revealed that there was a filter broadening as a consequence of its broken-stick nonlinearity stage, but this broadening predominantly affected the fre- quency responses outside the range defined by the 3-dB bandwidth used to derive the Q factors (see Fig. 4e). To illustrate the Q-factor transition when increasing the signal level in each model, the difference between Q factors obtained from 40- to 100-dB noises is shown in Figure 6d, where a decrease in Q factor with increasing signal level is represented by a positive Q-factor difference. Fi 6 Filt t i d lit f t Q f i Additionally, we observed that relanoiborra2019 and king2019 introduce a change in selectivity at overall higher levels compared to the biophysical and phenomeno- logical models. 4.1.2 Frequency selectivity: Filter tuning A closer look at this aspect revealed that this change occurs because relanoiborra2019 and king2019 only apply compression after the bandpass filtering and, therefore, lower level signals are used as input for their compression (broken-stick) module. Figure 6. Filter tuning expressed as quality factors Q for noises of 40, 70, and 100 dB SPL (panels a–c), and Q-factor difference obtained from the results of 40- and 100-dB noises (panel d). Literature: Figure 4 from [53] and Figure 4B from [12]. 4.1.3 Frequency selectivity: Number of filters The number of filters in a filter bank is relevant for sev- eral model applications because too few filters can lead to a loss of signal information (e.g., [84]) and too many filters may unnecessarily increase the computational costs. The number of filters is a free parameter in zilany2014/ bruce2018, but is fixed for verhulst2015 and verhulst2018 to yield an accurate precision of the trans- mission-line solver [85]. The remaining models use by default one ERB-wide bands (dau1997, king2019, and osses2021), or have an overlap every 0.5 ERBN (relanoiborra2019). meaning that the estimates were obtained from six statisti- cally-independent noise sections. The frequency response of thirty-two filters with CFs between 126 Hz (n = 396 in Eq. (3)) and 9587 Hz (n = 24 in Eq. (3)) at steps of n = 12 bins was obtained. For illustration purposes, we also included in this analysis the on-frequency CFs used in Figure 5 (CF = 502 Hz, n = 305; CF = 4013 Hz, n = 112), whose obtained responses are shown in Figure 4. For each filter response, a quality factor Q3 dB = CF/BW was obtained, where BW is the bandwidth defined by the lower and upper 3-dB down points of each filter transfer function (black dashed lines in Fig. 4). Here, we report the minimum number of filters that are required to obtain a filter bank with overlapping at 3-dB points of the individual filter responses. Using the empirical Q-factors of Figure 6, we assessed the number of filters that would be required to cover a frequency range between 126 Hz (n = 396 in Eq. (3)) and the first filter with its upper cut-off frequency equal or greater than 8000 Hz. The number of filters derived from the 40-dB and 100-dB fre- quency tuning curves (Fig. 6, panels c and d) are shown The frequency-selectivity simulations for each of the filter banks are shown in Figure 6 for noises of 40 (panel a), 70 (panel b), and 100 dB SPL (panel c). The ana- lytical filter tuning curves given by equations (1) and (2) are indicated as light and dark grey traces in Figure 6. Note that with this comparison, we assume that the Q factors A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 11 Figure 7. Simulated IHC responses to pure tones of different frequencies evaluated at the corresponding on-frequency bin. 4.1.3 Frequency selectivity: Number of filters The amplitudes were normalised with respect to their maximum value to allow a direct comparison across models. Literature: Figure 9 from [82] and Figure 7 from [83]. Figure 7. Simulated IHC responses to pure tones of different frequencies evaluated at the corresponding on-frequency bin. The amplitudes were normalised with respect to their maximum value to allow a direct comparison across models. Literature: Figure 9 from [82] and Figure 7 from [83]. in Table 2, including the average filter bandwidth in ERBN for the corresponding model. Figure 8. Ratio between simulated AC and DC components (VAC/VDC, see the text) in response to 80-dB pure tones. Literature: Figure 10 from [82] and Figure 8 from [83]. For the biophysical models, the filters were much wider at the higher level than for the other models, with average bandwidths being as wide as 3.05 ERBN for verhulst2015 and 2.30 ERBN for verhulst2018. This contrasts with the 1.57 ERBN for zilany2014 and bruce2018 and the 1.15 ERBN or less for the remaining models. These bandwidths are a consequence of the fast- acting (sample-by-sample) compression that is applied just before the transmission-line in the biophysical models and the slower-acting bandwidth control in zilany2014 (denoted as the “control path” in the chirp filter bank). While cochlear filters are generally wider at high sound levels (e.g., [76, 86]), the appropriate tuning must be evalu- ated depending on the species’ characteristics, the tested CFs, and the type of evaluated excitation signals. Figure 8. Ratio between simulated AC and DC components (VAC/VDC, see the text) in response to 80-dB pure tones. Literature: Figure 10 from [82] and Figure 8 from [83]. The DC potential was obtained as VDC = (Vpeak,max + Vpeak,min)/2  Vrest [82, 83]. 4.2 IHC processing: Phase locking to temporal fine structure 7) as a result of the applied half-wave rectification process. Furthermore, zilany2014/bruce2018 and verhulst2015 have Vpeak,min amplitudes of 66 mV and 4.7 mV, respectively. Despite the different range in their minimum voltages, there is a strong qualitative resemblance between waveforms (green and red traces in the figure). In fact, both these models use the same type of IHC nonlinearity (compare Eqs. (17)–(18) from [55] with Eqs. (4)–(5) from [34]) and the same LP filter implementation, albeit with a different filter order and cut-off frequency (see Tab. 2). VAC reductions of less than 76.0% (king2019: 59.7%- decrease from 3.12  103 to 1.26  103 a.u.; dau1997: 62.6%-decrease from 0.097 to 0.037 a.u.; and verhulst2018: 76.0%-decrease from 39.2 to 9.4 mV), while the other five models showed VAC reductions of at least 92.5%. From the low-frequency IHC waveforms (bottom-most waveforms in each panel), it can be seen that the simulated amplitudes of dau1997, king2019, relanoiborra2019, and osses2021 did not go below their Vrest (horizontal grid lines in Fig. 7) as a result of the applied half-wave rectification process. Furthermore, zilany2014/bruce2018 and verhulst2015 have Vpeak,min amplitudes of 66 mV and 4.7 mV, respectively. Despite the different range in their minimum voltages, there is a strong qualitative resemblance between waveforms (green and red traces in the figure). In fact, both these models use the same type of IHC nonlinearity (compare Eqs. (17)–(18) from [55] with Eqs. (4)–(5) from [34]) and the same LP filter implementation, albeit with a different filter order and cut-off frequency (see Tab. 2). ( ) The obtained AC/DC ratios are shown in Figure 8, where a reduction in phase locking is given by a lower ratio. For all models, the ratio decreased with increasing frequency. All AC/DC curves, except those for verhulst2018, overlap well at low frequencies with ratios between 2.1 and 5.9 (below 1000 Hz), decreasing to ratios between 0.06 (osses2021) and 0.83 (dau1997) at 4013 Hz. Although the AC/DC curve for verhulst2018 showed the highest overall ratios between 137.4 at 460 Hz down to 1.3 at 4013 Hz, due to its nearly zero DC voltages towards low frequencies (see the fairly symmetric waveforms around the horizontal grid in Fig. 7d), we still observed the systematic decrease in ratio with increasing frequency. 4.2 IHC processing: Phase locking to temporal fine structure If we further focus on the AC/DC curves in the frequency range between 600 and 1000 Hz, where the phase-locking is expected to start declining [82], all models showed monotonically decreas- ing curves starting from about 833 Hz (except for verhulst2018, that always showed a decreasing ten- dency). The lowest ratios were observed for osses2021, followed by the similarly steep curve of zilany2014. Finally, a similar AC/DC curve was obtained for relanoiborra2019 and verhulst2015. Figure 9. Simulated AN responses to a 4000-Hz pure tone of 70 dB SPL. For ease of visualisation, the responses from osses2021, verhulst2015, and the PSTHs are horizontally shifted by 20 ms. Literature: Figure 1 from [87] and Figures 3 and 10 from [36]. 4.3.1 Adaptation To illustrate the effect of auditory adaptation, we obtained AN model responses to a 4000-Hz pure tone of 70 dB SPL, duration of 300 ms, that was gated on and off with a cosine ramp of 2.5 ms. The obtained AN responses are shown in Figure 9. All responses had an amplitude overshoot just after the tone onset which then decreased to a plateau (e.g., between 300 and 340 ms, grey dashed lines). After the tone offset (t = 350 ms), the AN responses showed an undershoot with decreased amplitudes that subsequently returned to their resting level. This stereotypical behaviour is related to the AN adaptation process (e.g., [60]). 4.2 IHC processing: Phase locking to temporal fine structure The obtained IHC waveforms are shown in Figure 7. Within each panel, bottom to top waveforms represent on-frequency simulations for the test signals, from low to high frequency carriers, respectively. For some model out- puts, the four highest carriers (1870  fc  4013 Hz) were amplified by a factor of 3 to improve waveform visibility. The simulated voltages before the tone onset, i.e., the rest- ing potential Vrest, were equal to 0 for all models except for verhulst2018, where Vrest was 57.7 mV (not schema- tised in Fig. 7). It seems clear, however, that the decrease of peak-to-peak AC voltage towards high frequencies—a mea- sure of the residual amount of temporal fine structure—is significantly different across models. When increasing the CFs from 1099 to 4013 Hz, three models showed To illustrate the loss in phase locking to temporal fine structure with increasing stimulus frequency, we simulated IHC responses to pure tones with frequencies between 150 Hz (n = 387 in Eq. (3)) and 4013 Hz (n = 112 in Eq. (3)) spaced at n = 25 bins, resulting in 12 test frequen- cies. The tones were generated at 80 dB SPL, with a dura- tion of 100 ms, and were gated on and off with 5-ms raised- cosine ramps. The simulated waveforms, that are assumed to approximate the IHC potential, are displayed and described in terms of AC (fast-varying) and DC (average bias) components, and the simulated resting poten- tials (Vrest). The AC potential was assessed from the peak-to-peak amplitudes as VAC = Vpeak,max  Vpeak,min. A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 12 Figure 9. Simulated AN responses to a 4000-Hz pure tone of 70 dB SPL. For ease of visualisation, the responses from osses2021, verhulst2015, and the PSTHs are horizontally shifted by 20 ms. Literature: Figure 1 from [87] and Figures 3 and 10 from [36]. VAC reductions of less than 76.0% (king2019: 59.7%- decrease from 3.12  103 to 1.26  103 a.u.; dau1997: 62.6%-decrease from 0.097 to 0.037 a.u.; and verhulst2018: 76.0%-decrease from 39.2 to 9.4 mV), while the other five models showed VAC reductions of at least 92.5%. From the low-frequency IHC waveforms (bottom-most waveforms in each panel), it can be seen that the simulated amplitudes of dau1997, king2019, relanoiborra2019, and osses2021 did not go below their Vrest (horizontal grid lines in Fig. 4.3 AN firing patterns Simulations included AN responses to pure tones and to amplitude-modulated (AM) tones from which rate-level functions expressed as onset and steady-state responses were obtained. With these benchmarks we attempt to char- acterise model responses at the output of the AN synapse stage or their equivalent, with a particular interest in the phenomenon of adaptation [60, 75]. We comment on how adaptation is affected by the type of output of Stage 5, using either the approximations from the effective models, the average or instantaneous firing rate estimates of the phenomenological models (zilany2014, bruce2018), or the average rates of the biophysical models (verhulst2015, verhulst2018). The waveforms from effective models using the adaptation loops (dau1997, relanoiborra2019, osses2021) are shown in Figure 9a, where their ampli- tudes had values between 230.5 MU and 1440.2 MU A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 13 (at t  200 ms) while for verhulst2015 (red) the plateau is reached shortly after the tone onset. Figure 10. Simulated rate-level functions derived from the steady-state AN responses of 4000-Hz pure tones. For all models, average responses are shown (coloured traces). For the biophys- ical and phenomenological models, the responses for HSR, MSR, and LSR neurons are also shown (grey traces). Literature: Figure 7 from [36], Figure 5A from [35], and Figure 3 from [44]. Figure 11. Simulated rate-level functions derived from the onset (maximum) AN responses of 4000-Hz pure tones. The colour codes and legends are as in Figure 10. Literature: Figure 3 from [87]. Figure 10. Simulated rate-level functions derived from the steady-state AN responses of 4000-Hz pure tones. For all models, average responses are shown (coloured traces). For the biophys- ical and phenomenological models, the responses for HSR, MSR, and LSR neurons are also shown (grey traces). Literature: Figure 7 from [36], Figure 5A from [35], and Figure 3 from [44]. Figure 11. Simulated rate-level functions derived from the onset (maximum) AN responses of 4000-Hz pure tones. The colour codes and legends are as in Figure 10. Literature: Figure 3 from [87]. Figure 10. Simulated rate-level functions derived from the steady-state AN responses of 4000-Hz pure tones. For all models, average responses are shown (coloured traces). For the biophys- ical and phenomenological models, the responses for HSR, MSR, and LSR neurons are also shown (grey traces). Literature: Figure 7 from [36], Figure 5A from [35], and Figure 3 from [44]. Figure 11. 4.3 AN firing patterns Simulated rate-level functions derived from the onset (maximum) AN responses of 4000-Hz pure tones. The colour codes and legends are as in Figure 10. Literature: Figure 3 from [87]. (at t  200 ms) while for verhulst2015 (red) the plateau is reached shortly after the tone onset. (dau1997), with a strong onset overshoot and a resting position at 0 MU. For king2019 (Fig. 9b), a mild over- shoot was observed, whose maximum amplitude (1.52  103 a.u.) was higher in absolute value than that for the undershoot (1.08  103 a.u.). With an observed steady-state peak-to-peak amplitude of 0.87  103 a.u. king2019 is, at this stage, the model that preserves the most temporal fine structure. 4.3.3 AM model responses Figure 12. Simulated on-frequency AN responses to a 4000-Hz AM tone of 60 dB SPL (100% modulation, fmod = 100 Hz). Left: Onset responses. Right: Steady-state responses. Literature: Figure 12 from [75] and Figure 3C from [35]. Model responses were obtained for a 500-ms 4000-Hz pure tone that was sinusoidally modulated in amplitude (modulation index of 100%) at a rate fmod = 100 Hz, presented at 60 dB SPL, including up/down ramps of 2.5 ms. The initial (0–50 ms) and later (350–400 ms) por- tions of the simulated responses are shown in the left and right panels of Figure 12, respectively. In all models, the modulation rate of 100 Hz is visible as amplitude fluctua- tions with the corresponding periodicity of 10 ms. In addi- tion, adaptation was observed with stronger simulated responses immediately after the tone onset (left panels) than during the steady-state portion of the response (right panels). ) For the effective models with adaptation loops (dau1997, relanoiborra2019, osses2021), the maximum amplitudes (Fig. 12a, left) were much lower in osses2021 than for dau1997 and relanoiborra2019, due to the stronger overshoot limi- tation. For these models, it was also observed that their phases are not perfectly aligned due to the outer- and middle-ear filters that introduced a delay into relanoiborra2019 (black traces run “ahead” the blue traces of dau1997), while the group-delay compensation in osses2021 (Sect. 2.2) seemed to overcompensate the alignment of the simulated waveforms (purple traces run “behind” the blue traces). In the right panel, the dynamic range of relanoiborra2019 (black traces) is lower than for osses2021 and dau1997, which have very similar steady-state amplitudes. The reduced dynamic range in relanoiborra2019 is mainly due to the nonlinear cochlear compression of the filter bank that interacts further with the expansion stage. In king2019 (Fig. 12b), a small effect of adaptation was observed with a maximum onset response of 0.88  103 a.u. (left panel) that decreases to a local maximum amplitude of 0.24  103 a.u. during the steady-state response (right panel). Figure 12. Simulated on-frequency AN responses to a 4000-Hz AM tone of 60 dB SPL (100% modulation, fmod = 100 Hz). Left: Onset responses. Right: Steady-state responses. Literature: Figure 12 from [75] and Figure 3C from [35]. In relanoiborra2019, the simulated rates were between 70.2 and 83 MU for signal levels beyond 40 dB. 4.3.2 Rate-level functions Rate-level functions were simulated for a 4000-Hz pure tone presented at levels between 0 and 100 dB SPL with a duration of 300 ms, gated on and off with 2.5-ms cosine ramps. The obtained results are shown in Figures 10 and 11 for rate-level curves in the steady-state regime and for onset responses, respectively. For all models, average rates are shown (coloured traces) while for the phenomenological and biophysical models (panels c–h), the simulated response for the three types of neurons (HSR, MSR, and LSR) are shown (grey traces). For the phenomenological models (zilany2014 and bruce2018), the simulated waveforms using their two types of AN synapse outputs are shown in Figure 9c–d, based on a PSTH (dark green or brown curves) and mean-rate synapse output (light green or brown curves). The obtained PSTH and mean rate responses in zilany2014 differ in their steady-state values (lower values for the PSTH estimate), while for bruce2018 the difference is in their onset responses, with almost no onset adaptation in the simulated mean-rate output. For the biophysical models (Fig. 9e), the AN synapse outputs rep- resent mean firing rates where a stronger effect of adapta- tion was observed for verhulst2018 (sky blue), with a plateau after onset that was reached after about 150 ms For the phenomenological and biophysical models, the discharge curves in Figure 10c–h tend to saturate towards higher levels, which is in line with experimental evidence (e.g., [87]). One difference between these curves is that they start to increase at slightly higher levels for the biophysical (from ~20 dB SPL) than for the phenomenological models (from ~0 dB SPL). For the effective models (Fig. 10a and b), with the exception of relanoiborra2019, the simulated rates did not show saturation as a function of level. A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 14 4.3.3 AM model responses This saturation effect results from the combined action of the nonlinear cochlear filter (Stage 3) with the later expan- sion stage (Stage 5, Fig. 2) that precedes the adaptation loops. Despite the overall lack of saturation in the evaluated effective models when looking at the steady-state outputs, a different situation is observed for the onset responses of Figure 11, where the responses of the models using adapta- tion loops had a prominent onset saturation (dau1997: 1443 MU for levels above 50 dB; relanoiborra2019: 1435 MU for levels above 30 dB; osses2021: 614 MU for levels above 50 dB). Other interesting aspects to high- light are that: (1) almost no onset effect is observed in the mean-rate output of bruce2018; (2) king2019 does not account for any type of saturation as the signal level increases (Figs. 10b and 11b). It should be noted that although hard saturation (as in Fig. 11a) has not been experimentally observed for onset AN responses, a decrease in the rate of growth of onset-rate curves with level is expected [87], a condition that is not met in king2019 or verhulst2015. ( ) The AN responses produced by verhulst2015 and verhulst2018 (Fig. 12e) showed an overshoot reaching firing rates of 598.5 and 565.2 spikes/s, respectively. After the onset, the overshoot effect quickly disappeared in verhulst2015, reaching a maximum local rate of 251 spikes/s during the second modulation cycle and 222 spikes/s between 370 and 400 ms. In contrast, verhulst2018 adapted more slowly after the onset with a maximum rate of 319 spikes/s in response to the second modulation cycle, while the response continued adapting reaching a maximum rate of 176 spikes/s between times 370 and 400 ms. For zilany2014 (Fig. 12c) and bruce2018 (Fig. 12d), the mean-rate and PSTH outputs are shown as lighter and darker traces, respectively. It can be observed that in zilany2014, the AM modulations showed a sim- ilar mean-rate and PSTH excursions of about 100 spikes/s (Fig. 12b, right: mean rates between 194 and 295 spikes/s; PSTHs with rates between 94 and 196 spikes/s), but the PSTHs had overall lower rates. In bruce2018, a greater AM fluctuation is observed for the PSTH outputs A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 15 Figure 13. Simulated AN responses to a complex tone with three frequency components at 414, 650, and 1000 Hz. 4.3.4 Synchrony capture Model responses were obtained for a complex tone of 50 dB SPL formed by three sinusoids of equal peak amplitude and frequencies of 414 Hz (9.6 ERBN), 650 Hz (12.6 ERBN), and 1000 Hz (15.6 ERBN). This type of com- plex tone with more carriers and greater range of frequen- cies is commonly used in studies of profile analysis (e.g., [65]) and it is useful to explain an AN property named “synchrony capture” [57, 63] that is believed to play a rele- vant role in the neural coding of supra-threshold speech sounds [33, 63]. When synchrony capture occurs, the neural activity in on-frequency channels is driven primarily by one frequency component in the harmonic complex, such that there are minimal fluctuations due to the fundamental- frequency envelope, while off-frequency channels exhibit fluctuating AN patterns at the fundamental frequency. To illustrate whether the evaluated models account for synchrony capture, the model outputs in response to the described complex tone were obtained for frequencies between 415 Hz (n = 320 in Eq. (3)) and 1007 Hz (n = 245 in Eq. (3)) for CFs spaced at approximately 1 ERBN (Dn = 12 or 13), resulting in three on-CF and four off-CF channels. The obtained simulations are shown in Figure 13 for a 30-ms window (between 220 and 250 ms). For each waveform, a schematic metric of envelope fluctuation was obtained and shown as thick grey lines. Those envelope fluctuations were constructed by connect- ing consecutive local maxima that had amplitudes above the mean responses (onset excluded) of each simulated channel. Subsequently, the standard deviation of the obtained envelope estimate was (arbitrarily) divided by one thirtieth of the amplitude scales shown in the insets of each panel (e.g., divided by 800/30 MU for dau1997, relanoiborra2019, and osses2021). The obtained estimates were drawn as maroon circles and connected with dashed lines along the right vertical axes in Figure 13 Figure 13. Simulated AN responses to a complex tone with three frequency components at 414, 650, and 1000 Hz. The model simulations were obtained at on- and off-frequency CFs spaced at 1 ERBN. The thick grey lines represent the envelope of the AN responses. The maroon circle markers represent a metric that is proportional to the standard deviation of the corre- sponding envelope (see the text for details). Literature: Figures 7–8 from [90] and Figure 1 from [91]. 4.3.3 AM model responses The model simulations were obtained at on- and off-frequency CFs spaced at 1 ERBN. The thick grey lines represent the envelope of the AN responses. The maroon circle markers represent a metric that is proportional to the standard deviation of the corre- sponding envelope (see the text for details). Literature: Figures 7–8 from [90] and Figure 1 from [91]. (darker brown traces) with an excursion of 185 spikes/s (Fig. 12d, right: rates between 56 and 241 spikes/s) compared with the 40 spikes/s (rates between 121 and 161 spikes/s) of its mean-rate output. Additionally, bruce2018 showed a limited effect of adaptation in its mean-rate outputs, along with a shallower AM response in comparison to the obtained PSTH. We will not focus on the mean-rate output of this model, because (1) their authors validated the model primarily using PSTHs, recom- mending the use of the AN synapse output for further processing [36], (2) the model using PSTH outputs can be used as input for subcortical processing stages from the UR EAR toolbox [66], and (3) all the studies that we have so far identified using bruce2018 consistently used PSTH outputs [88, 89]. [ ] It should be noted that zilany2014, from the same model family, has been extensively validated using both mean-rate and PSTHs outputs. In fact, for studies where psychoacoustic aspects have been investigated (e.g., [65]) there is a tendency to use the mean-rate model outputs. 4.3.4 Synchrony capture Modulation transfer functions (MTFs) of a modu- lation filter with a BMF  80 Hz, assessed using 1000-Hz AM tones presented at 30 (panel a) or 70 dB SPL (panel b) that were sinusoidally modulated with fmod frequencies between 10 and 130 Hz. The MTFs are normalised to the maximum model response across the tested fmod frequencies. Literature: Figures 4–6 from [92] and Figures 1 and 4 from [93]. Figure 14. Modulation transfer functions (MTFs) of a modu- lation filter with a BMF  80 Hz, assessed using 1000-Hz AM tones presented at 30 (panel a) or 70 dB SPL (panel b) that were sinusoidally modulated with fmod frequencies between 10 and 130 Hz. The MTFs are normalised to the maximum model response across the tested fmod frequencies. Literature: Figures 4–6 from [92] and Figures 1 and 4 from [93]. Figure 15. Simulated IC responses using one modulation filter (BMF  80 Hz) to a click train of alternating polarity with a total duration of 1 s, repetition rate of 10 Hz and click duration of 100 ls. Only the responses to the two last clicks are shown, whose peak-to-peak amplitudes are indicated in Table 2. Liter- ature: Figure 1 from [94] and Figures 8–9 from [95]. Figure 15. Simulated IC responses using one modulation filter Simulated IC responses using one modulation filter Figure 15. Simulated IC responses using one modulation filter (BMF  80 Hz) to a click train of alternating polarity with a total duration of 1 s, repetition rate of 10 Hz and click duration of 100 ls. Only the responses to the two last clicks are shown, whose peak-to-peak amplitudes are indicated in Table 2. Liter- ature: Figure 1 from [94] and Figures 8–9 from [95]. 4.4.1 Modulation transfer function models, the MTFs were no longer bell-shaped and seemed to act as lowpass filters, which is inline with physiological evidence indicating that regions of “enhancement” in MTFs of low level-signals can become regions of “suppression” for higher presentation levels (see, e.g., Fig. 4 from [92]). The first set of figures represents a modulation transfer function (MTF) in response to 100% AM tones modulated at fmod rates between 10 and 130 Hz (steps of 5 Hz). The tones were centred at 1000 Hz, had a duration of 300 ms, included 5-ms up/down ramps, and were presented at 30 and 70 dB SPL. For this analysis, 100 ms in the last portion of the simulated responses were used (between times 190 and 290 ms). The MTFs were derived from the maximum of the simulated responses. The responses were normalised to the corresponding maximum estimate over the set of tested fmod values, so that the MTF of each model had a maximum value of 1. The resulting MTFs are shown in Figure 14. The effective models were more insensitive to the change in presentation levels. The only exception to this is relanoiborra2019, where a narrower MTF was obtained in Figure 14b (compared with panel a). The models dau1997, osses2021, and king2019 have MTFs that are qualitatively similar across presentation levels. 4.4 Subcortical neural processing We show two sets of figures to schematise the subcorti- cal processing of the evaluated models. 4.3.4 Synchrony capture (dimensionless scale with labels between 0 and 6, as indi- cated in panels a and b), where higher values indicate greater envelope fluctuation variability. The resulting envelope scale allows for a direct comparison between models. In Figure 13 it can be observed that for all models, the on-frequency channels had nearly flat envelope fluctua- tions. The variability estimate averaged across on- frequency bins (at 415, 662, and 1007 Hz) ranged between 0.11 (king2019) and 1.71 (bruce2018). The variability estimate across off-frequency bins (at 489, 574, 769, and 881 Hz) ranged between 0.74 (king2019) and 4.09 (relanoiborra2019, with a maximum deviation of 6.95 at CF = 881 Hz in Fig. 13g). For all models the off- CF variability was greater than the on-CF variability, with king2019 being the least sensitive model to code envelope fluctuations. A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 16 Figure 14. Modulation transfer functions (MTFs) of a modu- lation filter with a BMF  80 Hz, assessed using 1000-Hz AM tones presented at 30 (panel a) or 70 dB SPL (panel b) that were sinusoidally modulated with fmod frequencies between 10 and 130 Hz. The MTFs are normalised to the maximum model response across the tested fmod frequencies. Literature: Figures 4–6 from [92] and Figures 1 and 4 from [93]. Figure 15. Simulated IC responses using one modulation Figure 14. Modulation transfer functions (MTFs) of a modu- lation filter with a BMF  80 Hz, assessed using 1000-Hz AM tones presented at 30 (panel a) or 70 dB SPL (panel b) that were sinusoidally modulated with fmod frequencies between 10 and 130 Hz. The MTFs are normalised to the maximum model response across the tested fmod frequencies. Literature: Figures 4–6 from [92] and Figures 1 and 4 from [93]. Figure 15. Simulated IC responses using one modulation filter (BMF  80 Hz) to a click train of alternating polarity with a total duration of 1 s, repetition rate of 10 Hz and click duration of 100 ls. Only the responses to the two last clicks are shown, whose peak-to-peak amplitudes are indicated in Table 2. Liter- ature: Figure 1 from [94] and Figures 8–9 from [95]. Figure 14. 4.4.2 Response to clicks of alternating polarity This means for the current simulations, that the reported processing times of 122.9 and 319.5 s for verhulst2015 and verhulst2018, respectively, cannot be further reduced, even if the user requests the simulation of fewer CFs. In con- trast, in any model based on a parallel filter bank, including zilany2014 and bruce2018, each cochlear section is independent of each other, and a user-defined number of frequency channels can be simulated, which vastly reduces the computation time for different model configurations. For this processing, we used the default number of CFs for the biophysical and effective models, while for zilany2014 and bruce2018, 50 CFs were obtained between CFn = 133.7 Hz (n = 393, Eq. (3)) and CFn = 12010 Hz (n = 1), spaced at n = 8 bins to roughly meet the number of filters from Table 2. The obtained click responses are shown in Figure 15 and are illustrated for the last two clicks (of amplitudes A and A) of the test click train. The biophysical models provided click responses that had positive and negative amplitudes (Figs. 15e–f), which was not the case for the phenomenological models that also use the SFIE model. This is because verhulst2015 and verhulst2018 assume that a population response can be obtained from the sum of single neuron activity (as, e.g., in [56]), with no half-wave rectification in the SFIE model (a non-explicit choice of the authors [34, 35]) that, after scaling [35, 41], results in a simplified neural represen- tation that correlates with changes in electrical dipoles visible in scalp-recorded potentials [35]. The effective models, that use the modulation-filter- bank concept, showed only positive amplitudes for all filters with BMFs  10 Hz [31] due to their envelope extraction, a phase-insensitive (“venelope”) processing [37, 70]. For modulation frequencies below 10 Hz, the perceptual models preserve the phase information, something that is not illus- trated in Figure 15 (nor in Fig. 14). Due to the long processing time of the evaluated biophysical models, their implementations include an option of parallel processing (also available in the original implementation of bruce2018 [36]), where multiple input signals can be processed simultaneously. The number of signals that can be processed in parallel will depend on the number of threads of the host computer. 4.4.2 Response to clicks of alternating polarity The results in Figure 14 show that the models pro- duce bandpass-shaped MTFs with estimated BMFs between 35 Hz (zilany2014) and 70 Hz (dau1997, relanoiborra2019, and osses2021) that are below the theoretical BMFs (see Tab. 2). It is interesting to observe that the sharpest MTFs were obtained not only for dau1997 and osses2021 (both designed with Q = 2), but also for king2019 (which has a Q = 1), while a wider tuning was observed for the remaining models, including relanoiborra2019 (which has a Q = 2). The second set of figures focuses on simulating the response to a typical click train as used in the assessment of auditory brainstem responses (ABRs) [95]. We used a click train with a repetition rate of 10 Hz and a duration of 1 s (i.e., containing 10 clicks). The clicks had an alternat- ing polarity (amplitude A or A) and were presented at 70 dB peak-equivalent SPL (dB peSPL) [96], i.e., using A = 0.1789 Pa. Each individual click had a duration of 100 ls. For this processing, the simulated outputs of Stage 6 of each model (see Fig. 2) were averaged across CFs to obtain a broadband representation, i.e., all simulated repre- sentations were added together and then divided by the For the biophysical and phenomenological models, the MTFs obtained for the 70-dB AM tones (Fig. 14b) were different than those obtained for 30 dB (Fig. 14a). For these A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 17 was performed on a personal computer equipped with an Intel Core i5-10210UR, 1.6-GHz processor with 16 GB of RAM memory. number of CFs [34, 35]. This type of output can be used to derive a peak-to-peak or peak-to-trough amplitude correlate of the wave-V ABR component [95]. The results of the computational costs used by each model are given in the entry “Performance” of Table 2. The time required by the models to process one frequency channel ranged between ~0.02 s (osses2021, dau1997) and 2.5 s (bruce2018). For individual frequency channels, the biophysical models (verhulst2015 and verhulst2018) showed moderate calculation times between 0.3 and 0.8 s. However, these models always require (internally) the simulation of the whole discretised cochlea with 1000 cochlear sections, independent of the number of user-requested cochlear channels (default number of 401 for the Verhulst models). 4.4.2 Response to clicks of alternating polarity As a further solution to the long processing time, Stages 2-5 of verhulst2018 (transmission-line, IHC, and AN modules) and bruce2018 (generating mean PSTHs) have been approximated using deep neural networks in [99, 100] and [101], respectively. g ( g ) Finally, the simulated peak-to-peak amplitudes in response to the last positive and negative clicks of the pulse train (ninth and tenth click, shown in Fig. 15) are shown in the entries “Click A” and “Click A” of Table 2. From those amplitudes, it can be observed that there are models that have higher peak-to-peak amplitudes in response to positive clicks (zilany2014, verhulst2015, bruce2018, relanoiborra2019) and others where higher ampli- tudes are observed in response to the clicks of negative polarity (dau1997, verhulst2018, king2019, osses2021). Although we do not discuss the significance of this polarity sensitivity, this aspect has been a matter of discussion, in particular for electrical hearing, where it has been found that evoked potentials in response to positive and negative polarity clicks represent one of the differences between humans (e.g., [97]) and other mammals (e.g., [98]), whose responses are more sensitive to stimulation with clicks of negative and positive polarities, respectively. 5 Models in perspective The stimuli and comparison measures used in our evaluation (Sect. 4) were chosen to reflect relevant temporal and spectral properties of the models in a normal-hearing condition. Our evaluation provides an objective view, accompanied by a graphical representation of how the model responses reflect specific aspects of the hearing pro- cess in their model structure. The content of this study might be considered as a guideline for model selection, but the motivation was not to select a “winner” among the different evaluated models. We compared models which were verified in different exper- imental conditions or in connection to different hearing applications, and we only presented raw model outputs (Figs. 7, 9, 12, 13, 15) or outputs transformed to charac- terise specific hearing properties (Figs. 4–6, 8, 10, 11, 14). These outputs reflect model responses to a very specific dataset that may not be suitable to appropriately verify all models. Any model, though, to be informative, needs to be verifiable and falsifiable, such that it is possible to understand both its essential characteristics and features 5.1 Applications of the evaluated auditory models Dau1997 is a monaural model that has been used to simulate a number of psychoacoustic tasks including tone- in-noise and AM detection experiments using a forced- choice paradigm (e.g., [31, 44]). To enable the model for the comparison between two or more sounds, the output of Stage 6 (Fig. 2) is used as input to a decision back-end based on a signal-detection-theory (SDT) framework, the template-matching approach. This framework, extended to adopt two templates, has been recently validated to account for the perceptual similarity between two sounds using osses2021 [39]. 4.5 Computational costs The computational cost required to run each model was measured using the same click train as described in the pre- vious section. Therefore, we assessed the time required to process an input signal of 1-s duration between Stages 1 and 6 of each model (Fig. 2). This metric aims at providing a relative notion of the processing times across models. Note that some model implementations can use parallel process- ing, which was disabled in this evaluation. The assessment A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 18 limitations (suboptimal template matching strategies) or stochastic limitations such as internal additive noise, multi- plicative noise [109], and memory noise [72, 110]. The model can be adapted to simulate hearing impairment by modify- ing its compression parameters (knee point and compres- sion rate), and by increasing the bandwidth of the underlying cochlear filters. Despite the simplicity of this model—in fact one of its strengths—we have shown in this paper that the model can account for several of the compar- ison metrics, with the exception of the broadening of cochlear filters at higher presentation levels (Fig. 6), the adaptation saturation (Figs. 10–11), and the coding of fluctuation profiles (with minimal difference in amplitude fluctuations in Fig. 13). that explain the predictive power in a given range of conditions, as well as its limitations that make transparent where the model fails. In the following sections we provide a brief overview of the context in which each of the selected models has been used and include general recommendations for further applications. 5.2 Other applications of auditory models Apart from the listed applications, auditory models have also been used in several other applications such as sound quality assessment (e.g., [10, 111–113]), prediction of speech intelligibility (e.g., [103, 114]), and automatic speech recognition (e.g., [115]). The models zilany2014 and bruce2018 can account for elevated hearing thresholds due to OHC (“Cochlear gain loss” in Stage 3) or IHC impairment (“IHC loss” in Stage 4) [55]. The AN stage (Stage 5) includes two types of outputs: An actual spike generator and an analytical mean-rate synapse output. The spike generator has been primarily used to simulate physiological data, including the phenomenon of short- and long-term adaptation [75]. The mean-rate synapse output using zilany2014 has been used to simulate specific psychoa- coustic tasks [65, 102], including speech intelligibility pre- dictions [103]. In the context of this special issue on binaural hearing, it is worth mentioning a number of binaural applications that rely on the evaluated monaural auditory models: The low- pass modulation filter (similar to dau1997) [31, 44] served as the basis for a model of binaural masking that uses a decision stage based on the equalisation-cancellation theory [116]. This model was later extended to predict perceptual attributes of room acoustics [117–119]. The model zilany2014 has been used to predict (1) the sensitivity to interaural time and level differences by estimating the disparity between left and right AN responses using a deci- sion back-end based on shuffled cross-correlograms [120], and (2) the median-plane sound localisation for various pro- files of sensorineural hearing loss (OHC impairment) [121]. Finally, bruce2018 has been used to simulate the lateral- isation of high-frequency stimuli in a coincidence-counting model [88]. The models verhulst2015 and verhulst2018 were initially designed to simulate otoacoustic emissions [26] and can account for elevated hearing thresholds due to OHC impairment (“Cochlear gain loss” in Stage 3). Furthermore, they allow to study effects of the gradual disconnection of AN fibres, known as synaptopathy, on auditory brainstem responses [35, 104]. When coupled with a decision back-end, they have been used to simu- late psychoacoustic performance in simultaneous tone- in-noise and high-rate AM tasks (fmod ~100–120 Hz) [105, 106]. A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 y  In Section 4.1.3, we suggested a minimum number of filters for each filter bank to roughly meet a 3-dB filter crossing (Tab. 2, “40 dB: Number of bands”). The required number of bands may vary from applica- tion to application and depend on the type of sounds that are to be simulated. This choice can be particu- larly critical in models where the number of bands are a free parameter (here zilany2014 and bruce2018). For models that are used as front-ends to machine-learning applications, Lyon [22] suggested a “not-too-sparse set of channels” with about a 50% overlap between filters, i.e., twice the number of chan- nels that we recommend in Table 2. It is important to keep in mind, however, that our estimation was based on model responses to white noises, which are sus- tained signals in time and broadband in frequency. At higher presentation levels, where nonlinear filter banks act as compressors, similar estimations using sine tones (sustained narrowband signals, as in Fig. 5) or clicks (transient broadband sounds) may result in a different number of required bands. Diff t i l ti lt b t d h l It is important to note that the simplification of auditory models based on statistical methods or machine learning processes requires a careful interpretation. While these approaches might be well suited to achieve goals such as real-time processing (e.g., [99]) in applications of speech perception (e.g., [101]) or in the prediction of evoked poten- tials [89], they limit the modular comprehension of each auditory stage, especially if multiple model stages are approximated (as in [89, 101]). In a recent study [89], firing rates of cortical A1 neurons in ferrets were approximated using several time-frequency representations ranging from simple short-time Fourier transforms to more detailed models of AN synapses (includ- ing bruce2018) to which a linear-nonlinear (LNL) enco- der was used. Based on their separately-fitted encoders, the authors concluded that cortical processing in ferrets perform a “very simple signal transformation,” without dis- cussing how different the linear and nonlinear components in each of their encoders were. Despite the success of the authors in approximating neural responses in ferrets, we believe that it is difficult to know whether the “simple trans- formation” is indeed related to the underlying mechanisms of hearing (the cortical processing in ferrets) or rather is related to the complexity of operations in the fitted encoders. A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17  Different simulation results can be expected when eval- uating mean-rate and PSTH outputs of models includ- ing AN synapse stages as shown in Figures 9–12. The particular choice of the type of output depends on the target application of the model. The spike genera- tor is primarily used to simulate physiological data (e.g., [36, 75], while the mean-rate synapse output is typically used to simulate specific psychoacoustic tasks (e.g., [65, 102]).  The choice of a set of stimuli to test and validate a specific model is crucial. As we stated in Section 1, the simulation of “unseen” (arbitrary) sounds may produce model outputs that have not been previously validated (or at least not reported) by the model devel- opers. Actually, an unexpected model behaviour may not be strictly related to an unseen sound, but rather to an unseen sound property. For example, the models with adaptation loops have historically had an oversen- sitivity to transient sounds (e.g., [39, 125, 126]), lead- ing to model versions with limited onset responses to counteract this effect [31, 39] or have used stimuli with smoother onsets in their evaluation. A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 19  The results in Figures 6 and 14 show that there are nonlinear interactions between model stages as a func- tion of level and for different types of signals. This suggests that different sets of stimuli are required to characterise the behaviour of complex processes such as that of nonlinear filter banks. In other words, mod- els may not always act as a linear time invariant (LTI) system. using only a stage of envelope extraction followed by a modulation filter bank, omitting the stages of cochlear filtering and auditory adaptation. This model, however, is not thought to predict the performance in listening condi- tions where the omitted model stages do play a role as it is the case (in this example) for forward-masking tasks. Peripheral auditory models are often combined with a decision back-end module converting simulated responses into (1) a behavioural response that reflects detectability or discriminability of a sound (e.g., [2]), or into (2) perceptual metrics to estimate, e.g., loudness [28], perceived reverberation [117, 118], and sound-source localisation [122, 123]. For successful simulations, the decision stage should appropriately weight the information contained in the model representations. An analysis of weighted time- frequency representations (time, audio frequency, and/or modulation frequency) can reveal what portions of the simulated responses are more relevant (e.g., [39, 124]). y  In Section 4.1.3, we suggested a minimum number of filters for each filter bank to roughly meet a 3-dB filter crossing (Tab. 2, “40 dB: Number of bands”). The required number of bands may vary from applica- tion to application and depend on the type of sounds that are to be simulated. This choice can be particu- larly critical in models where the number of bands are a free parameter (here zilany2014 and bruce2018). For models that are used as front-ends to machine-learning applications, Lyon [22] suggested a “not-too-sparse set of channels” with about a 50% overlap between filters, i.e., twice the number of chan- nels that we recommend in Table 2. It is important to keep in mind, however, that our estimation was based on model responses to white noises, which are sus- tained signals in time and broadband in frequency. At higher presentation levels, where nonlinear filter banks act as compressors, similar estimations using sine tones (sustained narrowband signals, as in Fig. 5) or clicks (transient broadband sounds) may result in a different number of required bands. 5.3 Simplified auditory representations When an auditory model is used to broaden our under- standing of auditory processes [1, 2], it is required that the model be as complete as possible. More details in the model often come at the price of a more computationally- expensive implementation. Such a level of detail is repre- sented in the selected biophysical and phenomenological models, that attempt to shed light on the mechanisms behind the cellular and neural elements included in auditory processing. On the other hand, effective models have a more epistemic status providing an intelligible but simplified representation of the process. These models can guide the design of new experiments or facilitate the development of listener-targeted products. Such model simplification, however, potentially reduces the number of effects a model can account for, leading to an actual narrowing of its application field. An example of a successful model simplifi- cation is presented in [27], where MTFs were simulated The model relanoiborra2019 can predict speech intelligibility [38] when coupled with a decision back-end stage [38, 107]. Relying on the prediction power of earlier model implementations [77, 108], relanoiborra2019 should be able to (1) account for elevated thresholds based on OHC and IHC impairment [108], and (2) to predict a number of psychoacoustic tasks including simultaneous and forward masking and amplitude modulation [77]. Our results showed that relanoiborra2019 accounts well for hearing properties such as nonlinearities in the cochlear processing and auditory adaptation, including a saturation behaviour similar to that of the AN physiological models. The model king2019 was designed to simulate percep- tual tasks of amplitude- and frequency-modulation detec- tion, primarily at low modulation rates (fmod  20 Hz). The model’s decision back-end includes deterministic A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 Processing in Acoustics, Springer, 2008: 175–196. https://doi.org/10.1007/978-0-387-30441-0_12. 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We are grateful to several colleagues who participated in technical discussions during the writing process: Enrique Lopez-Poveda, Fotios Drakopoulos, Alessandro Altoè, Armin Kohlrausch, Thomas Biberger, and Richard Lyon. We are particularly grateful to Clara Hollomey, who provided immense support for the integration of all models into AMT 1.1. The authors AOV and LV received support from ANR (project: 17-EURE-0017), LHC received support from NIH (R01-DC010813), SV received support from the ERC project RobSpear (Grant No. 678120), and PM received support from the H2020 project SONICOM (EC Grant No. 101017743). 14. T. Anderson: A comparison of auditory models for speaker independent phoneme recognition. International Conference on Acoustics, Speech, and Signal Processing 2 (1993) 231–234. https://doi.org/10.1109/ICASSP.1993. 319277. 15. J. Breebaart, S. van de Par, A. 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In this study we compared eight monaural models of human auditory processing that simulate responses—with different levels of accuracy—up to the level of the inferior colliculus in the midbrain. We described and quantified the similarities and differences among model implementa- tions and derived a minimum number of filters required for those stages to ensure the preservation of auditory infor- mation based on our estimates of frequency selectivity. // / / 4. G. Bustamante, P. Danès, T. Forgue, A. Podlubne, J. Manhès: An information based feedback control for audio- motor binaural localization. Autonomous Robots 42 (2018) 477–490. https://doi.org/10.1007/s10514-017-9639-8. 5. R. Peng: Reproducible research in computational science. Science 334, 6060 (2011) 1226–1227. https://doi.org/ 10.1126/science.1213847. We showed that despite the differences in model design that result in more physiologically- (biophysical and phe- nomenological models) or perceptually-plausible approxi- mations (effective models), all the models can account for a number of basic hearing properties. Examples of these properties are the phase-locking reduction in inner-hair-cell processing and the phenomenon of auditory adaptation. Still, an in-depth understanding of each of the model stages is required when selecting a model for a specific application. We encourage future users to be explicitly aware of the specific datasets of sounds and experimental paradigms upon which their models have been evaluated, as well as of other underlying model limitations. To this end, a com- parison across model implementations provides a guideline for their selection and an excellent way to challenge the capabilities of different models. / 6. R. Patterson, M. Allerhand, C. 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Journal of the Acoustical Society of America 140 (2016) 1023–1038. https://doi.org/10.1121/1.4960574. 5.4 Considerations for further modelling work The following is a list of aspects that we recommend to keep in mind for further auditory modelling work, based on the general observations of this study:  If the evaluated sounds are assumed to be reproduced via loudspeakers, or supra-aural or circumaural head- phones, we recommend to use an outer-ear module as in relanoiborra2019, osses2021, or to apply an HRTF (as in [57]). Although we did not evaluate this effect, such an omission implicitly assumes that the outer ear (compare the grey and black lines for relanoiborra2019 and osses2021 in Fig. 3) does not influence the coding of incoming signals in the ascending auditory pathway. When using large datasets, where the stimuli are split into training and validation data (e.g., [16, 101, 112]), the stimuli should contain representative samples of the rele- vant sound properties that the model user wishes to test. A practice like this can help to support (or not) the appli- cability of a specific model to sounds that may have not been even validated before, the “unseen sounds”, reducing (or generating awareness of) the potential limitations of the test model. A. Osses Vecchi et al.: Acta Acustica 2022, 6, 17 20 1. R. Meddis, E. Lopez-Poveda, R. Fay, A. Popper (Eds.): Computational Models of the Auditory System. Springer Handbook of Auditory Research. Springer, 2010. 2. T. Dau, Auditory Processing Models. In: Havelock D., Kuwano S., Vorländer M., Eds. Handbook of Signal References Meddis: A human nonlinear cochlear filterbank. Journal of the Acoustical Society of America 110 (2001) 3107–3118. https://doi.org/10.1121/1.1416197. ( ) // / / 40. A. 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A B 0 5 10 15 20 0 200 400 600 800 Days post MC38 s.c. injection Tumor volume [mm3] WT ST2-/- * 0.0 0.5 1.0 1.5 2.0 Tumor mass [g] WT St2-/- * upplementary Figure S2. MC38 tumor growth is enhanced in St2-deficient hosts. C38 colon cancer cells were injected subcutaneously into either WT or ST2-/- mice. Tumor volume ove me (A) and tumor mass at endpoint (B) are shown. Data presented is pooled from 3 independent expe ents (WT n=11 and St2-/- n=9). Graphs show Mean ± SEM and unpaired student t-test was performed: statistical significant (p-values <0.05). A B 0 5 10 15 20 0 200 400 600 800 Days post MC38 s.c. injection Tumor volume [mm3] WT ST2-/- * 0.0 0.5 1.0 1.5 2.0 Tumor mass [g] WT St2-/- * upplementary Figure S2. MC38 tumor growth is enhanced in St2-deficient hosts. C38 colon cancer cells were injected subcutaneously into either WT or ST2-/- mice. Tumor volume ove me (A) and tumor mass at endpoint (B) are shown. Data presented is pooled from 3 independent expe ents (WT n=11 and St2-/- n=9). Graphs show Mean ± SEM and unpaired student t-test was performed: statistical significant (p-values <0.05). A B 0 5 10 15 20 0 200 400 600 800 Days post MC38 s.c. injection Tumor volume [mm3] WT ST2-/- * 0.0 0.5 1.0 1.5 2.0 Tumor mass [g] WT St2-/- * A 0 5 10 15 20 0 200 400 600 800 Days post MC38 s.c. injection Tumor volume [mm3] WT ST2-/- * B 0.0 0.5 1.0 1.5 2.0 Tumor mass [g] WT St2-/- * B A ary Figure S2. MC38 tumor growth is enhanced in St2-deficient hosts. Supplementary Figure S2. MC38 tumor growth is enhanced in St2-deficient hosts. MC38 colon cancer cells were injected subcutaneously into either WT or ST2-/- mice. Tumor volume over time (A) and tumor mass at endpoint (B) are shown. Data presented is pooled from 3 independent experi- ments (WT n=11 and St2-/- n=9) Graphs show Mean ± SEM and unpaired student t test was performed: * MC38 colon cancer cells were injected subcutaneously into either WT or ST2-/- mice. Tumor volume over time (A) and tumor mass at endpoint (B) are shown. Data presented is pooled from 3 independent experi- ments (WT n=11 and St2-/- n=9). Graphs show Mean ± SEM and unpaired student t-test was performed: * = statistical significant (p-values <0.05).
https://openalex.org/W2773258323
https://www.shs-conferences.org/articles/shsconf/pdf/2017/07/shsconf_ies2017_01014.pdf
English
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Ukrainian-Chinese collaboration: Prospects of development
SHS web of conferences
2,017
cc-by
4,692
Ukrainian-Chinese collaboration: Prospects of development Oleh Kratt1*, Kateryna Pryakhina2, and Maryna Bilyk3 1Doctor of Economics, professor, Kremenchuk Mykhailo Ostrohradskyi National University, Ukraine 2PhD candidate, Kremenchuk Mykhailo Ostrohradskyi National University, Ukraine 3PhD student, Kremenchuk Mykhailo Ostrohradskyi National University, Ukraine Abstract. In article the current state of the Chinese-Ukrainian relations is analysed, the priority directions of strategic cooperation are considered. The analysis of economic development of Ukraine and China with definition of positive and negative tendencies is carried out. It is certain that there is a mutual aspiration of the parties before increase in volumes of bilateral trade, diversification of its structure, development of long-term forms of economic cooperation, deepening of investment interaction between Ukraine and China. The analysis of the study is conducted during 2010-2016 years. We have identified prospective investment sectors within the framework of cooperation between Ukraine and China - Transport Infrastructure, Agriculture, Renewable energy, Telecommunications, IT, educational sphere. The current dynamics of the trade balance between Ukraine and the People's Republic of China, taking into account the considerable predominance of imports, requires a thorough review of trade policy measures: to improve the structure of trade, introducing new forms of trade relations, To develop the institutional framework for cooperation, To search for ways to reduce the energy intensity of export industries, To stimulate the means of tariff and non-tariff regulation of imports into Ukraine of mainly investment high-tech goods. The forecast of possible risks according to each perspective direction of development is made. Key words: collaboration, development, analysis, economic relations, prospects * Corresponding author: katerinapryakhina@gmail.com © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). SHS Web of Conferences 39, 01014 (2017) IES2017 SHS Web of Conferences 39, 01014 (2017) IES2017 DOI: 10.1051/shsconf/20173901014 3 Results To date, a significant number of Ukrainian scholars view the Chinese vector of Ukraine's foreign policy as one of the highest priority. Works by Pron [1] devoted to the history of the formation and development of Ukrainian-Chinese relations. The author believes that countries have deep and strong historical traditions that cover multidectoral spheres of politics, economics, and culture. Among the latest publications, it is worth highlighting articles by Holod [2], which explores China's investment expansion in terms of prospects for developing relations with Ukraine. Vysotskaya [3] in her studies carried out a thorough analysis of the main trends and prospects for the development of Ukrainian-Chinese economic relations. In the scientific work of Rogovuy [4] discusses the prospects and risks of developing Ukrainian-Chinese trade and economic relations. Ukrainian-Chinese economic cooperation in the context of foreign economic security it took place in scientific works by Levkivskyi [5]. The mechanism of international economic relations between the two countries is considered and the priority promising areas for further development of foreign economic relations between China and Ukraine are singled out. Grodsky [6] researches modern trade and investment cooperation between Ukraine and China. The strategic guidelines for intensify cooperation between Ukraine and China in the context of globalization are outlined. On the special role of bilateral cooperation to strengthen economic presence of Ukraine in the Asian region is focused [6]. Position of Ukraine in the PRC’s system of foreign economic relations it took place to be in scientific works by Vlasenko L. In the article foundations of Chinese theory of international relations have been considered, differences between Chinese and classical Western concepts of international interactions were highlighted. Existing hierarchy of PRC’s strategic partnership was analyzed [7]. In scientific work from Cheng [8], the choice and strategic opportunities of China in the context of «Ukrainian crisis» was analyzed the determining factors that influenced the choice of China in the context of «Ukrainian Crisis», taking into account the geo-economic and geo-political interests of China. It is also proved that contrary to the European Union and the United States of America, who often use political tools to resolve conflicts (which often leads to disputes and conflicts escalation), China offers economic settlement mechanisms that are acceptable to all stakeholders. Halperina [9] is studying a question about System adaptation of social and economic model of China to conditions of global instability. 2 Data and Methods The scientific article uses general scientific methods: statistical analysis for studying, grouping, comparison of indices of total turnover of countries, graphic method - for visual representation of tables and schemes. Promising directions for the development of relations between China and Ukraine are analyzed and the risk forecast for investors is synthesized. 1 Introduction For this time People's Republic of China is one of the world economic leaders whose value in high gear grows in many spheres of the international life. The certificate of it is election of national monetary unit of China - yuan - world reserve currency on the basis of which the cost of special drawing right of the IMF is calculated. Ukraine, in turn, has rather high economic potential, world achievements in some fields of science and technology, an advantageous geopolitical position which does it by a zone of certain economic and political interests, in particular for People's Republic of China. The mutual understanding of these realities also became the main prerequisite of formation and development of the Ukrainian-Chinese relations. The analysis of the study is conducted during 2010- SHS Web of Conferences 39, 01014 (2017) IES2017 SHS Web of Conferences 39, 01014 (2017) DOI: 10.1051/shsconf/20173901014 2016 years, import and export indicators and the balance of foreign trade balance used. The forecast of possible risks according to each perspective direction of development is made. 2016 years, import and export indicators and the balance of foreign trade balance used. The forecast of possible risks according to each perspective direction of development is made. The purpose of article is the research of character and the prospects of development of the modern Ukrainian-Chinese economic relations. 3 Results Dynamics of foreign trade turnover of Ukraine with China for 2010-2016, million USD Year 2010 2011 2012 2013 2014 2015 2016 , % Export 1316,6 2180 1777,2 2726,7 2674,1 2399,1 1832,5 5,6 Imports 4700,4 6268,3 7899,6 7903,2 5411,0 3771,0 4687,7 -1 Balance -3383,8 -4088,3 -6122,4 -5176,6 -2736,8 -1371,9 -2855,2 -3 Total turnover 6017 8448,3 9676,8 10629,9 8085,1 6170,1 6520,2 1 The indicators of Table 1 show that the average growth rate of export is 5.6%, which indicates an increase in exports every year by 5.6%. In this case, the import rate decreases by 1% and Balance in 3%. The total turnover is increased every year by 1%. The highest indicator of Ukrainian export operations is for 2013 ($ 2726.7 million, Fig. 1). For the period from 2014 to 2016 there is a significant reduction in the volumes of exported goods, which is explained by the political situation in the south and east of Ukraine, under the influence of Russian aggression. This also applies to imported Chinese goods to Ukraine (a significant reduction in 2015 to $ 3771 million). The presence of a negative balance in bilateral trade with China puts Ukraine ahead of the task of finding ways to increase the volume of Ukrainian exports. Table 1. Dynamics of foreign trade turnover of Ukraine with China for 2010-2016, million USD Year 2010 2011 2012 2013 2014 2015 2016 , % Export 1316,6 2180 1777,2 2726,7 2674,1 2399,1 1832,5 5,6 Imports 4700,4 6268,3 7899,6 7903,2 5411,0 3771,0 4687,7 -1 Balance -3383,8 -4088,3 -6122,4 -5176,6 -2736,8 -1371,9 -2855,2 -3 Total turnover 6017 8448,3 9676,8 10629,9 8085,1 6170,1 6520,2 1 Table 1. Dynamics of foreign trade turnover of Ukraine with China for 2010-2016, million USD The indicators of Table 1 show that the average growth rate of export is 5.6%, which indicates an increase in exports every year by 5.6%. In this case, the import rate decreases by 1% and Balance in 3%. The total turnover is increased every year by 1%. The highest indicator of Ukrainian export operations is for 2013 ($ 2726.7 million, Fig. 1). For the period from 2014 to 2016 there is a significant reduction in the volumes of exported goods, which is explained by the political situation in the south and east of Ukraine, under the influence of Russian aggression. This also applies to imported Chinese goods to Ukraine (a significant reduction in 2015 to $ 3771 million). 3 Results The features of the system adapt social and economic model of China to the conditions of global instability. Thesis determined new conditions of socio-economic model of China. In the years of Ukraine’s independence, these relations have generally followed an upward trend and reached a peak in December 2013 when the Treaty of Friendship and 2 SHS Web of Conferences 39, 01014 (2017) IES2017 SHS Web of Conferences 39, 01014 (2017) DOI: 10.1051/shsconf/20173901014 Cooperation was signed.China’s interest in cooperation with Ukraine is also linked to the decision of the Chinese leadership to create “foreign food bases”. In particular, it includes the use of Ukraine’s agricultural opportunities combined with China’s investment and technological capacities. The current Ukraine-China relations are largely influenced by China’s growing political and economic interests regarding the countries of the EU and New Eastern Europe (Belarus, Ukraine, Moldova, Latvia, Lithuania, and Estonia). China plans to implement its global strategy to increase exports and investment in potential markets located between Russia and the EU. Ukraine could become an important place to promote Chinese products and brands, gain access to new markets, and acquire strategic assets. Despite China’s interest towards Ukraine, these relations were put on hold since the beginning of the Revolution of Dignity. Likely, this is because rapprochement with Ukraine can go only as far as China will be able to maintain its safe balance in the geopolitical US- Russia-China triangle and in the world [10]. It costs to mark, that world economic relations represent a combination of different forms of business interaction that occurs between countries of the world, and primarily include trade and economic cooperation, the flow of investment flows, scientific and technical ties, the exchange of technologies and intellectual property. Therefore, it is advisable to look more closely at each of the forms of interaction between Ukraine and the People's Republic of China at the present time. According to the General Customs Administration of the People's Republic of China, in 2016, the trade turnover between Ukraine and the PRC amounted to $ 6520.2 million. At the same time, Chinese exports to Ukraine - 4687.7 million. The US (increase by 32.2%), Chinese imports from Ukraine amounted to $ 1,832.5 million (decrease by 24.1%). Balance of bilateral trade in favor of PRC amounted to $ 2855.2 million [11]. The average growth rate was used to substantiate the evaluation of the change in indicators. Table 1. 3 Results The presence of a negative balance in bilateral trade with China puts Ukraine ahead of the task of finding ways to increase the volume of Ukrainian exports. 3 3 SHS Web of Conferences 39, 01014 (2017) IES2017 DOI: 10.1051/shsconf/20173901014 Fig. 1. Indicators of export-import operations of Ukraine with China for the period 2010-2016 Fig. 1. Indicators of export-import operations of Ukraine with China for the period 2010-2016 Most specific gravity in the structure of export for today is occupied by supplying with mineral products, grain-crops, fats and oils of animal or vegetable origin. It is possible to mark, that the Ukrainian export to Republic of China has raw material character mostly. The main reason for this is to orient the Chinese market to self-sustaining and stimulate the domestic producer, correspondingly reducing the number of industries in which, given the lack of Chinese counterparts, significant imports would be available (Fig. 2). Fig. 2. Indicators of exports of Ukrainian products to China in 2016 Fig. 2. Indicators of exports of Ukrainian products to China in 2016 The basis for imports from China in 2016 was: electric machines – 22.5%; nuclear reactors, boilers and machines – 17,1%; plastics, polymeric materials – 6.1%; ferrous metals – 4.4%; organic chemical compounds – 3.7%; various chemical products – 3.7%; footwear – 3.5%; articles of ferrous metals – 3% [12]. Summarizing the above-mentioned, it is possible to state that the export-import operations and development cooperation directions of the last years have shown that the increase in commodity turnover will occur due to increase in import to the Ukraine of Chinese products and services. Thus the volumes of home export in PRC have saltatory 4 SHS Web of Conferences 39, 01014 (2017) IES2017 DOI: 10.1051/shsconf/20173901014 character (Fig. 1) and hardly will exceed a monthly index in the 250 million дол USA in a prospect. character (Fig. 1) and hardly will exceed a monthly index in the 250 million дол USA in a prospect. p p In 2015 Ukraine joined initiative “One belt, signing one way” from PRC “Protocol about a collaboration within the framework of the Silk way”, and now, actually, the searches of deepening and activations of trade and economic relation are conducted between countries within the framework of this protocol. A bilateral investment collaboration does not answer possibilities of China and necessities of Ukraine as yet. 3 Results The increase of volumes of import from PRC in 2016 is not accompanied by activation of investment collaboration (0,48% in the general volume of the attracted lines of foreign investments), the Ukrainian investments in PRC are practically absent also [11]. Priority for development of mutually beneficial investment collaboration are such spheres, as transport, agricultural, creation of infrastructural objects, space, innovative, aviation, scientific, technical and other spheres. Basic investment projects are within the framework of collaboration between Ukraine and China. 1. Mechanical engineering. The Beijing Skyrizon Aviation Industry Investment Co Ltd company created with Ukrainian “Motor Sіch” joint venture which is based already in China. Construction of a factory for the production and maintenance of aircraft engines was started in Chongqing City. At the AirshowChina 2016 exhibition the Beijing Skyrizon company declared a possibility of license production by joint venture of the D-136 helicopter engines, MC-500B and TV3-117VMA-SBM1V and engines for planes of general purpose A-450C, turbojet D436-148FM and D-18T, etc. Beijing Skyrizon finances production start, and the Ukrainian side provides technologiesA certain risk for our state is the lack of the strategic directions from use and loan of the Chinese technologies and innovations that could become a push to improvement of the technologies which are available in Ukraine and attraction of foreign experience. 2. Renewable energy. The CNBM International Corporation in Ukraine owns the 10 largest solar power plants located in the Mykolayiv and Odessa regions. Their capacity is 267 MW, while the power of all Ukrainian solar power plants reaches 500 MW. According to CNBM, the investment amounted to about $ 1 billion. 2. Renewable energy. The CNBM International Corporation in Ukraine owns the 10 largest solar power plants located in the Mykolayiv and Odessa regions. Their capacity is 267 MW, while the power of all Ukrainian solar power plants reaches 500 MW. According to CNBM, the investment amounted to about $ 1 billion. 3. Agro-industrial complex The Chinese corporation COFCO Agri launched in 2016 in the Mykolaiv Sea Commercial Port a transshipment complex of grain and oilseeds with an annual capacity of 2.5 million tons. The investment project cost $ 75 million, it is already working properly, and from the Ukrainian water area there are steamships with grain for the People's Republic of China. Several elevators and an oil extraction plant are also controlled by COFCO Agri in Ukraine. 3. 3 Results Agro-industrial complex The Chinese corporation COFCO Agri launched in 2016 in the Mykolaiv Sea Commercial Port a transshipment complex of grain and oilseeds with an annual capacity of 2.5 million tons. The investment project cost $ 75 million, it is already working properly, and from the Ukrainian water area there are steamships with grain for the People's Republic of China. Several elevators and an oil extraction plant are also controlled by COFCO Agri in Ukraine. Beginning in 2013 around the village of Naumovka, located in the Chernihiv region in the north of Ukraine is a farm company, which is founded by the large Chinese Agricultural State Corporation Huangfantsyu. Today, In Venture Investment Group is leading a major player in the soybean market and soybean oil production in China, which is interested in purchasing or building refineries in Ukraine. 4. Sphere of telecommunications. The office of the Chinese company Xinwei Group, one of the leading telecommunication companies in the world, is opened in Kyiv in 2014. The Ukrainian telecommunications operator Prostate builds a 4-generation mobile broadband multimedia communications network based on the McWiLL technology of the Xinwei Group. 150 base stations have already been installed, which covered Kyiv and significant territory from Kharkiv to Mariupol, and the "strip" coverage runs through 4 eastern regions of Ukraine. Further informatization is an important tool for the development of all social spheres of Ukraine. It is in the common interest of Ukraine and China to continue to work on this issue [13]. 5 SHS Web of Conferences 39, 01014 (2017) IES2017 DOI: 10.1051/shsconf/20173901014 A number of investment projects which the Chinese investors in Ukraine intend to realize is perspective. From the last: waste recycling plant in the Lviv region, processing of waste in Ukraine, thermal power plant in the Zhytomyr region, the belt road near Kiev, the bridge in Kremenchuk, the airport in Zhytomyr and other infrastructure facilities. Ukraine and China actively develop cooperation in the cultural and humanitarian sphere. The development of cooperation between Ukrainian universities, educational and scientific organizations of the Republic of China has positive dynamics and significant prospects for further growth. One of the priority areas of cooperation is the training of Chinese citizens in Ukrainian higher education institutions. The practice of creating joint institutes will test new teaching methods. Preparation and conducting internships for students, graduate students and young scientists is one of the priority areas of bilateral cooperation. 3 Results It should also be noted the intensification of the Ukrainian-Chinese dialogue at the expert level. The Ukrainian and Chinese experts often meet, communicate and exchange opinions at different conferences, meetings, forums A significant amount of expert meetings shows the mutual aspiration to find the solution for the existing problems. Expert discussions help to find compromises, to overcome differences, to strengthen bilateral dialogue and to develop a further strategy for the development of relations. We hope that current expert work will be implemented at the political and economic level in concrete intergovernmental agreements. Therefore, in our opinion, directions of cooperation between Ukraine and China in the following areas are promising (Table 2). 6 SHS Web of Conferences 39, 01014 (2017) IES2017 DOI: 10.1051/shsconf/20173901014 Table 2. Directions of cooperation between Ukraine and China Sphere of cooperation Development prospects Risk forecast to investors Transport Infrastructure - development of a network of multimodal transport corridors using water (maritime) and rail links in the North-South and West- East directions. (One of the promising projects in the context of the development of trade routes is a deep sea port project that could actually double the capacity of the Ukrainian port infrastructure for the needs of the New Silk Road) - legislation of Ukraine is imperfect in the field of customs, tax, investment regulation, - the renewal of the fleet of rolling stock of Ukrainian carriers is slow, - there is a low activity in the implementation of projects in Ukraine for the construction of high-speed roads (railways, motor- car), including paid, introduction and development of multimodal transport and logistics. Agriculture - export of meat and dairy products, seeds of oilseeds and nuts, - cooperation in the development of infrastructure and logistics, more active and close cooperation in the field of veterinary and phytosanitary, - cooperation in the field of geodatabase and geospatial data generation, - development of smart farming, - the construction of plants on the production of means of plant and fertilizer protection on the territory of Ukraine, the use of new technologies for raising the productivity and productivity of cattle breeding and poultry farming. - the technical condition of agricultural enterprises does not meet the needs of production, - monopolization of the corporate agro-sector of the markets of resources, especially financial, channels of product sales, - restriction of the circulation of agricultural land. 3 Results Renewable energy - energy efficiency and rational use of nature: (the branch of alternative energy - solar, river, wind, etc.). - the risks of changing the legislative framework for a green tariff and the lack of an updated strategy for renewable energy in Ukraine by 2035, - the complexity of the tax system. Telecommu- nications, IT - new prospects opens for Ukraine a project for the creation of a new all-Ukrainian 4G network with the help of the Chinese side, - cooperation in the field of information technologies on the basis of Ukrainian IT Start-Up. - the existence of legal and organizational barriers, - the complexity of the tax system, bureaucracy insufficient reliability of information infrastructure. Educational sphere - signing an agreement on the mutual increase of student quotas for exchange, - concluding agreements on cooperation and implementation of the China-Ukrainian double diploma program, - preparation and internship of graduate students and young scientists on the basis of mutual exchange, - conducting international educational forums, including The Forum of Rectors of Ukrainian and Chinese Universities, in order to expand the direct cooperation of universities. - language barriers - deepening mutual learning of Chinese and Ukrainian languages, - the beginning of reforms in the educational sphere. S2017 stock of Ukrainian carriers is slow, - there is a low activity in the implementation of projects in Ukraine for the construction of high-speed roads (railways, motor- car), including paid, introduction and development of multimodal transport and logistics. - the technical condition of agricultural enterprises does not meet the needs of production, - monopolization of the corporate agro-sector of the markets of resources, especially financial, channels of product sales, - monopolization of the corporate agro-sector of the markets of resources, especially financial, channels of product sales, - restriction of the circulation of agricultural land. - restriction of the circulation of agricultural land. 7 SHS Web of Conferences 39, 01014 (2017) IES2017 DOI: 10.1051/shsconf/20173901014 4 Discussion and Conclusion Thirdly, China is interested to invest the investments in economy of Ukraine, considering its branched transport system, existence of a powerful scientific and industrial complex, access to the markets of the European Union. It is confirmed by a number of already realized investment projects (the creation of Beijing Skyrizon Aviation Industry Investment Co Ltd and Motor Sich joint venture, tart in 2016 in the Nikolaev sea trade port transshipment facility of grain and oil-bearing crops, etc). And today the complementary nature of economic systems, the presence of a significant potential for cooperation in the trade, economic, scientific and technical, and humanitarian sectors create the necessary prerequisites for the progressive development of mutually beneficial Ukrainian-Chinese relations. 4 Discussion and Conclusion Considering the conducted research it is possible to draw the following conclusions. Firstly, Ukraine has an important geopolitical and geo-economic situation, making it an important transport and logistics hub within the framework of the Silk Road Infrastructure Project, which aims to connect Europe and Asia more closely. The Silk Road initiative gives Ukraine the technical ability to bypass the restrictions imposed by Russia on the transit of its products to the countries of Central AsiaMoreover, our country is interesting to China as a production site for further export to the EU. This aspect has a positive impact on the development of Ukrainian-Chinese economic relations. Secondly, China is one However, the trade balance for Ukraine is negative. Imports of goods from China to Ukraine in 2016, according to state statistics, grew by 24.3% (to $ 4687.7 million), exports decreased by 24.1% compared to 2015 (before $ 1832.5 million). However there is a mutual aspiration of the parties before increase in volumes of bilateral trade, diversification of its structure, development of long-term forms of economic cooperation, deepening of investment interaction between Ukraine and China. The deepening of trade relations with the PRC positively affects Ukraine's economic growth, however, the current dynamics of the trade balance between Ukraine and the PRC, given the significant predominance of imports, requires careful review of trade policy measures. Therefore, our proposals are as follows: p p 1. To improve the structure of trade, introducing new forms of trade relations, in particular industrial cooperation, cooperation on a compensation basis and the organization of joint productions through the purchase of licenses. p p 1. To improve the structure of trade, introducing new forms of trade relations, in particular industrial cooperation, cooperation on a compensation basis and the organization of joint productions through the purchase of licenses. j p g p 2. To search for ways to reduce the energy intensity of export industries as it significantly affects the competitiveness of Ukrainian exports. 3. To develop the institutional framework for cooperation, which should include not only interstate macroeconomic, but also a number of sectoral bodies for establishing effective cooperation in specific areas. p p 4. To stimulate the means of tariff and non-tariff regulation of imports into Ukraine of mainly investment high-tech goods. y g g 5. Creation of a common investment fund on parity terms. 2. V. Holod, China Investment Expansion. Prospects for Developing Relations with Ukraine, Chinese research studies, 38-44 (2012) 3. M. Vysotskaya, Analysis of the main trends of development between Ukraine and China Strategy of development of Ukraine. Economics, sociology, law, 13-18 (2013) 1. S. Pron, China: Foreign Policy and Diplomacy in the Second Half of the 20th Century. Monograph Nikolaev, 147, (2012) g p , ( ) 2. V. Holod, China Investment Expansion. Prospects for Developing Relations with Ukraine, Chinese research studies, 38-44 (2012) 3. M. Vysotskaya, Analysis of the main trends of development between Ukraine and 1. S. Pron, China: Foreign Policy and Diplomacy in the Second Half of the 20th Century. Monograph Nikolaev, 147, (2012) 2. V. Holod, China Investment Expansion. Prospects for Developing Relations with Ukraine, Chinese research studies, 38-44 (2012) 3. M. Vysotskaya, Analysis of the main trends of development between Ukraine and China Strategy of development of Ukraine. Economics, sociology, law, 13-18 (2013) References 1. S. Pron, China: Foreign Policy and Diplomacy in the Second Half of the 20th Century. Monograph Nikolaev, 147, (2012) 2. V. Holod, China Investment Expansion. Prospects for Developing Relations with Ukraine, Chinese research studies, 38-44 (2012) 3. M. Vysotskaya, Analysis of the main trends of development between Ukraine and China Strategy of development of Ukraine. Economics, sociology, law, 13-18 (2013) 8 SHS Web of Conferences 39, 01014 (2017) IES2017 DOI: 10.1051/shsconf/20173901014 4. V. Rogovuy, Prospects and risks of Ukrainian-Chinese trade and economic relations development. Bulletin of Zhytomyr State Technological University, 68 (2016) 4. V. Rogovuy, Prospects and risks of Ukrainian-Chinese trade and economic relations development. Bulletin of Zhytomyr State Technological University, 68 (2016) 5. V. Levkivskyi. Ukrainian-Chinese economic cooperation in the context of foreign economic security. Scientific herald of the Chernihiv State Institute of Economics and Management, Series 1: Economics, 95-103 (2013) 6. S. Grodsky, Modern trade and investment cooperation between Ukraine and China Economic analysis, 202-206 (2013) 7. L. Vlasenko, Position of Ukraine in the PRC’s system of foreign economic relations. Scientific herald of Uzhgorod National University. Series: International Economic Relations and World Economy, 15-19 (2017). 8. H. Cheng, The choice and strategic opportunities of China in the context of Ukrainia crisis. Foreign Trade: Economics, Finance, Law, 117-128 (2015). 9. L. Halperina, System adaptation of social and economic model of China to conditions of global instability. [online], Available at: http://ir.kneu.edu.ua/ (2016) 9. L. Halperina, System adaptation of social and economic model of China to conditions of global instability. [online], Available at: http://ir.kneu.edu.ua/ (2016) 10. Euromaidan Press [online], Available at: http://euromaidanpress.com/ (2017) 11. Embassy of Ukraine in the People's Republic of China and Mongolia (in combination) [online], Available at: http://china.mfa.gov.ua/ua/ukraine-cn/trade (2017) 11. Embassy of Ukraine in the People's Republic of China and Mongolia (in combination) [online], Available at: http://china.mfa.gov.ua/ua/ukraine-cn/trade (2017) 12. Official site of the State Statistics Service of Ukraine [online], Available at: http://www.ukrstat.gov.ua (2017) 12. Official site of the State Statistics Service of Ukraine [online], Available at: http://www.ukrstat.gov.ua (2017) 13. Silk Road of Ukraine [online], Available at: http://silkroad.in.ua/ (2017) 13. Silk Road of Ukraine [online], Available at: http://silkroad.in.ua/ (2017) 9 9
https://openalex.org/W2324300685
https://zenodo.org/records/1516322/files/article.pdf
English
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Body Temperature of Newly Hatched Chicks
Poultry science
1,921
public-domain
2,486
BODY T E M P E R A T U R E ; O F N E W L Y H A T C H E D C H I C K S IvESiviE E. CARD Cornell University That the normal body temperature of the adult domestic fowl is fairly high (io6° to I07°F.) is generally known, but so far as the writer has been able to determine, there are no statements in the literature as to the normal temperature of chicks when first hatched. A beginning was made on a study of this problem in 1918 while the writer was connected with the Poultry Depart- ment of the Storrs Agricultural Experiment Station. This paper reports certain phases of that .study. at University of Arizona on June 11, 2016 http://ps.oxfordjournals.org/ Downloaded from at University of Arizona on June 11, 2 http://ps.oxfordjournals.org/ Downloaded from Data were secured on a total of 119 S. C. White Leghorn chicks. These were pedigree-hatched, June 21, 1918, in Prairie State Incubators. Upon being taken from the hatching bags each chick was marked with a numbered leg-band in order that all data might be studied with reference to the parentage of the chicks. The chicks were grouped into four lots, on a basis of parent- age, with as nearly equal numbers as possible in each lot. Lot one contained all chicks which hatched from hens 115, 118, 132 and 135 ; lot two all chicks from hens 117 119, 133 and 136; and so on. The distribution of chicks according to sex and female parentage is shown in the following table. All chicks were sired by the same male. TABLE I Lot No. I I I I I 2 2 2 2 2 3 3 3 3 3 4 4 4 4 4 All i/i?« No. 115 i i 8 132 13s — 117 119 133 136 — 122 125 127 128 — 138 1 4 0 1 4 1 144 — — TABLE I Male Chicks Female Chicks 4 I 5 6 16 3 3 6 6 18 6 3 5 4 18 5 5 6 2 18 70 2 4 4 2 12 4 4 4 0 12 2 6 4 I 13 3 3 4 2 12 49 Total 6 5 9 8 28 7 7 10 6 30 8 9 9 5 31 8 8 10 4 3 0 119 Lots one and three received fine chick grain three times a day on the 3rd, 4th, and 5th days. Lots two and four received no feed at any time.' None of the chicks received water or milk. It is the opinion of the writer, altho he has no specific data on the point in question, that the physical condition of lots two and four, and consequently their temperature curves, would have been considerably different if they had been supplied with fresh water, even tho they received no feed. At the end of the five day period all chicks were killed and their sex determined by post mortem examination. DATA SECURED Chicks were held in incubators thruout the test. All chicks were weighed to the nearest tenth of a gram at 2-3 p. M. on the day they were hatched. All still living were similarly weighed at 2-3 p. M. on the fifth day after hatching. All chicks in lots three and four were weighed at 2-3 p. M. on each of the four intervening days. Body temperature of all chicks in lots one and two was re- corded night and morning, at 7-8 o'clock, on each of the first five days after hatching, except that in the case of lot two the chicks were so weak and so low in vitality at the end of the test that in many cases their temperature could not be recorded on the thermometer used. The lower limit of the scale was at 95°F. The records for lot two cease with the morning of the fifth day. Temperature observations were made on the chicks in lots three and four once daily at 1-2 p. M. throughout the five day period. POUL TRY SCIENCE 10 APPARATUS AND TECHNIQUB In weighing the chicks use was made of a triple beam balance made by the Central Scientific Company. Little difficulty was experienced in keeping the chicks quieten the pan fora suffi- cient time to read their weights. By having an assistant record the weights this operation was performed very rapidly. TEMPERA rURE OF NE WL Y HA TCHED CHICKS \\ In determining temperatures a small size, sixty second, clinical thermometer was used. Temperatures were taken in the rectum and the operation was greatly facilitated by touching the tip of the thermometer bulb in castor oil before attempting to insert it. Chicks were taken from the incubator one at a time in order to eliminate the possibility of the room temperature lowering the natural body temperature thru chilling. This was necessary be- cause of the length of time necessary to read and record tem- peratures on all the chicks. In other words, the body tempera- tures as recorded were those maintained by the chicks when kept in an environmental temperature of 98-ioo°F. It was not possible to read and record temperatures at a rate of more than forty-five chicks an hour, and the average rate was about forty. This is readily appreciated when one considers that it was necessary to take a chick from the machine, close the door, dip the bulb of the thermometer in oil, insert the bulb in the rectum, read the band number, wait sixty seconds for the mer- cury to reach a constant level, read and record the temperature, place the chick in a cloth lined box or another incubator, shake down the mercury, and then open the door of the machine and catch another chick. Without the use of castor oil in which to dip the point of the mercury bulb it was impossible to proceed at a rate of more than twenty-five chicks an hour. OBJECTS The purpose of the test was to determine the range limits of and variation in body temperature of chicks on each of the first five days after hatching, and to determine whether temperature tended to be constant for an individual, or to fluctuate. If the latter were true, how did it fluctuate ? What effect, if any, on temperature would result from withholding food beyond the usual limit of seventy-two hours after hatching ? This was ex- pected to throw some additional light on the old question of when chicks should have their first feed and on the justification for the postal ruling requiring the delivery, of chicks shipped by parcel post, within seventy-two hours. Another point for study was the difference between the sexes with respect to body tem- perature, and the possibility of determining sex by observing this character. PO UL TR Y SCIENCE 12 TALLE II. TEMPERATURE IN DEGREES FAHRENHEIT jst day 106.8 104.7 106.0 103.0 lOI.O roi.9 107,0 104.4 105.6 102.8 101.2 102.1 106 8 104.8 106.0 102.7 101.4 102.1 107.0 104.6 105.9 103.6 100.8 102.3 106.4 104.6 105.6 106.4 104.6 105-5 106.8 104.0 105.9 106.5 104.1 105.4 2d day 105.3 103-4 104.3 104.8 102.4 103-5 104.8 103.4 104.1 104.7 102.6 103-5 105.4 103.5 104.3 104.4 ΙΟΙ.6 103-6 105-ο I03.2 104.1 104.2 I02.4 I03.7 io6.8 103-4 104.7 107.3 102.6 104.0 107.2 103.2 105.4 105.6 103.7 104.4 Sd day 105.0 103.4 104.1 105.8 102.6 103.8 104.8 102.7 104.0 105.7 102.6 104.7 104.8 103.3 103.9 103.6 100.6 102.5 104.6 ΙΟΙ.7 I03.6 103,4 ΙΟΙ.ο 102.2 Ίο5.ο Ι03-4 104.2 Ι05.0 103.6 104.2 104.2 102.1 103.4 104-3 102.0 Ι03.6 Males—lot one Highest morning Lowest morning Mean (16 males) Highest evening . TEMPERA rURE OF NE WL Y HA TCHED CHICKS I 3 OBJECTS Lowest evening Mean (16 males) Females—lot one Highest morning Lowest morning Mean (12 females) Highest evening Lowest evening Mean (12 females) Males—lot two Highest morning Lowest morning Mean (18 males) Highest evening Lowest evening Mean (18 males) Females—lot two Highest morning Lowest morning Mean (12 females) Highest evening Lowest evening Mean (12 females) Males—lot three Highest mid-day Lowst mid-day Mean (18 males) Females—lot three Highest mid-day Lowest mid-day Mean (13 females) Males—lot four Highest mid-day Lowest mid-day Mean (18 males) Females—lot four Highest mid-day Lowest mid-day Mean (12 females") 106.0 103.8 105.0 105.0 102.2 104.0 106.3 104.6 105.5 105.2 102.6 104.0 104.0 102.2 103.0 103.4 100.3 101.9 103.8 102.4 103.0 103.4 101.1 102.4 106.0 104.0 105.0 105.6 104.0 104.8 104.7 101.8 103.4 104.8 102.4 i o ^ 8 105.8 100.2 104-7 105-8 103.2 104.9* 105-9 103.8 105.1 106.0 102.8 104.5 101.6 96.2 99-7* 101.6 97-8 99-9 105.4 100.8 103-5 106.0 99-8 103.9* 102.0 98.5 100.4 102.9 100.0 101.4 *One chick died before the last records were taken. Fifth day mean based on one less than the indicated number of chicks. TABLE I I I . BODY W E I G H T IN GRAMS When TABLE L o t t h r e e Av. of 18 males Av. of 13 females __ L o t four Av. of 18 males Av. of 12 females __ I I I . BODY W E I G H T IN GRAMS When Hatched 35-2 35-5 37-0 37-0 ist day 3 2 4 32.6 34-4 .\3-8 2d day 30,0 29.9 32.0 31.4 3d day 27.7 27.4 29.4 28.9 4th day 25.5 25-6 26.6 26,2 ^th day 23.8 24.0 24.6 24.4 TABLE I I I . BODY W E I G H T IN GRAMS TEMPERA rURE OF NE WL Y HA TCHED CHICKS SUMMARY OF THË DATA lu the tables are presented the highest and lowest observed temperatures, together with the mean temperatures morning and evening, for males and females in lots one and two. Similarly, there are presented for the males and females in lots three and four, the highest and lowest observed temperatures taken near the middle of the day, and the mean for males and females sepa- rately. It should be remembered that lots one and three were fed on the 3rd, 4th, and 5th days while lots two and four re- ceived no food. at University of Arizona on June 11, 2016 http://ps.oxfordjournals.org/ Downloaded from at University of Arizona on June 11, 2 http://ps.oxfordjournals.org/ Downloaded from at University of Arizona on June 1 http://ps.oxfordjournals.org/ Downloaded from The mean daily weight for males and females in lots three and four is also presented. It will be noted that there was in each lot a fairly uniform decrease in weight during the five day period, the amount of this decrease being approximately one-third of the original weight. Feeding did not prevent this loss of weight. In the author's opinion much of this loss can be traced to the fact that the chicks received no water or milk. Further obser- vations are necessary to determine whether chicks actually lose weight during the first few days when under normal brooding conditions. Graphs are presented to show the changes in successive morning, evening, and early afternoon temperatures for males and females in each of the lots. To each of the curves of ob- served mean temperatures a straight line has been fitted to show the general trend. It is of interest to note that, in general, morning and mid-day temperatures tend to fall thru the five day period while evening temperatures tend to rise. The three upper graphs refer to male chicks, the three lower to females. Solid lines refer to chicks in lots one and three that were fed broken lines to chicks in lots two and four that were not fed. The equations of the fitted straight lines are as follows : Lot one—morning temperatures of male chicks : J'=io5-39—•19·*' Lot two—morning temperatures of male chicks : j/==i07.50—1.38ΛΓ Lot one—morning temperatures of female chicks : jc= 104.74-|-. SUMMARY OF THË DATA o/^x Lot two—morning temperatures of female chicks : J/^107.23—Ι.,^ΙΛΤ Lot one—evening temperatures of male chicks : j/=ioj.67+.65J: Lot two—evening temperatures of male chicks : j)/^io2.95 —.17ΛΓ J/^107.23—Ι.,^ΙΛΤ Lot one—evening temperatures of male chicks : j/=ioj 67+ 65J: j/=ioj.67+.65J: Lot two—evening temperatures of male chicks : 14 POULTRY SCIENCE Lot one—evening temperatures of female chicks : j)'^io2.i6-f-.54Jtr Lot two—evening temperatures of ^female chicks : j)'= 102.95—Λ2Χ ' Lot three—mid-day temperatures of male chicks ; >=ιο5-77-·39·=^ Lot four—mid-day temperatures of male chicks : j)/=:I07.60 Ι.Τ,ΟΧ Lot three—mid-day temperatures of female chicks : y-\-\0$.20—.2ilfX Lot four—mid-day temperatures of female chicks : jf=io6.30--.86^r j)'= 102.95—Λ2Χ ' Lot three—mid-day temperatures of male chicks ; > ιο5 77 39 ^ >=ιο5-77-·39·= Lot four—mid-day temperatures of male chicks : j)/=:I07 60 Ι Τ ΟΧ j)/ :I07.60 Ι.Τ,ΟΧ Lot three—mid-day temperatures of female chicks : y-\-\0$.20—.2ilfX Lot four—mid-day temperatures of female chicks : jf=io6.30--.86^r r—mid-day temperatures of female chicks : y p jf=io6.30--.86^r CONCLUSIONS CONCLUSIONS at University of Arizona on June 11, 2 http://ps.oxfordjournals.org/ Downloaded from i) The normal temperature of Single Comb White Leghorn chicks the first day after hatching is io6°F. in the morning and I02°F. in the evening. In early afternoon it is nearly half a degree below the morning temperature. 2) Morning temperature decreases, on the average, during the first five days after hatching. y g 3) Evening temperature tends to rise during these five days, 4) Mid-day temperature tends to fall more rapidly than morn­ ing temperature from the first to the fifth day. 5) There is no evidence to indicate that sex could be deter­ mined with anything like practical accuracy by observing body temperature of chicks when hatched. 6) On the evening of the third day after hatching there is a difference of about eight-tenths of a degree Fahrenheit between the mean temperature of males and females. On the morning of the fourth day the corresponding difference is half of one degree Fahrenheit. 7) The morning temperature of chicks not given food or water for five days after hatching falls very rapidly thruout the period. p 8) The evening temperature of chicks not given food or water begins to fall after the second day. 9) This would indicate that chicks should be given food (or water, or both food and water) before they are seventy-two hours old. 10) It would appear that a limit of seventy-two hours for the time of chicks in transit is entirely reasonable. A summary of the foregoing material was presented at the annual meeting of the American Association of Instructors and Investigators in Poultry Husbandry held at Ithaca, New York, July, 1918. O s Ο κ, ^ ^ ^ 1 ε 3 * J " 1 t 3 4 δ " i ζ ttorNÎNQ Tètipçrafures-Males-Lol5i&a hewNg Tèiipcrotures-flots-lots ι &z flid-da^ ΈnpeΓ ^ ^ ^ ^ ^ t i S * S i1orNiN^¥iipa-ûfurcs-f?/fofes - lofs i&z tvmmgTf'/^perafures-JcMoÎe -io/s /&a flidcfOY Έ/ BOPV TEMPERATURE ΟΓ SC WEflTE LEGHORN CMIX Obscrvcd HeoN TenperatuKS & Tilted s/rai^ht /iNes. Solid llNes-Chix fed (^raiN i'.-p&s'doys. at University of Arizona on June 11, 2016 djournals.org/
https://openalex.org/W2067730578
https://www.jstage.jst.go.jp/article/jea1991/12/2/12_2_112/_pdf
English
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Meta-analysis on the Therapeutic State of Hypertensive Population in Japan: Focusing on the Impact of New Diagnostic Criteria of Japanese Guideline for the Management of Hypertension 2000.
Journal of epidemiology
2,002
cc-by
4,589
Received December 21, 2001; accepted January 31, 2002. 1 Department of Health Care Policy , National Institute of Health Services Management. 2 Research Department , Institute for Health Economics and Policy. 1 Department of Epidemiology , Research Institute for Brain and Blood Vessels Akita. Address for correspondence : Toshihiko Hasegawa, MD, MPH, Department of Health Care Policy, National Institute of Health Services Management, 2-3-6 Minami, Wako, Saitama 351-0104, Japan. Toshihiko Hasegawa 1 Yoko Hori 1, Hiroyuki Sakamaki 2 and Kazuo Suzuki 9 A Meta-analysis on the therapeutic state of hypertensive population in Japan is performed by the three nation-wide governmental surveys focusing on the impact of new diagnostic criteria described in the Guidelines for the Management of Hypertension in Japan 2000. These surveys are the National Survey of Circulatory Disorders, National Nutrition Survey and Patient Survey in 1990. The meta-analysis approach is used to evaluate the validity and reliability of these three national data sets, particularly the National Nutrition Survey. The population with history of hypertensive treatment and without previous diagnosis was calculated using the old and new diagnostic criteria. The results of three national surveys are fairly consistent. National Nutrition survey can be used to monitor the overall therapeutic status of Japanese population if the definition is considered judiciously. The impact of new diagnostic criteria is extensive as demonstrated by the results of the analysis on the National Nutrition Survey of 1999. The hypertensive population doubled and one half of the Japanese population over the age of 30 is now defined as hypertensive. A policy to manage this newly diagnosed hypertensive population is urgently needed to lessen the burden on Japanese health care system. JEpidemioi, 2002 ; 12 : 112-119 hypertension, meta-analysis, national nutrition survey, national survey of circulatory disorders, guidelines for the management of hypertension in Japan (JSH2000) Vol. 12, No. 2 March Vol. 12, No. 2 March Journal of Epidemiology Meta-analysis on the Therapeutic State of Hypertensive Population in Japan: Focusing on the Impact of New Diagnostic Criteria of Japanese Guideline for the Management of Hypertension 2000 hypertension, meta-analysis, national nutrition survey, national survey of circulatory disorders, guidelines for the management of hypertension in Japan (JSH2000) Meta-analysis on the Therapeutic State of Hypertensive Population in Japan: Focusing on the Impact of New Diagnostic Criteria of Japanese Guideline for the Management of Hypertension 2000 Toshihiko Hasegawa 1 Yoko Hori 1, Hiroyuki Sakamaki 2 and Kazuo Suzuki 9 oshihiko Hasegawa 1 Yoko Hori 1, Hiroyuki Sakamaki 2 and Kazuo Suzuki RESULTS For National Nutrition Survey, data is classified according to drug treatment status. No drug treatment groups were also classified by blood pressure, systolic pressure of more than one 160 mm Hg, the diastolic pressure more than 95 (Note 4, Table 1). For the population with history of medication, three groups, namely those that stopped taking medicine, those taking it occasionally, those taking it daily, were identified. Nation-wide estimate is calculated by similar method as the case of National Survey of Circulatory Disorders. 1) The Estimate of Hypertensive Population by National Survey of Circulatory Disorders 1990 and National Nutrition Survey 1990 According to the 1990National Survey of Circulatory Disorders, population ever treated for hypertension is 18,792,000 including those under admission numbering 16,000 (Figure 1). Outpatient visiting population is 11,151,000. Population with no visit is 6,000,000. Others is 1,627,000. Among the population without previous visit to a medical facility 5,218,000 are hypertensive when using the old diag- nostic criteria. Another 11,029,000 are classified as hyperten- sive when using the new diagnostic criteria. 13,702,000 are normal high when the new diagnostic criteria are applied. Adding these numbers up, there are 22,953,000 hypertensives in the population using the old criteria and 34,982,000 accord- ing to the new criteria. According to the National Nutrition Survey 1990, population ever treated by medication is 14,187,000, including those taking daily medication at 9,843,000 and those taking medication occasionally at 932,000 and those that have stopped medication at 3,413,000. Among the population with no previous history of medication 7,149,000 are hypertensive based on old diagnostic criteria, and 12,631,000 are hypertensive based to the new diagnostic criteria with another 14,412,000 for normal high. 2. Meta-analvsis between National Survey of Circulatory Disorders and Patient Survey INTRODUCTION National Nutrition Survey (Note 1), National Survey of Circulatory Disorder (Note 2) and medical facility based sur- veys, like Patient Survey (Note 3). Application of the results of this study is to authenticate the validity and reliability of National Nutrition Survey and Patient Survey, since National Survey of Circulatory Disorders is done only once every ten years. National Nutrition Survey and Patients Survey on the other hand, is available for continuous monitoring because they are done every year and every three years. Then the impact of new diagnostic criteria of Guidelines for the Management of Hypertension in Japan 2000 (Note 4, Table 2) is evaluated using National Nutrition Survey of 1999 "). Hypertension is an important disease since it is a major con- tributing risk factor to other circulatory disease and health care cost 1.2). In Japan, in particular stroke was the No. l killer in the past and now has become the largest cause of elderly disability. Health care costs of hypertension treatment can be considered as a good investment if hypertension is controlled well. It is important to evaluate the therapeutic status of the hypertensive population. But the estimation of therapeutics status by differ- ent nation-wide government surveys is conflicting '-5. The objective of this paper is to estimate the therapeutic status of hypertensive population in Japan by Meta-analysis comparing two different kind of survey, a population based survey such as 112 Meta-Analysis on Hypertension in Japan 113 3. The Estimate of the Impact by New Diagnostic Criteria 3. The Estimate of the Impact by New Diagnostic Criteria By using the National Nutrition Survey 1999 the same cal- culation was done to estimate the total number of patients by age and sex group. For population without any previous med- ication, the three categories of population are estimated by age and sex group using new diagnostic criteria. One group com- posed of those with blood pressure values of more than systolic 160 or diastolic 95 (Note 4, Table 1). Next was the group of the newly diagnositic hypertensives. Lastly there was a group for the "high normal". Nation wide population was calculated for high normal, new mild hypertensive and traditional undiag- nosed hypertensive population. Among treated groups there were those that were taking medication occasionally or daily. Control fraction of treated population were calculated by using 5 year age and sex group fraction multiplied by each segment population. The therapeutic status measured by National Survey of Circulatory Disorders and National Nutrition Survey is orga- nized around two different principles. The National Survey of Circulatory Disorders is based upon a visit to health care facili- ty, and the National Nutrition Survey is based on drug treat- ment. The estimation of hypertensive population in both sur- veys is calculated through the prevalence rate in 5 year-age and sex groups and multiplied by each group population. From the National Survey of Circulatory Disorders, data were divided by the presence of past history of hypertension. For the no past history group, hypertensive population was identified by using the old criteria i.e. the systolic pressure of more than 160 mm Hg or the diastolic pressure more than 95 nun Hg (Note 4, Table 1). For the population with a previous history, three groups, namely, no visit, one visit over more than a one month period, and one visit in less than one month period, and others probably indicating irregular visit were identified. 2) Comparative Estimate of Hypertensive Population under Medical Care by National Survey of Circulatory Disorders 1990 and the Patients Survey 1990 2) Comparative Estimate of Hypertensive Population under Medical Care by National Survey of Circulatory Disorders 1990 and the Patients Survey 1990 Estimate using 1990 Patients Survey is 9,422,000 (95%C.I. 9,177,000 - 9,667,000) which is 1,729,000 (15.5%) smaller than the estimate by the 1990 National Survey of Circulatory Disorders. But most of the difference comes from the patients MATERIALS AND METHODS Estimates of those two databases are compared by five-year age and sex groups. 1. Meta-analysis between National Survey of Circulatory Disorders and National Nutrition Survey 1. Meta-analysis between National Survey of Circulatory Disorders and National Nutrition Survey 3. The Estimate of the Impact by New Diagnostic Criteria 2. Meta-analvsis between National Survey of Circulatory Disorders and Patient Survey For National Survey of Circulatory Disorders 1990, patient with previous treatment is selected and classified into three groups, namely no current visit to medical facility, visit once more than one month, and visit once less more than one month. The data were classified into five-year age group and sex. The ratio was calculated for each group and multiplied by the general population for each segment to estimate the total number of patients. For Patient Survey, the 1990 database was used. For each five-year age group and sex group, total patient were calculated using the following formula used by Japanese Government Statistics 9). Total patient = In-patient number + Out-patient number of one day visit X 6/7 Total patient = In-patient number + Out-patient number of 2) Comparative Estimate of Hypertensive Population under Medical Care by National Survey of Circulatory Disorders 1990 and the Patients Survey 1990 2) Comparative Estimate of Hypertensive Population under Medical Care by National Survey of Circulatory Disorders 1990 and the Patients Survey 1990 Visit interval (day) Visit interval (day) Estimate using 1990 Patients Survey is 9,422,000 (95%C.I. 9,177,000 - 9,667,000) which is 1,729,000 (15.5%) smaller than the estimate by the 1990 National Survey of Circulatory Disorders. But most of the difference comes from the patients Estimate using 1990 Patients Survey is 9,422,000 (95%C.I. 9,177,000 - 9,667,000) which is 1,729,000 (15.5%) smaller than the estimate by the 1990 National Survey of Circulatory Disorders. But most of the difference comes from the patients Main and sub diagnosis of hypertension, visit interval more than one month are included for patients. 95% confidence interval (CI) was calculated according to sample size. 114 3) Estimation of the Impact of New Diagnostic Criteria Using during the National Nutrition Survey of 1999 3) Estimation of the Impact of New Diagnostic Criteria Using during the National Nutrition Survey of 1999 visiting less than once a month , that is, 1,115,000 (95%C.I. 7,853,448 - 13,240,288) by age and sex group (Figure 2) . The largest discrepancy is among males aged between 42-60 years visiting medical care facility less than once a month . This dis- crepancy is generally smaller for females . Same methods as described above were used to estimate the patients ever treated previously with medication in the hyper- tensive population without previous drug treatment, using the old and new criteria. Sex and five year age groups were used to Figure 1. T. Hasegawa, et al, 2) Impact of New Diagnostic Criteria Hypertensive population using the old criteria in the 1999 survey was estimated at 25.3 million cases. This is an incre- ment of 2.8 million since 1990 (Figure 1). Undiagnosed popu- lation increased to 8.1 million with a 1 million increment since the 1990 survey. The fraction of undiagnosed patients is about 30% using the old criteria and this was not different from the 1990 survey. But caution has to be exercised because this frac- tion could be smaller depending on the definition. Applying the new criteria, another 21.7 million were included in the hypertensive population. This totals to 47.0 million, which is 47.7% of population over 30 years of age. The application of the new criteria almost doubles the hypertensive population and now one out of every two Japanese is considered to be hypertensive. If the "high normal" population is also included, then 65% of population is at some risk according to the new diagnostic criteria. The fraction of hypertensive population increases as population ages. The males are always higher than females and at the age over 75, the hypertensive population is about 80% when using the new criteria and about 60 % when using even the old criteria. For estimating the therapeutic status using the old criteria, 8.1 million people are undiagnosed and another 6.3 million people diagnosed previously as hyperten- sive are not under control (Figure 3). The therapeutic strategy of expanding diagnostics criteria by JSH 2000 leads to the inclusion of 21 million new patients. This may not be practical because the large hypertensive population by old criteria still has not been controlled well. Lastly, white-coat hypertension i.e. transient hypertension upon meeting the medial profession is well known" 15) 10 to 40% of hypertension could be in this category. At least 10% of the 8 million undiagnosed hyperten- sive population using old criteria and another 21 million using new criteria could be actually an overestimation, because the measurement of blood pressure for National nutrition survey is only at one occasion not particularly time. At least 10% of the 15 million people who are treated with medication by National nutrition survey 1990 population might have been treated unnecessarily. On other hand, false negative blood pressure values have been reported as well 16). To examine those false (positive and negative) in value requires continuous blood pressure monitoring or home based blood pressure assessments using better measurement methods. DISCUSSION pliance, as middle aged workers tend to attend medical care facility very infrequently 5). The patients treated with medica- tion have an overall uncontrolled rate of 46.5%, but can be as high as 68% in males aged 55 to 49 years old (Figure 4). In the 1999 survey, the controlled rate was brought down in numbers, particularly in the old age group. However when the new diag- nostic criteria were applied, the uncontrolled rate became 79.4%. Since the new guidelines had not been developed until year 2000, target for treatment then was not the new criteria. 1) Meta-analysis for Validity and Reliability of Different Data Sets Discrepancy of estimated number of patients under current treatment between national cardiovascular diseases survey and National Nutrition Survey is very small 0.38 million in 1990 (3.4% of treated population) (Figure 1). This can be due to a random error or a non-pharmacologically treated population. But discrepancy between population never treated and the pop- ulation ever treated is 46 million that is significant. It is possi- ble that National Survey of Circulatory Disorders included cur- rently no visiting population. 30% of population with previous diagnostic history of hypertension but currently not visiting a medical facility stopped medication or at least has never taken medication. So about 4 million patients could have been white- coat hypertension or had been controlled by no-pharmacologi- cal treatment since about half of this population is normoten- sive. On the other hand, there is the real hypertensive popula- tion because hypertension is a life long disease. Undiagnosed population according to the National Survey of Circulatory Disorders is 5.2 million. This is 7.1 million according to the National Nutrition Survey. The discrepancy is 1.7million but it is probably due to hypertensive fraction of the population who is not visiting to medical care facility. Total was 24.0 million vs. 22.5 million hypertensive population when using the old criteria. This discrepancy is only 1.5 million (6%). However, when the new criteria is used, the differences is small 0.6 mil- lion. The undiagnosed fraction of hypertensive population is 21.8% by National Survey of Circulatory Disorders and 31.8% by National Nutrition Survey. This is a 10% difference. Therefore, the total number of the hypertensive population is not different according to the 2 surveys, but the undiagnosed population fraction is different due to the application of differ- ent definitions. 2. Meta-analvsis between National Survey of Circulatory Disorders and Patient Survey Hypertensive population estimate by different classifications and surveys . Figure 1. Hypertensive population estimate by different classifications and surveys . Male Female Figure 2. Comparative estimate of hypertensive patients by 10 year age, sex group and visit per month . Figure 2. Comparative estimate of hypertensive patients by 10 year age, sex group and visit per month Meta-Analysis on Hypertension in Japan 115 calculate. Population ever treated was 17,195,000 including those already stopped medication at 2,498,000 (Figure 1). Among the population never treated with drugs, 8,111,000 were hypertensive based on the old diagnostic criteria. Another 21,724,000 were hypertensive based on the new criteria with an additional 14,675,000 as "high normal". When all of these numbers are added together, the hypertensive population totals 25,306,000 applying the old criteria (31.0% of total population calculate. Population ever treated was 17,195,000 including those already stopped medication at 2,498,000 (Figure 1). Among the population never treated with drugs, 8,111,000 were hypertensive based on the old diagnostic criteria. Another 21,724,000 were hypertensive based on the new criteria with an additional 14,675,000 as "high normal". When all of these numbers are added together, the hypertensive population totals 25,306,000 applying the old criteria (31.0% of total population age over 30) and 47,030,000 (47.7% of total population age over 30) applying the new criteria. If the "high normal" are also added, hypertensive and high normals amount to 53,593,577 (65.7% of total population age over 30). The population frac- tion of hypertensive population by sex and age group is shown in the Figure 3. The uncontrolled blood pressure fraction among treated hypertensive population is calculated and is shown (Figure 4). Figure 3. Hypertensive population rate by 5 years and sex group, by old and new diagnostic criteria with 95% confidence interval. Figure 3. Hypertensive population rate by 5 years and sex group, by old and new diagnostic criteria with 95% confidence interval. Figure 3. Hypertensive population rate by 5 years and sex group, by old and new diagnostic criteria with 95% confidence interval. Figure 3. Hypertensive population rate by 5 years and sex group, by old and new diagnostic criteria with 95% confidence interval. Figure 4. Undiagnosed and uncontrolled rate by 5 years and sex group, by old diagnostic criteria. Figure 4. Undiagnosed and uncontrolled rate by 5 years and sex group, by old diagnostic criteria. T. Hasegawa, et al. 116 DISCUSSION Caution is therefore required to interpret the undiagnosed fraction when National Nutrition Survey is used. About one third of such a population was once diagnosed as hypertensive but has not been followed up. 2) Impact of New Diagnostic Criteria It may be difficult to use those methods for nation-wide study but the result of a pilot For the patients who have been under treatment, the discrep- ancy between National Survey of Circulatory Disorders and Patient Survey is 1.6million (16.4%). Most of this discrepancy can be explained by the discrepancy in middle age group of the male hypertensive population (Figure 2). The discrepancy could be due to a random error but also the bias due to the sampling methods of survey. Those middle age males are usu- ally the working population. If the clinic at working place is excluded for Patient Survey, the number could be underesti- mated. But on the other hand, the Patients Survey is supposed to be randomized at the clinic level 10). The other possibility is the recall-reporting bias of National Survey of Circulatory Disorders because it is a self-reported survey done by the med- ical professions as patients usually responds favorably. Nevertheless, the most likely scenario is the problem of com- Meta-Analysis on Hypertension in Japan 117 study in certain areas could be useful to adjust a major survey results'. metrical measurement and bloody chemistry test were per- formed on each person. Blood pressure was measured by aus- cultation using a mercury sphygmomanometer. As a rule, mea- surements were taken from the right arm with the subjects. If the first measurement values were outside the normal range, the measurements were repeated. Sample number is 17,986 in 1990 and 12,763 in 1999. Survey stated in 1951 and to include hypertension treatment status after 1986. CONCLUSION By meta-analysis of three national data set for hypertension, the estimate is consistent provided definition of classification has been taken into account. Discrepancy between National Survey of Circulatory Disorders and National Nutrition Survey seems to be due to the populations who are diagnosed hyper- tensive but not treated currently. However, the overall hyper- tensive population is quite similar. National Nutrition Survey can be used for annual follow up and evaluation. Discrepancies between National Survey of Circulatory Disorders and Patients Survey are mainly due to middle-aged male hypertensive pop- ulation. The reason could be random errors and recall bias. A detailed study is required to reveal the main reason. Nevertheless the overall trend is consistent and the Patient Survey can be very good basis to analyze treatment behavior of hypertensive patients. The impact of the new diagnostic cri- teria is tremendous. The number of patients becomes double and more than half of the population of aged over 30 in Japan is labeled as hypertensive. The undiagnosed population increases from 22.3% up to 50%. Blood pressure controlled fraction of treated patients decreased from about 60% to 20%. This change will add a large new treatment burden to medical facility and financial burden to social insurance. 2. The National Survey of Circulatory Disorders The National Survey of Circulatory Disorders was done about every ten years 1971, 1980,1990, 2000; the most recent available data was 1990. A Nation wide random sample aged over 30 was taken from the chosen 300 living areas based upon the areas defined by the Comprehensive Survey of Living Condition of the People on Health and Welfare. New interview questions was added to ask the past history and treatment of circulatory disease in addition to the nutrition survey. Sample size was 10,956 in 1990. ACKNOWLEDGMENT The Authors would like to thank the following persons for kind discussion and suggestion. 3. National Patient Survey Patient Survey has been done since 1953. After 1984, survey was done every 3 years with larger sample size. Particular, after 1993, sample was increased up to 70%. Survey consists of two parts. First part is to measure the prevalence of in- patient and outpatient by one-day survey. Second part is as the discharge survey during the month of September. 20% of hos- pitals and 5% of clinics were selected using a random sample of prefecture in 1990. The survey consisted of age and sex, visit interval, age and sex, main diagnosis and sub diagnosis. 4. Diagnostic Criteria The old diagnostic criteria of WHO (Table 1) has been used for a long time until recently 16.17, 18). Japanese Ministry of Health & Welfare and Japanese Medical Association devel- oped the first guideline based on the WHO criteria in 1990 under the recommendation of Ministry of Health & Welfare. The Japanese Society of Hypertension developed new guide- line based on new WHO criteria in 2000 19.20) and the sixth report of the joint National committee on Prevention, Deletion, Evaluation, and Treatment of high blood pressure (JNC-IV) (Table 2) 22). Syunroku Baba, National Cardiovascular Center. Hirotsugu Ueshima, Department of Health Science, Shiga University of Medical Science. lkao Saito, Health Center, Keio University. Yutaka Imai, Department of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Medicine and Pharmaceutical Science. And the technical support Kunichka Matsumoto and Yoshihiro Kitamura, National Institute of Health Services Management. g This paper is funded by Grants-in-Aid for Scientific Research from the Ministry of Health, Labor and Welfare, Research on the technology assessment of hypertension arrangement (No. H12-hyo-002) and Research on the promo- tion and evaluation of Healthy Japan 21 (No. H12-Kenko- 003). Table 1. Old criteria by old WHO guideline. 1999 Report. Tokyo, 1999. (in Japanese) 1999 Report. Tokyo, 1999. (in Japanese) 10. Kyoto City Institute of Health and Environmental Sciences. Status of access to medical services for hypertensives in Kyoto City based on patient's survey in 1990. Annu Rep Kyoto City Inst Health Environ Sci, 1995; 61: 96-101. (in Japanese) 1. Okayama A, Kita Y, Ueshima H. Hypertension as a risk factor for cardiovascular disease, finding from epidemio- logical studies. Sogo Rinsho, 1997; 46: 1695-1701. (in Japanese) 2. Famett L, Mulrow CD, Linn WD, Lucey CR, Tuley MR. The J-curve phenomenon and the treatment of hyperten- sion. Is there a point beyond which pressure reduction is dangerous? JAMA, 1991; 265: 489-495. 11. Saito I, Takeshita E, Hayashi S et al. Comparison of clin- ic and home blood pressure levels and the role of the sympathetic nervous system in clinic-home differences. Am J Hypertens, 1990; 3: 219-224. 3. Kimura Y, Fukiyama K. Comprehensive management of hypertension. Epidemiology of hypertension in Japan. Junkankika, 1999; 46 (Suppl): 152-163. (in Japanese) 12. Saito I, Takeshita E, Murata K, Kawabe H, Saruta T. Serum cortical in the white-coat phenomenon. Blood Press Monit, 1996; 1: 381-383 4. Saito S, Ohnishi H, Takagi S, Shimamoto K. Epidemiology of hypertension in Japanese. Nippon Rinsho, 2000; 58: 593-596. (in Japanese) 13. Saito I, Murata K, Tsujioka M, Kawabe H, Saruta T. Long-term changes in clinic blood pressure in patients with white-coat hypertension. Blood Press Monit, 1998; 3: 97-100. 5. Baba S, Pan WH, Ueshima H et al. Blood pressure levels, related factors, and hyprtension control status of Japanese and Americans. J Hum Hypertens, 1991; 5: 317-332. 14. Staessen JA, Fagard RH, Lijnen PJ et al. Mean and range of the ambulatory pressure in normotensive subjects from a meta-analysis of 23 studies. Am J Cardiol, 1991; 67: 723-727. 6. Saruta T. The Japanese new guideline for the manage- ment of hypertension-background of its preparation and characteristics of the new guideline. Nippon Rinsho, 2001; 59: 837-840. (in Japanese) 15. Staessen JA, O'Brien ET, Atkins N, Amery AK. Short report: ambulatory blood pressure in normotensive com- pared with hypertensive subjects. The Ad-Hoc Working Group. J Hypertens, 1993; 11: 1289-1297. 7. Fujishima M. Management of hypertension in Japan cur- rent state and clinical issue. Nippon Rinsho, 2001; 59: 892-899. (in Japanese) 16. Glen SK, Elliott HL, Curaio JL, Lees KR, Reid JL. White- coat hypertension as a cause of cardiovascular dysfunc- tion. Lancet, 1996; 348: 654-657. 8. 1. National Nutrition Survey 300 living area was randomly selected based upon area of the Comprehensive Survey of Living Condition of the People on Health and Welfare. Interview, blood pressure anthropo- 118 T. Hasegawa, et al. Table 2. Diagnostic criteria: JSH (2000), JNC-VI (1997), WHO/ISH (1999). Table 2. Diagnostic criteria: JSH (2000), JNC-VI (1997), WHO/ISH (1999). Table 2. Diagnostic criteria: JSH (2000), JNC-VI (1997), WHO/ISH (1999). 1999 Report. Tokyo, 1999. (in Japanese) Guidelines Subcommittee of the Japanese Society of Hypertension. Japanese society of hypertension guide- lines for the management of hypertension. Tokyo, 2000. (in Japanese) 17. Ohkubo T, Imai Y. Epidemiology of hypertension in Japan. Ohasako study. Ketsuatsu, 2000; 7: 457-462. (in Japanese) 9. Statistics and Information Department, Minister's Secretariat, Ministry of Health and Labor. Patient Survey 18. MacMahon S, Peto R, Cutler J et al. Blood pressure, stroke, Meta-Analysis on Hypertension in Japan 119 and coronary heart disease. Part 1, Prolonged differences in blood pressure: prospective observational studies cor- rected for the regression dilution bias. Lancet, 1990; 335: 765-774. and coronary heart disease. Part 1, Prolonged differences in blood pressure: prospective observational studies cor- rected for the regression dilution bias. Lancet, 1990; 335: 765-774. 21. Anonymous. 1999 World Health Organization-International Society of Hypertension Guidelines for the Management of Hypertension. Guidelines Subcommittee. J Hypertens, 1999; 17: 151-183. 19. WHO Expert Committee. Arterial hypertension. WHO Technical Report Series, 1978: 628. 22. Anonymous. The sixth report of the Joint National Committee on prevention, detection, evaluation and treatment of high blood pressure. Arch Intern Med, 1997; 157: 2413-2446. 20. Guidelines for Management of Mild Hypertension. Memorandium from a WHO/ISH meeting. ISH hyperten- sion special edition, 1993.
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Two Opposing Functions of Angiotensin-Converting Enzyme (ACE) That Links Hypertension, Dementia, and Aging
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  Citation: Le, D.; Brown, L.; Malik, K.; Murakami, S. Two Opposing Functions of Angiotensin-Converting Enzyme (ACE) That Links Hypertension, Dementia, and Aging. Int. J. Mol. Sci. 2021, 22, 13178. https://doi.org/10.3390/ijms 222413178 Citation: Le, D.; Brown, L.; Malik, K.; Murakami, S. Two Opposing Functions of Angiotensin-Converting Enzyme (ACE) That Links Hypertension, Dementia, and Aging. Int. J. Mol. Sci. 2021, 22, 13178. https://doi.org/10.3390/ijms 222413178 Keywords: aging; age-related comorbidities; angiotensin-converting enzyme; amyloid-degrading enzyme; Alzheimer’ s disease; dementia; hypertension; life extension; stress resistance Received: 29 October 2021 Accepted: 5 December 2021 Published: 7 December 2021 Review Two Opposing Functions of Angiotensin-Converting Enzyme (ACE) That Links Hypertension, Dementia, and Aging Duc Le, Lindsay Brown, Kundan Malik and Shin Murakami * Duc Le, Lindsay Brown, Kundan Malik and Shin Murakami * Department of Basic Sciences, Master of Science in Medical Health Sciences, College of Osteopathic Medicine, Touro University California, Vallejo, CA 94592, USA; dle5@student.touro.edu (D.L.); lbrown25@student.touro.edu (L.B.); kmalik@student.touro.edu (K.M.) * Correspondence: shin.murakami@tu.edu Abstract: A 2018 report from the American Heart Association shows that over 103 million American adults have hypertension. The angiotensin-converting enzyme (ACE) (EC 3.4.15.1) is a dipeptidyl carboxylase that, when inhibited, can reduce blood pressure through the renin–angiotensin system. ACE inhibitors are used as a first-line medication to be prescribed to treat hypertension, chronic kidney disease, and heart failure, among others. It has been suggested that ACE inhibitors can alleviate the symptoms in mouse models. Despite the benefits of ACE inhibitors, previous studies also have suggested that genetic variants of the ACE gene are risk factors for Alzheimer’s disease (AD) and other neurological diseases, while other variants are associated with reduced risk of AD. In mice, ACE overexpression in the brain reduces symptoms of the AD model systems. Thus, we find two opposing effects of ACE on health. To clarify the effects, we dissect the functions of ACE as follows: (1) angiotensin-converting enzyme that hydrolyzes angiotensin I to make angiotensin II in the renin–angiotensin system; (2) amyloid-degrading enzyme that hydrolyzes beta-amyloid, reducing amyloid toxicity. The efficacy of the ACE inhibitors is well established in humans, while the knowledge specific to AD remains to be open for further research. We provide an overview of ACE and inhibitors that link a wide variety of age-related comorbidities from hypertension to AD to aging. ACE also serves as an example of the middle-life crisis theory that assumes deleterious events during midlife, leading to age-related later events.   Citation: Le, D.; Brown, L.; Malik, K.; Murakami, S. Two Opposing Functions of Angiotensin-Converting Enzyme (ACE) That Links Hypertension, Dementia, and Aging. Int. J. Mol. Sci. 2021, 22, 13178. https://doi.org/10.3390/ijms 222413178 Academic Editor: Pavel Hozák Received: 29 October 2021 Accepted: 5 December 2021 Published: 7 December 2021 International Journal of Molecular Sciences Review 1. Introduction ACE plays a major role in the angiotensin–renin system that regulates blood pressure and salt balance. ACE was first reported as a hypertensin-converting enzyme [1]. In 1958, the term “hypertensin” was revised to “angiotensin” [2]. ACE inhibitors had been initially reported as a medication to treat hypertension [3,4]. The history of angiotensin and ACE have been described elsewhere [5]. The ACE gene encodes a protein with 732 amino acid residues and a molecular weight of 80,073 [6]. Clinically, it is an important target for the treatment of hypertension. Current first-line medications for hypertension include ACE inhibitors, angiotensin receptor blockers (ARBs), beta-blockers, and calcium channel blockers [7]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Primary hypertension, or essential hypertension, is the condition of having high blood pressure. Although no clear cause for primary hypertension is known, a combi- nation of genetics, poor diet, and lack of exercise is thought to play a role. In contrast, secondary hypertension is high blood pressure caused by another medical condition. Dia- betes, Cushing’s syndrome, pheochromocytoma, hyperparathyroidism, hypokalemia, or primary hyperaldosteronism are several examples of conditions that may cause secondary hypertension [8]. Secondary hypertension can be diagnosed following a series of screening and lab tests to identify underlying factors and impacted organs [9]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 13178. https://doi.org/10.3390/ijms222413178 Int. J. Mol. Sci. 2021, 22, 13178 2 of 10 2 of 10 Our recent studies suggested that the ACE gene is associated with a broad range of neurological diseases, including AD, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson’s disease (PD), and schizophrenia [10,11], which is consistent with the findings from other groups [12,13]. ACE inhibitors are well-established medications for hyperten- sion, which is also a major risk of cognitive impairment and dementia [14] and of death by COVID-19 [15]. Here, we overview the underlying mechanisms of ACE relevant to AD and age-related comorbidities. We hope to provide the links between ACE and hypertension, AD, neurological diseases, and aging. 2. Mechanism of ACE ACE has a dipeptidyl carboxypeptidase activity that can hydrolyze and cleave the c-terminal dipeptide of angiotensin I (ten amino acid residues) to make angiotensin II (eight amino acid residues). ACE has another activity as an amyloid-degrading enzyme (ADE) that can hydrolyze beta-amyloid (discussed in Section 3.4). As shown in Figure 1, the N and C domains contain active sites with a zinc ion-binding site. Previous studies have reported that inhibition of the C domain can manage blood pressure, while inhi- bition of the N domain has little or no impact [16,17]. The conversion of angiotensin I to angiotensin II is prevented by binding to the C domain, leading to disruption of the renin–angiotensin–aldosterone system (RAAS). Angiotensin II acts as a vasoconstrictor by stimulating Gq proteins on the vascular smooth muscle, activating an IP3-dependent pathway that increases intracellular calcium levels and causes constriction. Circulating ACE ACE N-catalytic domain ACE C-catalytic domain Cleavage site ACE-secretase Cytosol Extracellular space Membrane C-terminal Cytosolic domain Transmembrane domain Active site Active site Active site Active site Zn2+ Zn2+ Zn2+ Zn2+ Figure 1. A diagram representing angiotensin-converting enzyme (ACE) and ACE-secretase. Phos- pholipid bilayer of the cell membrane (indicated by yellow), ACE-secretase bound to the cell mem- brane (indicated by red), and ACE (indicated by blue). The figures for ACE depict two major domains, the N- and C-catalytic domains, which are active sites that bind zinc ions. ACE-secretase cleaves at a site nearby the N-catalytic domains to form circulating ACE. This circulating and non-circulating ACE can hydrolyze and cleave angiotensin I to form angiotensin II in the mechanisms described in the text. ACE N-catalytic domain Figure 1. A diagram representing angiotensin-converting enzyme (ACE) and ACE-secretase. Phos- pholipid bilayer of the cell membrane (indicated by yellow), ACE-secretase bound to the cell mem- brane (indicated by red), and ACE (indicated by blue). The figures for ACE depict two major domains, the N- and C-catalytic domains, which are active sites that bind zinc ions. ACE-secretase cleaves at a site nearby the N-catalytic domains to form circulating ACE. This circulating and non-circulating ACE can hydrolyze and cleave angiotensin I to form angiotensin II in the mechanisms described in the text. Int. J. Mol. Sci. 2021, 22, 13178 3 of 10 3 of 10 Angiotensin II has two major receptors that it can interact with to produce homeostatic actions (Figure 2). 2. Mechanism of ACE Angiotensin II type 1 receptor (AT1R) has well-documented functions in vasoconstriction, cellular proliferation, inflammation, and fibrosis. Due to its significant role in increasing blood pressure, AT1R is a major target for anti-hypertensive drugs, specifically ARBs. Conversely, angiotensin II type 2 receptor (AT2R) does not have as clearly defined of a role as AT1R and is an ongoing subject of research [18]. Past studies have suggested that AT2R primarily functions as an antagonist for AT1R, inducing contradicting effects such as vasodilation and inhibition of cell proliferation, inflammation, and fibrosis. In addition, studies have shown that the Mas receptor, which responds to angiotensin (1-7), induces similar effects to AT2R. Due to this observed function of regulating the proinflammatory effects of angiotensin II, studies have tested and demonstrated the impact of the receptor Mas axis on macrophage function and inflammation diseases [19]. Thus, a defect in the Mas receptors, or a deficiency in ACE2, has been shown to cause an increase in inflammatory responses within the CNS and the vascular systems. REVIEW 4 of 11 Figure 2. A schematic diagram of the Renin-Angiotensin System (RAS) pathway and the points in which inhibition may occur. The three key organs of the RAS pathway include the lungs, liver, and kidney. The RAS pathway revolves around the production of angiotensinogen, the precursor to angiotensin originating from the liver. Through renin produced in the kidney, followed by ACE produced in the lungs, angiotensin II can subsequently be formed. By inhibiting either renin or ACE, the progress of the RAS pathway can effectively be halted. Below the schematic is a summary of the final physiolog- ical effects followed by the binding of each target is listed at the bottom of the diagram. Angiotensin II will produce vaso- constriction, proliferation, inflammation, and fibrosis through binding to the AT1 receptor (AT1R). Binding to AT2 recep- tor or MasR, following conversion to Angiotensin-(1-7), will result in opposing physiologic effects. This conversion to Angiotensin-(1-7) is known as alternative RAS activation and is enabled by the angiotensin-converting-enzyme-2 (ACE2) and neprilysin (NEP) a protease abundantly found in the kidneys Figure 2. A schematic diagram of the Renin-Angiotensin System (RAS) pathway and the points in which inhibition may occur. The three key organs of the RAS pathway include the lungs, liver, and kidney. The RAS pathway revolves around the production of angiotensinogen, the precursor to angiotensin originating from the liver. 2. Mechanism of ACE Through renin produced in the kidney, followed by ACE produced in the lungs, angiotensin II can subsequently be formed. By inhibiting either renin or ACE, the progress of the RAS pathway can effectively be halted. Below the schematic is a summary of the final physiological effects followed by the binding of each target is listed at the bottom of the diagram. Angiotensin II will produce vasoconstriction, proliferation, inflammation, and fibrosis through binding to the AT1 receptor (AT1R). Binding to AT2 receptor or MasR, following conversion to Angiotensin-(1-7), will result in opposing physiologic effects. This conversion to Angiotensin-(1-7) is known as alternative RAS activation and is enabled by the angiotensin-converting-enzyme-2 (ACE2) and neprilysin (NEP), a protease abundantly found in the kidneys. Figure 2. A schematic diagram of the Renin-Angiotensin System (RAS) pathway and the points in which inhibition may occur. The three key organs of the RAS pathway include the lungs, liver, and kidney. The RAS pathway revolves around the production of angiotensinogen, the precursor to angiotensin originating from the liver. Through renin produced in the kidney, followed by ACE produced in the lungs, angiotensin II can subsequently be formed. By inhibiting either renin or ACE, the progress of the RAS pathway can effectively be halted. Below the schematic is a summary of the final physiolog- ical effects followed by the binding of each target is listed at the bottom of the diagram. Angiotensin II will produce vaso- constriction, proliferation, inflammation, and fibrosis through binding to the AT1 receptor (AT1R). Binding to AT2 recep- tor or MasR, following conversion to Angiotensin-(1-7), will result in opposing physiologic effects. This conversion to Angiotensin-(1-7) is known as alternative RAS activation and is enabled by the angiotensin-converting-enzyme-2 (ACE2) d il i (NEP) t b d tl f d i th kid Figure 2. A schematic diagram of the Renin-Angiotensin System (RAS) pathway and the points in which inhibition may occur. The three key organs of the RAS pathway include the lungs, liver, and kidney. The RAS pathway revolves around the production of angiotensinogen, the precursor to angiotensin originating from the liver. Through renin produced in the kidney, followed by ACE produced in the lungs, angiotensin II can subsequently be formed. By inhibiting either renin or ACE, the progress of the RAS pathway can effectively be halted. 3.2. ACE as a Link to AD and Diverse Neurological Diseases 3.2. ACE as a Link to AD and Diverse Neurological Diseases In humans, ACE polymorphisms are associated with AD. Two groups conducted population studies that suggested an association with late-onset AD (LOAD) by inves- tigating an insertion/deletion polymorphism of the ACE gene [25,26]. However, earlier studies have been controversial: 74 case–control studies and 6 family-based studies have investigated the genetic association between ACE and AD, mixed with 26 positive results, 31 negative results, 16 not applicable, and the remaining being inconclusive, according to the AlzGene database (Accessed 15 November 2021) [27]. A series of meta-analysis studies have been necessary and showed the significance of the genetic association between the ACE gene and AD [10,11,28–32]. More details of ACE gene variations are discussed in the next section. The ACE gene is associated with a wide range of neurological diseases, including AD, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Parkinson’s disease (PD), and schizophrenia [10–13]. The neurological diseases have a strong association with ACE, MTHFR, and TNF [10]. The studies have significant clinical implications, namely that patients with a risk factor gene of AD may show diverse neurological symptoms such as ALS, MS, PD, and schizophrenia. Thus, patients with AD may show diverse symptoms in addition to those directly relevant to AD. The nature of the foundation of the relationship between AD and age-related comor- bidities is unclear. However, hypertension and strokes are independent risk factors of cognitive impairment and dementia [14,33]. The onset of cognitive impairment is common after long-term hypertension and stroke [14]. Chronic inflammation is commonly seen in patients with comorbidities including hypertension and diabetes prior to the development of AD [34,35]. Chronic inflammation in the brain may be an intermediary component in the progression of AD and becomes more common with the aging process. The understanding of age-related comorbidities should open a new avenue for research to understand the mechanisms that link a wide variety of pathophysiology in aging. 2. Mechanism of ACE Below the schematic is a summary of the final physiological effects followed by the binding of each target is listed at the bottom of the diagram. Angiotensin II will produce vasoconstriction, proliferation, inflammation, and fibrosis through binding to the AT1 receptor (AT1R). Binding to AT2 receptor or MasR, following conversion to Angiotensin-(1-7), will result in opposing physiologic effects. This conversion to Angiotensin-(1-7) is known as alternative RAS activation and is enabled by the angiotensin-converting-enzyme-2 (ACE2) and neprilysin (NEP), a protease abundantly found in the kidneys. Int. J. Mol. Sci. 2021, 22, 13178 4 of 10 3. ACE as a Gene That Links Hypertension, AD, and Aging 3.1. ACE as a Link to Hypertension 3. ACE as a Gene That Links Hypertension, AD, and Aging 3.1. ACE as a Link to Hypertension Hypertension develops with increasing age. Despite varying definitions, a threshold of early-onset hypertension is at the age of fewer than 55 years old [20] when recom- mending first-line medications, such as ACE inhibitors [21]. ACE generates angiotensin II, which has an effect on vasoconstriction and increases blood pressure. ACE inhibitors can reduce angiotensin II, leading to vasodilation and a subsequent decrease in blood pres- sure (i.e., vaso-protection against hypertension). ACE inhibitors may also downregulate sympathetic activity, leading to less cardiovascular burden. Other cardiovascular effects of ACE inhibitors include the promoted renal excretion of sodium and water, leading to a further decrease in blood volume. ACE inhibitors and angiotensin II receptor blockers (ARBs) are also known to reduce the risk of hospitalization for heart failure in an ethnicity- dependent manner [22]. Thus, ACE inhibitors and ARBs are first-line medications for treating hypertension, chronic kidney disease, and heart failure. g yp y ACE has been a point of interest in previous studies, which have shown that hyper- tension is a strong risk factor for developing cognitive impairment and dementia [14]. Abnormalities such as chronically elevated blood pressure or a series of mini strokes have been associated with impaired cognitive function and the onset of various forms of dementia, including AD. A wide variety of cardiovascular diseases are also sensitive to hypertension [23,24]. 3.3. Genetic Variations of ACE That Link to AD Genetic variants in ACE have been reported that are associated with the risk of AD, including the insertion/deletion variant and single nucleotide polymorphisms (SNPs). The insertion/deletion variants have an Alu repeat present (insertion) or absent (dele- tion) [36], which initially showed an association with AD [28,37,38]. More than seven Int. J. Mol. Sci. 2021, 22, 13178 5 of 10 SNPs are associated with AD, including rs4291 [31,32,39,40], rs138190086 [37], rs4343, and rs1800764 [39]. Four variants, rs4968782, rs4459609, rs4316, and rs4343, are associated with decreased risk of AD; rs4316 and rs4343 are synonymous substitutes but are predicted to affect transcription factor binding [41]. It has also been suggested that ACE may also inhibit Aβ aggregation by the amyloid-degrading function [42]. It is also consistent with the implication of clinical studies described below. Interestingly, a rare penetrant ACE variant, rs4980 (R1279Q), seems to cause AD, since the variant can accelerate Aβ-induced neurodegeneration in mice [43] (discussed in Section 3.4). Thus, there are two opposing types of variants: variants associated with increased risk of AD and variants associated with reduced risk of AD. Clinical studies suggest that ACE polymorphisms affect its level and activity in the cerebrospinal fluid (CSF) and serum, with some inconsistent findings. In previous studies, the insertion/deletion polymorphisms and three SNPs (rs4291, rs4343, and rs1800764) show an increased risk of AD, increased CSF Aβ, and an earlier age of AD onset [39,44]. CSF ACE activity is increased in AD, while CSF ACE levels are reduced. Aβ1-42 can increase the ACE level and activity in the cultured neurons in vitro. Thus, ACE may be induced by Aβ. In another study, of seven SNPs associated with CSF ACE levels, four SNPs (rs4316, rs4343, rs4459609, and rs4968782) are associated with decreased risk of AD and increased ACE levels in CSF and plasma [41]. However, the finding in rs4343 is not consistent with the previous studies showing reduced ACE levels [39,44]. Nonetheless, the clinical studies support the protective function of ACE in the AD pathology, which may involve ACE as an amyloid-degrading enzyme. 3.4. Two Opposing Health Effects of ACE on AD As discussed above, two opposing effects of ACE have been observed. ACE inhibitors can be used as treatment options for hypertension and other health conditions, while alterations of the ACE gene are associated with AD. In addition, there are two types of ACE variants: variants with increased risk of AD, and variants with reduced risk of AD. Why do the seemingly opposing health effects of ACE occur? We differentiate the effects into two functions: We differentiate the effects into two functions: We differentiate the effects into two functions: (1). ACE as an angiotensin-converting enzyme that makes angiotensin II in the renin– angiotensin system. The action of ACE is described in Section 2. ACE inhibitors reduce the production of angiotensin II, which results in vasodilation and reduced blood pressure. In this case, inhibiting the activities as an angiotensin-converting enzyme would relieve hypertension and hypertension-sensitive conditions, includ- ing heart failure, chronic kidney disease, or diabetes mellitus [45]. In mice, the role of the ACE/angiotensin II signaling has been investigated. A previous study suggests that a brain-penetrant ACE inhibitor, Captopril, reduces AD symptoms in the mouse AD model (Tg2576) which overexpresses a Swedish APP mutation (KM670/671NL) [46]. In another mouse AD model (5XFAD) that has five AD-linked mutations, ACE variant rs4980 (R1279Q) causes aging-dependent, Aβ-accelerated selective hippocampal neuron vulnerability and female susceptibility [43]. The Aβ- induced hippocampal neurodegeneration is rescued by the ACE inhibitor (Captopril) and AT1R inhibitor/ARB (Losartan) that can penetrate the brain [43]. The studies suggest that ACE/angiotensin II signaling causes Aβ-induced neurodegeneration and that brain-penetrant ACE inhibitor/ARB can protect the neurons. Although clinical studies remain to be completed, it is reasonable to conclude that ACE inhibitors may be beneficial to health under specific conditions. (2). p (2). ACE as an amyloid-degrading enzyme (ADE) that can hydrolyze beta-amyloid and decrease amyloid toxicity. ADE represents a group of broadly defined en- zymes, currently, including 14 enzymes: ACE, acyl peptide hydrolase, aminopep- tidase A, cathepsin B, endothelin-converting enzyme, glutamate carboxypeptidase II, insulin-degrading enzyme, MBP, MMP-2, MMP-9, NEP2, neprilysin, plasmin, and PreP [47,48]. ACE has an activity of beta-amyloid amyloid-degrading enzymes (2). ACE as an amyloid-degrading enzyme (ADE) that can hydrolyze beta-amyloid and decrease amyloid toxicity. ADE represents a group of broadly defined en- zymes, currently, including 14 enzymes: ACE, acyl peptide hydrolase, aminopep- tidase A, cathepsin B, endothelin-converting enzyme, glutamate carboxypeptidase II, insulin-degrading enzyme, MBP, MMP-2, MMP-9, NEP2, neprilysin, plasmin, and PreP [47,48]. 3.4. Two Opposing Health Effects of ACE on AD ACE has an activity of beta-amyloid amyloid-degrading enzymes (2). ACE as an amyloid-degrading enzyme (ADE) that can hydrolyze beta-amyloid and decrease amyloid toxicity. ADE represents a group of broadly defined en- zymes, currently, including 14 enzymes: ACE, acyl peptide hydrolase, aminopep- tidase A, cathepsin B, endothelin-converting enzyme, glutamate carboxypeptidase II, insulin-degrading enzyme, MBP, MMP-2, MMP-9, NEP2, neprilysin, plasmin, and PreP [47,48]. ACE has an activity of beta-amyloid amyloid-degrading enzymes Int. J. Mol. Sci. 2021, 22, 13178 6 of 10 (ADEs) that can hydrolyze and convert Aβ1-42 to Aβ1-40 in homogenates of the mouse Tg2576 AD model and human AD autopsy [47], which is consistent with its dipeptidyl carboxypeptidase activity that cleaves the c-terminal two amino acid residues. ACE can also cleave Aβ1-40 into two smaller peptides (Aβ1-7 and Aβ8- 40) [49–51]. In mice, inhibitions of ACE can worsen the accumulation of Aβ1-42 in the mouse model [47]. Consistently, overexpression of ACE in the brain resident microglia, peripheral myelomonocytes, and macrophages can alleviate the symptoms of the double-transgenic APPSWE/PS1∆E9 (AD+) mice [52,53]. Thus, in the AD model system in mice, the ADE activity can reduce Aβ -induced pathologic problems. The efficacy of the ADE activity seems to be consistent with the clinical studies with increased ACE and reduced risk of AD. ACE is also known to have broad specificity to substrates, including enkephalins, C-terminal extended proenkephalins, luteinizing hormone-releasing hormone, substance P and a protected chemotactic tripeptide [54], cholecystokinin-8, gastrin analogues [55], and kinins [56]. However, it is not clear how the ACE substrates are relevant to AD, and thus we do not discuss them further. 3.5. ACE as a Link to Aging (Life Extension Model Systems) Previous studies have investigated the effects of ACE inhibitors and homologs of ACE in model species. In the nematode, Caenorhabditis elegans, the acn-1 gene, a homolog of nematode ACE, was used to explore the relation between the use of ACE inhibitors and longevity [57]. The application of the ACE inhibitor, Captopril, reduces the acn-1 activity, leading to life extension, increased stress resistance, and a delay in age-related degenerative changes measured by pharyngeal pumping. Further analysis indicated that the lifespan effects of Captopril are additive to other known life-extending interventions (and genes), including the insulin/IGF-1 pathway (age-1 and daf-2), caloric restriction (eat-2), a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase (sir-2.1), heat shock (hsf-1), and mitochondria insufficiency (isp-1). y In the fruit fly, Drosophila melanogaster, another ACE inhibitor, Lisinopril, was used to study the impacts of Ance, an ortholog of ACE, in the three genotypes from the reference panel lines [58]. Mean lifespan was increased following the application of Lisinopril in three inbred lines from a natural population (DGRP73, CGRP229, and DGRP304). The effect of lisinopril showed a genotype-specific manner with the degree of change to lifespan (the highest effect in DGRP304), age-specific speed (the highest effect in DGRP229), endurance (the highest effect in DGRP229), and strength (the highest effect in DGRP229). The inbred lines that had an improvement of physical performance while on Lisinopril had a reduction in the age-related aggregation of protein in skeletal muscle, suggesting a role of stress resistance in lifespan extension. The results showed a significant involvement of skeletal muscle Ance in the lifespan of Drosophila species and that there is genetic variation in the phenotypic responses to ACE inhibitors. Mouse models have also been used to study the effect of ACE inhibitors on lifespan. A study that used the combined application of statin, simvastatin, and ACE inhibitor, Ramipril, resulted in an increased mean lifespan for a group of isocalorically fed mice subjects [59]. 3.6. ACE as a Link to Stress Resistance and the Middle-Life Crisis Theory on Aging ACE inhibitors and mutations are associated with life extension and stress resistance (discussed above). Increased resistance to multiple forms of stresses has been shown as a component of life extension in the model species, including yeasts, nematodes, fruit flies, and mice [60–67]. ACE as an angiotensin-converting enzyme has been used as a drug target to control hypertension and related complications. In contrast, ACE as an amyloid-degrading enzyme has been used to control more advanced phase accumulation of beta-amyloid. The different activities of ACE support the notion of aging stages from the transition state to the more advanced pathological state [68–70]. Furthermore, those are consistent with a previous study that indicates the underlying mechanisms of the human Int. J. Mol. Sci. 2021, 22, 13178 7 of 10 7 of 10 AD genes, including lipid metabolism, amyloid-related pathways, and neural and immune systems [10]. We see that age-related comorbidities occur during the middle of lifespan when the onset of hypertension and associated health conditions occur. At the stage of the lifespan, the ACE function as an angiotensin-converting enzyme is beneficial to the health condition, while the function as an amyloid-degrading enzyme is more important when an abnormal accumulation of beta-amyloid starts. ACE provides an example for the middle-life crisis theory on aging in which middle-life events result in aging and age- related diseases in late life. In the theory, the middle-life events explain the transition to aging from normal to more advanced age-related changes [69,70]. 4. Conclusions and Perspective Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. g gg g 3. Gavras, H.; Brunner, H.R.; Laragh, J.H.; Sealey, J.E.; Gavras, I.; Vukovich, R.A. An Angiotensin Converting-Enzyme Inhibitor to Identify and Treat Vasoconstrictor and Volume Factors in Hypertensive Patients. N. Engl. J. Med. 1974, 291, 817–821. [CrossRef] [PubMed] 1. Skeggs, L.T.; Kahn, J.R.; Shumway, N.P. The preparation and function of the hypertensin-converting enzyme. J. Exp. Med. 1956, 103, 295–299. [CrossRef] 4. Conclusions and Perspective Hypertension is major comorbidity that develops over time. First-line medications include ACE inhibitors that have been well established in medicine. In contrast, growing evidence suggests that ACE genetic variants are risk factors for AD and other neurological diseases. This seemingly controversial finding has been an intriguing topic but has never been discussed and clarified. To this end, we dissected the functions of ACE and explained this seemingly controversial finding. ACE inhibitors are beneficial to reduce hypertension and associated health conditions. Later in life, ACE as an amyloid-degrading enzyme starts to play a role in fighting against AD pathology, which is supported by clinical studies with increased ACE and reduced risk of AD (see Section 3.3). This later-life ACE function remains to be explored in humans, since it could provide a promising target for the treatment of AD and other related health conditions. The current understanding is that hypertension medication should be used to prevent vascular, stroke, and mixed dementia [71], while the treatment for AD-specific symptoms requires considerations. Although it has been suggested that ACE inhibitors may reduce the amyloid-degrading activity of ACE [39], ACE inhibitors can inactivate abnormal ACE/angiotensin II signaling that causes Aβ-induced neurodegeneration (Section 3.4). ACE inhibitors have been shown to reduce the symptoms of the AD mouse model [43,46]. Importantly, inhibition of ACE can extend lifespans in model systems, including the nematode, the fruit fly, and the mouse, suggesting potential health benefits during lifespans. Thus, we reason that ACE inhibitors may be beneficial until the accumulation of amyloids causes health problems. The areas of ACE inhibitors and ACE receptor inhibitors are promising fields for further exploration. Taken together, the current status of understanding ACE in medications still has open questions specific to hypertension, other age-related comorbidities, and types of dementia. Author Contributions: D.L., L.B. and K.M. were all involved in the literature search, selection, and manuscript preparation. S.M. has been involved in direction and all aspects of preparation of this manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We thank the members of the Murakami laboratory for useful discussion and technical assistance. , [ ] 2. Braun-Menendez, E.; Page, I.H. Suggested Revision of Nomenclature—Angiotensin. Science 1958, 127, 242. 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Influence of Nano-Hydroxyapatite on the Metal Bioavailability, Plant Metal Accumulation and Root Exudates of Ryegrass for Phytoremediation in Lead-Polluted Soil
International journal of environmental research and public health/International journal of environmental research and public health
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Influence of Nano-Hydroxyapatite on the Metal Bioavailability, Plant Metal Accumulation and Root Exudates of Ryegrass for Phytoremediation in Lead-Polluted Soil Ling Ding 1, Jianbing Li 2, Wei Liu 1, Qingqing Zuo 1 and Shu-xuan Liang 1,* Ling Ding 1, Jianbing Li 2, Wei Liu 1, Qingqing Zuo 1 and Shu-xuan Liang 1,* 1 College of Chemistry and Environmental Science, Hebei University, Key Laboratory of Analytical Science and Technology of Hebei Province, Baoding 071002, China; 18730271511@163.com (L.D.); auhlw80@126.com (W.L.); sunqc710@163.com (Q.Z.) 2 Environmental Engineering Program, University of Northern British Columbia, Prince George, BC V2N4Z9, Canada; Jianbing.Li@unbc.ca * Correspondence: liangsx168@126.com; Tel.: +86-312-507-9749; Fax: +86-312-507-9739 Academic Editor: Susanne Charlesworth g * Correspondence: liangsx168@126.com; Tel.: +86-312-507-9749; Fax: +86-312-507-9739 Academic Editor: Susanne Charlesworth Academic Editor: Susanne Charlesworth Received: 11 December 2016; Accepted: 25 April 2017; Published: 16 May 2017 Abstract: Lead is recognized as one of the most widespread toxic metal contaminants and pervasive environmental health concerns in the environment. In this paper, the effects of nano-hydroxyapatite (NHAP) on remediation in artificially Pb-contaminated soils and ryegrass were studied in a pot experiment. The addition of NHAP decreased the water- and acid-soluble, exchangeable, and reducible fractions of Pb, extracted using the Community Bureau of Reference (BCR) method, whilst greatly increasing the residual fraction of Pb. Oxidizable Pb was increased slightly. No significant increase in soil pH was caused by the application of NHAP. Compared to conditions without NHAP, the addition of NHAP decreased the Pb content in ryegrass shoots and roots by 13.19–20.3% and 2.86–21.1%, respectively. Therefore, the application of NHAP reduced the mobility and bioavailability of Pb in the soil. In addition, the application of NHAP improved the fresh weight of shoots and roots, and promoted the growth of ryegrass. NHAP played a positive role in stimulating ryegrass to secrete tartaric acid. Keywords: nano-hydroxyapatite; ryegrass; Pb-polluted soil Keywords: nano-hydroxyapatite; ryegrass; Pb-polluted soil International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health Int. J. Environ. Res. Public Health 2017, 14, 532; doi:10.3390/ijerph14050532 1. Introduction Soil pollution by heavy metals has become a serious concern in many developing countries due to intense industrialization and urbanization. Heavy metals are more complex than other environmental pollutants because they can be toxic to all living organisms. They are not biodegradable and tend to accumulate in tissues [1]. Lead is recognized as one of the most widespread toxic metal contaminants and pervasive health concerns in the environment [2]. It is generated from the natural weathering of rocks and industrial activities, including mining and lead ore smelting, lead-acid battery manufacturing, lead-based paints, etc. [3]. In addition, Pb is not an essential nutrient in the metabolic processes of plants and animals, and it can accumulate to high levels and have biological toxicity to organisms [4,5]. The limit for Pb content is 35 mg/kg, according to the environmental quality standards for soils [6]. Therefore, the development of remediation strategies for Pb-contaminated soils is very important for human health and ecological protection. y p g p Currently, most researchers focus on the use of chemical remediation and phytoremediation to control soil heavy metal pollution [7,8]. There have been a number of studies on in situ immobilization of Pb-contaminated soils using hydroxyapatite, and two different mechanisms were Int. J. Environ. Res. Public Health 2017, 14, 532; doi:10.3390/ijerph14050532 www.mdpi.com/journal/ijerph www.mdpi.com/journal/ijerph 2 of 9 Int. J. Environ. Res. Public Health 2017, 14, 532 mainly found: Dissolution–precipitation and ion exchange (between Pb2+ in solution and Ca2+ on hydroxyapatite lattice) [9]. The influence of both is dependent on pH and pore solution chemistry [10]. In general, hydroxyapatite has a better effect in acidic soil (pH ~5), but the soil in our experiment was alkaline soil (pH ~8). Therefore, hydroxyapatite did not apply in our experiment. Nanomaterials have a higher reactivity and adsorption capacity than ordinary-sized materials. Nano-hydroxyapatite (NHAP) is an important mineral component of human hard tissues, such as bones and teeth, and is the less soluble form of phosphate. It is an ideal material for the immobilization of heavy metals because of its high sorption capacity for heavy metals, low water solubility, high stability under reducing and oxidizing conditions, availability, and cost effectiveness [11]. At present, the application of nano-hydroxyapatite on Pb-contaminated soils is limited. The purpose of this study was to evaluate the effectiveness of nano-hydroxyapatite in immobilizing Pb in contaminated soils. 1. Introduction Compared with most hyperaccumulators, ryegrass is preferentially used for phytoremediation because it is extensively grown, easy to be managed, and has a high biomass, therefore, it is economical to use it for phytoremediation [12]. Ryegrass can accumulate a large amount of toxic substances, and has a high tolerance to heavy metals [13]. Thus, for this experiment we selected ryegrass as a phytoremediation plant. Our previous work studied the effect of 0.5% (w/w) NHAP on Pb-polluted soil, and the results showed that NHAP reduced the Pb contents of ryegrass [14]. However, at present, there are few studies on the immobilization of Pb using NHAP; especially, there is no research about the combination of NHAP and ryegrass, and the reasons for the reduced mobility and bioavailability of Pb caused by NHAP have not been fully investigated. In this paper, we studied the effects of higher NHAP applications at different Pb contents in the remediation of Pb-contaminated soil. The aim of this study was to investigate the effects of NHAP on the changes in the form of Pb in soil, and the accumulation of Pb in ryegrass and the growth of ryegrass. 2.1. Design of the Pot Experiment The tested soil (0–20 cm) was farm field soil extracted from Baoding City, Hebei Province, China. The soil was air-dried, crumbled and then milled (2 mm). The soil had a pH of 7.87, 23.28 g/kg organic matter, 8.43 g/kg total nitrogen (TN), 7.62 g/kg total phosphorus (TP), and a cation exchange capacity (CEC) of 1.40 mol/kg. The pH was measured on a 2:5 (w/v) water suspension of the soil sample after stirring for 10 min [15]. TN in the soil was determined using the Kjeldahl method [16]. The organic matter in the soil was determined using the potassium dichromate volumetric method. Total phosphorus was determined using the molybdate–ascorbic acid procedure at 700 nm [17]. Cation exchange capacity was determined using the compulsive exchange method with 1 mol/L of ammonium acetate (pH 7) [18]. The background value of Pb in the soil was 58.28 mg/kg. Pb was applied to the soil as Pb(NO3)2 at four concentrations (0, 400, 800, and 1200 mg/kg of dried soil). Lead-spiked soil was aged in a greenhouse for one month. Nano-hydroxyapatite was purchased from the Nanjing Emperor Nano Material Company (Nanjing, China) and had a purity greater than 96%. The pH of NHAP was 8.11, and the specific surface area was 154 m2/g. The pH was measured on a 1:20 (w/v) water suspension of the nano-hydroxyapatite samples after stirring for 1 h [19]. The Brunauer–Emmett–Teller (BET) surface area of the NHAP was determined by N2 sorption analysis at 77 K in a surface analyzer after degassing. The pot experiments were conducted in a greenhouse with an air temperature of 22–25 ◦C at Hebei University. The design of the pot experiments for the different treatments is listed in Table 1. Twenty seeds of ryegrass were sown per pot, which was filled with 0.15 kg soil (60% moisture content) and 1.5 g NHAP. Pots without NHAP were used as a control. Three replicates were set for each treatment. Thirty days after germination, the samples of ryegrass were harvested by cutting the shoots at the soil surface, and the roots were carefully separated from the soil. Plants were thoroughly washed 3 of 9 Int. J. Environ. Res. Public Health 2017, 14, 532 with running water, followed by distilled water, and then dried at 105 ◦C for 1 h, and then at 65 ◦C in an oven (BGZ-30, Shanghai Boxun Industry and Commerce Company, Shanghai, China) until completely dry. 2.3. Determination of pH Four grams of soil sample were put into plastic centrifuge tubes. Then, 10 mL of distilled water was added to the tubes. The mixture of soil and solution was stirred for 10 min and then allowed to settle for 30 min. The pH value was measured using a pH meter (HACH, Loveland, CO, USA). 2.2. Pb Content Determination Shoot or root dry matter (0.1 g) was digested using 5 mL HNO3 and 2 mL H2O2. Soil samples (0.1 g) were digested with 5 mL HNO3, 2 mL H2O2 and 2 mL HF. The Pb concentration in the digested solutions was determined using an A3 atomic absorption spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China). The standard reference material (GBW 07411, National Institute of Metrology, Beijing, China) was analyzed with the samples during the course of the analyses. The linear correlation coefficient of the Pb standard solution was r > 0.999. The mean recovery of the Pb standard reference material was 98%. The range of the Pb standard solution was 1–15 mg/L. 2.1. Design of the Pot Experiment They were finally weighed, and the dry weight of the plants was recorded [20]. The soil was air-dried, crumbled and then milled (2 mm). Table 1. Design of the pot experiments for the different treatments. Table 1. Design of the pot experiments for the different treatments. Treatment Pb-Spiked Content (mg/kg) Addition Amount of NHAP (g) 0 mg/kg 0 0 0 mg/kg + NHAP 0 1.5 400 mg/kg 400 0 400 mg/kg + NHAP 400 1.5 800 mg/kg 800 0 800 mg/kg + NHAP 800 1.5 1200 mg/kg 1200 0 1200 mg/kg + NHAP 1200 1.5 NHAP: nano-hydroxyapatite. Table 1. Design of the pot experiments for the different treatments. 2.6. Statistical Analysis All values are the means of three replicates. Data are presented as the mean value ± standard deviation. To verify the statistical significance of differences among the treatments, data were analyzed using SPSS statistical software (IBM Nederland BV, Amsterdam, The Netherlands) using one-way ANOVA and Duncan’s multiple-range test. Differences were considered significant at p < 0.05. 2.4. Organic Acids Analysis A portion of the ryegrass rhizosphere soil was collected during the collection of the plant samples and kept at 4 ◦C for analysis. For organic acid extraction, 1.0 g of soil was extracted by 10 min of agitation at 200 rpm with 10 mL 0.1 mol/L H3PO4, and the extracts were filtered using a 0.2 mm filter membrane. The separation of organic acids was carried out on a system consisting of an analytical high-performance liquid chromatography (HPLC) unit (Waters 1525 Binary HPLC Pump, Waters 2998 Photodiode Array Detector, Waters, Milford, MA, USA) with a Cosmosil packed column (C18-PAQ, 4.6 mm I. D., Nacalai Tesque, Kyoto, Japan), in conjunction with a column heating device set at 30 ◦C. Elution was carried out isocratically at a solvent flow rate of 1.0 mL/min of 0.02 mol/L NaH2PO4 and chromatographic-grade acetonitrile (98:2). The injection volume was 20 µL. Detection was performed with a UV detector set at 213 nm. The standard solution was prepared by mixing eight low molecular weight organic acids: tartaric acid, lactic acid, acetic acid, citric acid, pyruvic acid, oxalic acid, succinic acid, and L-malic acid. Organic acid identification was performed by comparison of the retention times with those of authentic standards. The peaks in the chromatograms were integrated using a default baseline construction technique. The organic acid was quantified by the peak area. Int. J. Environ. Res. Public Health 2017, 14, 532 4 of 9 2.5. Community Bureau of Reference Sequential Extraction Tests 2.5. Community Bureau of Reference Sequential Extraction Tests .5. Community Bureau of Reference Sequential Extraction Tests The Community Bureau of Reference (BCR) was used to extract different fractions of Pb [21]. One gram of dried specimen of soil sample was added to a polypropylene centrifuge tube; the sequential extraction procedures are listed in Table 2. Table 2. Sequential extraction procedure for soil Pb. Fraction Reagent Shaking Time and Temperature Exchangeable (F1) 40 mL of 0.11 mol/L CH3COOH 16 h at 25 ◦C Reducible (iron/manganese oxyhydroxides) (F2) 40 mL of 0.5 mol/L NH2OH·HCl 16 h at 25 ◦C Oxidizable (organic matter and sulfides) (F3) 10 mL of 8.8 mol/L H2O2, twice, cool and add 50 mL of 1 mol/L NH4Ac 1 h at 25 ◦C, 1 h at 85 ◦C, 1 h at 85 ◦C, 16 h at 25 ◦C Residual (R) HNO3-H2O2-HF Microwave digestion 2 6 Statistical Analysis Table 2. Sequential extraction procedure for soil Pb. 3.2. Effect of Nano Hydroxyapatite on the pH of Soil The pH of soil is an important parameter th 3.2. Effect of Nano-Hydroxyapatite on the pH of Soil soil. Metal solubility and mobility decreased with the increase in pH. According to Table 3, the addition of NHAP increased soil pH by 0.02–0.13 units, compared with the control group. However, this difference was not significant. The result was consistent with the finding that the application of NHAP can increase the soil pH value [3]. NHAP was dissolved in the soil solution, releasing PO43−, and PO43− to react with the H+ in the soil-generated HPO42− and H2PO4− [28]. Soil pH decreased with the rise of Pb content in soil, but there were almost no significant differences. For the 1200 mg/kg treatment, the pH of the soil had a larger reduction. In addition, the roots of plants can also affect soil pH by secreting protons and organic acids. The contents of heavy metals in soil have impacts on the secretion of plant roots [29]. Therefore, the observed tendency in this study might be the result of factors such as plant secretions, NHAP, and Pb content. More in-depth research to determine the specific impact of each factor on the pH of soil is needed. Table 3. Effects of NHAP on rhizosphere soil pH. The different letters in the table represent significant differences between treatments at p < 0.05. Exogenous Pb The Rhizosphere Soil pH The pH of soil is an important parameter that affects metal immobilization and dissolution in soil. Metal solubility and mobility decreased with the increase in pH. According to Table 3, the addition of NHAP increased soil pH by 0.02–0.13 units, compared with the control group. However, this difference was not significant. The result was consistent with the finding that the application of NHAP can increase the soil pH value [3]. NHAP was dissolved in the soil solution, releasing PO43−, and PO43− to react with the H+ in the soil-generated HPO42−and H2PO4−[28]. Soil pH decreased with the rise of Pb content in soil, but there were almost no significant differences. For the 1200 mg/kg treatment, the pH of the soil had a larger reduction. In addition, the roots of plants can also affect soil pH by secreting protons and organic acids. The contents of heavy metals in soil have impacts on the secretion of plant roots [29]. Therefore, the observed tendency in this study might be the result of factors such as plant secretions, NHAP, and Pb content. 3.1. Speciation Analysis of Pb in Soil Exogenous Pb Concentration (mg/kg) The Rhizosphere Soil pH Without NHAP With NHAP 0 8.64 ± 0.10 a,b 8.66 ± 0.03 a 400 8.69 ± 0.04 a 8.73 ± 0.02 a 800 8.51 ± 0.20 b 8.64 ± 0.03 a,b 1200 8.12 ± 0.01 d 8.20 ± 0.01 c a, b, c, d: The different letters in the table represent significant differences between treatments at p < 0.05. and (R) residual. N: nano-hydroxyapatite. 3.2. Effect of Nano-Hydroxyapatite on the pH of Soil The pH of soil is an important parameter that affects metal immobilization and dissolution in soil. Metal solubility and mobility decreased with the increase in pH. According to Table 3, the addition of NHAP increased soil pH by 0.02–0.13 units, compared with the control group. However, this difference was not significant. The result was consistent with the finding that the application of NHAP can increase the soil pH value [3]. NHAP was dissolved in the soil solution, releasing PO43−, and PO43− to react with the H+ in the soil-generated HPO42−and H2PO4−[28]. Soil pH decreased with the rise of Pb content in soil, but there were almost no significant differences. For the 1200 mg/kg treatment, the pH of the soil had a larger reduction. In addition, the roots of plants can also affect soil pH by secreting protons and organic acids. The contents of heavy metals in soil have impacts on the secretion of plant roots [29]. Therefore, the observed tendency in this study might be the result of factors such as plant secretions, NHAP, and Pb content. More in-depth research to determine the specific impact of each factor on the pH of soil is needed. Table 3. Effects of NHAP on rhizosphere soil pH. The different letters in the table represent significant differences between treatments at p < 0.05. Exogenous Pb Concentration (mg/kg) The Rhizosphere Soil pH Without NHAP With NHAP 0 8.64 ± 0.10 a,b 8.66 ± 0.03 a 400 8.69 ± 0.04 a 8.73 ± 0.02 a 800 8.51 ± 0.20 b 8.64 ± 0.03 a,b 1200 8.12 ± 0.01 d 8.20 ± 0.01 c a, b, c, d: The different letters in the table represent significant differences between treatments at p < 0.05. 3.1. Speciation Analysis of Pb in Soil Public Health 2017, 14, 532 produced a low solubility type of lead phosphate. Nanoparticles have a very large micro-interface, with a strong surface complexation ability with respect to heavy metals, which accelerates the rate of dissolution, shortening the equilibrium time between dissolution and sedimentation. The acidity required by dissolved nanoparticles was lower than larger particles, which can reduce the probability of acidification as secondary pollution in the processes of hydroxyapatite to immobile Pb. Therefore, nano-scale materials are expected to improve the remediation effect. It was reported that the bioavailability of heavy metals in soil are linearly related to their biotoxicities [27]. The results showed that the application of NHAP could alleviate the biotoxicity of Pb and lower its mobility in soil to ensure the healthy growth and development of plants. Int. J. Environ. Res. Public Health 2017, 14, 532 5 of 9 Therefore, nano-scale materials are expected to improve the remediation effect. It was reported that the bioavailability of heavy metals in soil are linearly related to their biotoxicities [27]. The results showed that the application of NHAP could alleviate the biotoxicity of Pb and lower its mobility in soil to ensure the healthy growth and development of plants. Figure 1. Lead partitioning in Pb-spiked soil with and without NHAP application. The values of 0, 400, 800, 1200 respectively stand for the addition of Pb content (0, 400, 800, 1200 mg/kg); 0 + N, 400 + N, 800 + N, 1200 + N respectively stand for the addition of Pb content (0, 400, 800, 1200 mg/kg) and NHAP (1.5 g). The operationally defined soil fractions were: (F1) exchangeable, (F2) reducible (iron/manganese oxyhydroxides), (F3) oxidizable (organic matter and sulfides), and (R) residual. N: nano-hydroxyapatite. 3 2 Effe t of Na o Hyd o ya atite o the H of Soil Figure 1. Lead partitioning in Pb-spiked soil with and without NHAP application. The values of 0, 400, 800, 1200 respectively stand for the addition of Pb content (0, 400, 800, 1200 mg/kg); 0 + N, 400 + N, 800 + N, 1200 + N respectively stand for the addition of Pb content (0, 400, 800, 1200 mg/kg) and NHAP (1.5 g). The operationally defined soil fractions were: (F1) exchangeable, (F2) reducible (iron/manganese oxyhydroxides), (F3) oxidizable (organic matter and sulfides), and (R) residual. N: nano-hydroxyapatite. Figure 1. Lead partitioning in Pb-spiked soil with and without NHAP application. 3.1. Speciation Analysis of Pb in Soil The use of sequential extraction furnished detailed information regarding the origin, mode of occurrence, biological and physicochemical availability, mobilization, and transport of heavy metals [22]. In this paper, BCR was used to evaluate the effect of NHAP on the changes of Pb fractions in the soil. It has been reported that conventional hydroxyapatite can react with Pb to form chloropyromorphite during the sequential extraction process (especially in the non-steady amended state) [23]. However, at present, the sequential extraction method is still a universally-applied method for the determination of heavy metal fractions. We attempted to avoid this possible error in the experiments by strictly controlling the experimental conditions, and placing the results of the BCR measurements in uniform and comparable conditions. According to the degree of heavy metal bioavailablity in different metal fractions, metal species were divided into three categories: bioavailable, potentially bioavailable, and bio-unavailable. The bioavailable category included the water soluble and exchangeable fractions. The content of this heavy metal portion was small, but had excellent mobility, and was most likely to be absorbed and utilized by organisms [24]. The mobility of heavy metals was directly related to water solubility, and the high water solubility of heavy metals can result in a high leaching risk for groundwater and can threaten the health of organisms [25]. As shown in Figure 1, the amounts of Pb present in F1 and F2 were noticeably lower after NHAP was added compared with the control group, declining by 21.69–66.08% and 25–52.02%, respectively. The residual fraction of Pb increased by 124.67% compared with the control group. After adding NHAP, the Pb content of F3 increased by 6.83% on average. Any changes for F3 were not as obvious as those of the other three Pb fractions. The results showed that the application of NHAP can change the fraction of Pb from bioavailable to bio-unavailable. NHAP significantly reduced the mobility and availability of Pb in soil. The portions of the four Pb fractions extracted using BCR were almost the same as soil with different applied Pb levels. The formation of pyromorphite from Pb was the most important effect of NHAP application [26]. This led the transformation of Pb from non-residual to residual fractions by changing its dissolution–precipitation mechanism. NHAP was first dissolved in soil solution which released phosphate ions, and then phosphate ions and lead ions in the soil solution 5 of 9 Int. J. Environ. Res. 3.1. Speciation Analysis of Pb in Soil The values of 0, 400, 800, 1200 respectively stand for the addition of Pb content (0, 400, 800, 1200 mg/kg); 0 + N, 400 + N, 800 + N, 1200 + N respectively stand for the addition of Pb content (0, 400, 800, 1200 mg/kg) and NHAP (1.5 g). The operationally defined soil fractions were: (F1) exchangeable, (F2) reducible (iron/manganese oxyhydroxides), (F3) oxidizable (organic matter and sulfides), and (R) residual. N: nano-hydroxyapatite. Figure 1. Lead partitioning in Pb-spiked soil with and without NHAP application. The values of 0, 400, 800, 1200 respectively stand for the addition of Pb content (0, 400, 800, 1200 mg/kg); 0 + N, 400 + N, 800 + N, 1200 + N respectively stand for the addition of Pb content (0, 400, 800, 1200 mg/kg) and NHAP (1.5 g). The operationally defined soil fractions were: (F1) exchangeable, (F2) reducible (iron/manganese oxyhydroxides), (F3) oxidizable (organic matter and sulfides), and (R) residual. N: nano-hydroxyapatite. nano-hydroxyapatite. 3.2. Effect of Nano-Hydroxyapatite on the pH of Soil The pH of soil is an important parameter that affects metal immobilization and dissolution in soil. Metal solubility and mobility decreased with the increase in pH. According to Table 3, the addition of NHAP increased soil pH by 0.02–0.13 units, compared with the control group. However, this difference was not significant. The result was consistent with the finding that the application of NHAP can increase the soil pH value [3]. NHAP was dissolved in the soil solution, releasing PO43−, and PO43− to react with the H+ in the soil-generated HPO42− and H2PO4− [28]. Soil pH decreased with the rise of Pb content in soil, but there were almost no significant differences. For the 1200 mg/kg treatment, the pH of the soil had a larger reduction. In addition, the roots of plants can also affect soil pH by secreting protons and organic acids. The contents of heavy metals in soil have impacts on the secretion of plant roots [29]. Therefore, the observed tendency in this study might be the result of factors such as plant secretions, NHAP, and Pb content. More in-depth research to determine the specific impact of each factor on the pH of soil is needed. Table 3. Effects of NHAP on rhizosphere soil pH. The different letters in the table represent significant differences between treatments at p < 0.05. 3.4. Plant Growth and Biomass I h d 3.4. Plant Growth and Biomass In this study, ryegrass grew rapidly and healthily, with no visual symptoms of necrosis or whitish-brown chlorosis during plant growth. After the plants were harvested, the height of ryegrass and the fresh weight were measured, and the results are shown in Figure 3. The average heights of the ryegrass shoots in the control group were 16.4 cm, 23.8 cm, and 29.2 cm, and the heights of those with added NHAP were 16.6 cm, 24.4 cm, and 29.8 cm, respectively. The addition of Pb did not cause obvious toxicity to the growth of ryegrass; only a slight inhibition. The total fresh weight of ryegrass with NHAP showed a significant increasing trend compared with the control group, and there was a higher increase in root weight compared to shoot weight. The fresh weight of shoot increased by an average of 12.35%, while that of the root was 32.76%. Ryegrass has very large and dense fibrous roots which spread to the entire soil core in the pots during the experimental period. NHAP reduced the mobility and bioavailability of Pb, and alleviated the high toxicity of Pb to ryegrass. The P content in the soil was elevated after the addition of NHAP, which could promote plant growth and increase biomass. Thus, NHAP did not hinder the growth of ryegrass, but had a positive role in promoting its growth. In this study, ryegrass grew rapidly and healthily, with no visual symptoms of necrosis or whitish-brown chlorosis during plant growth. After the plants were harvested, the height of ryegrass and the fresh weight were measured, and the results are shown in Figure 3. The average heights of the ryegrass shoots in the control group were 16.4 cm, 23.8 cm, and 29.2 cm, and the heights of those with added NHAP were 16.6 cm, 24.4 cm, and 29.8 cm, respectively. The addition of Pb did not cause obvious toxicity to the growth of ryegrass; only a slight inhibition. The total fresh weight of ryegrass with NHAP showed a significant increasing trend compared with the control group, and there was a higher increase in root weight compared to shoot weight. The fresh weight of shoot increased by an average of 12.35%, while that of the root was 32.76%. Ryegrass has very large and dense fibrous roots which spread to the entire soil core in the pots during the experimental period. 3.3. Effect of Nano-Hydroxyapatite on Pb Accumulation in Ryegrass 3.3. Effect of Nano Hydroxyapatite on Pb Accumulation in Ryegrass The Pb content in shoots and roots are shown in Figure 3.3. Effect of Nano-Hydroxyapatite on Pb Accumulation in Ryegrass 3.3. Effect of Nano Hydroxyapatite on Pb Accumulation in Ryegrass The Pb content in shoots and roots are shown in Figure The Pb content in shoots and roots are shown in Figure 2. The results showed that the metal content in shoots and roots was altered by the addition of Pb to the soil, as well as the addition of NHAP. Increasing concentrations of Pb in soil led to an increase of Pb content in roots and shoots. Compared to the control group, the addition of NHAP led to an approximately 2.86–21.1% decrease in Pb concentrations in the roots, and a 13.19–20.3% decrease in the shoots. There was a significant decrease in shoots with NHAP treatments compared to the control group, while the decrease caused by NHAP was not significant in roots, except at the highest concentration of Pb contamination. Plant-available Pb was highly correlated with water-soluble Pb (r = 0.812 for shoots, p < 0.05; r = 0.870 for roots; p < 0.01). Thus, the application of NHAP decreased the Pb concentration of roots and shoots, because NHAP converted the bioavailable fractions of lead into bio-unavailable fractions. The Pb content in shoots and roots are shown in Figure 2. The results showed that the metal content in shoots and roots was altered by the addition of Pb to the soil, as well as the addition of NHAP. Increasing concentrations of Pb in soil led to an increase of Pb content in roots and shoots. Compared to the control group, the addition of NHAP led to an approximately 2.86–21.1% decrease in Pb concentrations in the roots, and a 13.19–20.3% decrease in the shoots. There was a significant decrease in shoots with NHAP treatments compared to the control group, while the decrease caused by NHAP was not significant in roots, except at the highest concentration of Pb contamination. Plant- available Pb was highly correlated with water-soluble Pb (r = 0.812 for shoots, p < 0.05; r = 0.870 for roots; p < 0.01). Thus, the application of NHAP decreased the Pb concentration of roots and shoots, because NHAP converted the bioavailable fractions of lead into bio-unavailable fractions. Figure 2. Effects of NHAP on Pb contents in shoots (A) and roots (B). a–h: The different letters in the figure represent significant differences between treatments at p < 0.05. Figure 2. 3.3. Effect of Nano-Hydroxyapatite on Pb Accumulation in Ryegrass 3.3. Effect of Nano Hydroxyapatite on Pb Accumulation in Ryegrass The Pb content in shoots and roots are shown in Figure Effects of NHAP on Pb contents in shoots (A) and roots (B). a–h: The different letters in the figure represent significant differences between treatments at p < 0.05. Figure 2. Effects of NHAP on Pb contents in shoots (A) and roots (B). a–h: The different letters in the figure represent significant differences between treatments at p < 0.05. Figure 2. Effects of NHAP on Pb contents in shoots (A) and roots (B). a–h: The different letters in the figure represent significant differences between treatments at p < 0.05. 3.2. Effect of Nano Hydroxyapatite on the pH of Soil The pH of soil is an important parameter th 3.2. Effect of Nano-Hydroxyapatite on the pH of Soil More in-depth research to determine the specific impact of each factor on the pH of soil is needed. Concentration (mg/kg) Without NHAP With NHAP 0 8.64 ± 0.10 a,b 8.66 ± 0.03 a 400 8.69 ± 0.04 a 8.73 ± 0.02 a 800 8.51 ± 0.20 b 8.64 ± 0.03 a,b 1200 8.12 ± 0.01 d 8.20 ± 0.01 c a, b, c, d: The different letters in the table represent significant differences between treatments at p < 0.05. Table 3. Effects of NHAP on rhizosphere soil pH. The different letters in the table represent significant differences between treatments at p < 0.05. Exogenous Pb Concentration (mg/kg) The Rhizosphere Soil pH Without NHAP With NHAP 0 8.64 ± 0.10 a,b 8.66 ± 0.03 a 400 8.69 ± 0.04 a 8.73 ± 0.02 a 800 8.51 ± 0.20 b 8.64 ± 0.03 a,b 1200 8.12 ± 0.01 d 8.20 ± 0.01 c a, b, c, d: The different letters in the table represent significant differences between treatments at p < 0.05. ( g g) 0 8.64 ± 0.10 a,b 8.66 ± 0.03 a 400 8.69 ± 0.04 a 8.73 ± 0.02 a 800 8 51 ± 0 20 b 8 64 ± 0 03 a b Table 3. Effects of NHAP on rhizosphere soil pH. The different letters in the table represent significant differences between treatments at p < 0.05. Int. J. Environ. Res. Public Health 2017, 14, 532 Int. J. Environ. Res. Public Health 2017, 14, 532 6 of 9 6 of 9 3.5. Organic Acid Response to Nano-Hydroxyapatitein the Ryegrass Rhizosphere 3.5. Organic Acid Response to Nano-Hydroxyapatitein the Ryegrass Rhizosphere Organic acids are widely present in plants and in the rhizosphere Organic acids are widely present in plants and in the rhizosphere environment [30]. Under environmental stresses, such as heavy metals, organic acids secreted by plants were found to be significantly increased [31]. In this study, the low molecular weight organic acids in rhizosphere soil were measured after harvest at day 30. Tartaric acid was detected in perennial ryegrass rhizosphere soil, while other organic acids were below the limits of detection. As shown in Figure 4, tartaric acid content increased significantly along with the increase in Pb content in the soil. The tartaric acid contents were significantly positively correlated to the soil Pb contents in all treatments. This showed that secretion of tartaric acid by ryegrass was sensitive to Pb stress. The application of NHAP caused an increase in tartaric acid content by an average of 98.82% compared with the treatments without the addition of NHAP. The probable cause for this was that NHAP administration promoted the growth of ryegrass. It has been reported that plant root secretion of organic acids can improve the mobility and bioavailability of heavy metals in soil [32,33]. The increase of tartaric acid content also increased the likelihood that ryegrass absorbed Pb from rhizosphere soil. The objective of NHAP application was to lower the bioavailability of Pb, which seemed to be inconsistent with the role of tartaric acid. It has been reported that low molecular weight organic acids, including acetic acid, malic acid, citric acid, and oxalic acid, promoted the adsorption of Pb2+ on the surface of NHAP [34]. Therefore, the increase in tartaric acid could be considered as a beneficial aspect of NHAP for plant growth and reducing the bioavailabiliy of Pb. Tartaric acid had no inhibition effect on the remediation results of NHAP Organic acids are widely present in plants and in the rhizosphere environment [30]. Under environmental stresses, such as heavy metals, organic acids secreted by plants were found to be significantly increased [31]. In this study, the low molecular weight organic acids in rhizosphere soil were measured after harvest at day 30. Tartaric acid was detected in perennial ryegrass rhizosphere soil, while other organic acids were below the limits of detection. As shown in Figure 4, tartaric acid content increased significantly along with the increase in Pb content in the soil. The tartaric acid contents were significantly positively correlated to the soil Pb contents in all treatments. 3.5. Organic Acid Response to Nano-Hydroxyapatitein the Ryegrass Rhizosphere 3.5. Organic Acid Response to Nano-Hydroxyapatitein the Ryegrass Rhizosphere Organic acids are widely present in plants and in the rhizosphere This showed that secretion of tartaric acid by ryegrass was sensitive to Pb stress. The application of NHAP caused an increase in tartaric acid content by an average of 98.82% compared with the treatments without the addition of NHAP. The probable cause for this was that NHAP administration promoted the growth of ryegrass. It has been reported that plant root secretion of organic acids can improve the mobility and bioavailability of heavy metals in soil [32,33]. The increase of tartaric acid content also increased the likelihood that ryegrass absorbed Pb from rhizosphere soil. The objective of NHAP application was to lower the bioavailability of Pb, which seemed to be inconsistent with the role of tartaric acid. It has been reported that low molecular weight organic acids, including acetic acid, malic acid, citric acid, and oxalic acid, promoted the adsorption of Pb2+ on the surface of NHAP [34]. Therefore, the increase in tartaric acid could be considered as a beneficial aspect of NHAP for plant growth and reducing the bioavailabiliy of Pb. Tartaric acid had no inhibition effect on the remediation results of NHAP. environmental stresses, such as heavy metals, organic acids secreted by plants were found to be significantly increased [31]. In this study, the low molecular weight organic acids in rhizosphere soil were measured after harvest at day 30. Tartaric acid was detected in perennial ryegrass rhizosphere soil, while other organic acids were below the limits of detection. As shown in Figure 4, tartaric acid content increased significantly along with the increase in Pb content in the soil. The tartaric acid contents were significantly positively correlated to the soil Pb contents in all treatments. This showed that secretion of tartaric acid by ryegrass was sensitive to Pb stress. The application of NHAP caused an increase in tartaric acid content by an average of 98.82% compared with the treatments without the addition of NHAP. The probable cause for this was that NHAP administration promoted the growth of ryegrass. It has been reported that plant root secretion of organic acids can improve the mobility and bioavailability of heavy metals in soil [32,33]. The increase of tartaric acid content also increased the likelihood that ryegrass absorbed Pb from rhizosphere soil. The objective of NHAP application was to lower the bioavailability of Pb, which seemed to be inconsistent with the role of tartaric acid. 3.4. Plant Growth and Biomass I h d 3.4. Plant Growth and Biomass NHAP reduced the mobility and bioavailability of Pb, and alleviated the high toxicity of Pb to ryegrass. The P content in the soil was elevated after the addition of NHAP, which could promote plant growth and increase biomass. Thus, NHAP did not hinder the growth of ryegrass, but had a positive role in promoting its growth. 7 of 9 7 of 9 o Int. J. Environ. Res. Public Health 2017, 14, 532 Int. J. Environ. Res. Public Health 2017, 14, 532 J , , Figure 3. Effect of NHAP in different treatments on ryegrass shoot (A) and root (B) biomass. a, b, c, d: The different letters in the table represent significant differences between treatments at p < 0.05. Figure 3. Effect of NHAP in different treatments on ryegrass shoot (A) and root (B) biomass. a, b, c, d: The different letters in the table represent significant differences between treatments at p < 0.05. Figure 3. Effect of NHAP in different treatments on ryegrass shoot (A) and root (B) biomass. a, b, c, d: The different letters in the table represent significant differences between treatments at p < 0.05. 3.5. Organic Acid Response to Nano-Hydroxyapatitein the Ryegrass Rhizosphere Figure 3 Effect of NHAP in different treatm on ryegrass shoot (A) and root (B) biomass. a, b, c, Figure 3. Effect of NHAP in different treatments on ryegrass shoot (A) and root (B) biomass. a, b, c, d: The different letters in the table represent significant differences between treatments at p < 0.05. Figure 3. Effect of NHAP in different treatments on ryegrass shoot (A) and root (B) biomass. a, b, c, d: The different letters in the table represent significant differences between treatments at p < 0.05. d: The different letters in the table represent significant differences between treatments at p < 0.05. 5 Organic Acid Response to Nano Hydroxyapatitein the Ryegrass Rhizosphere 3.5. Organic Acid Response to Nano-Hydroxyapatitein the Ryegrass Rhizosphere 3.5. Organic Acid Response to Nano-Hydroxyapatitein the Ryegrass Rhizosphere Organic acids are widely present in plants and in the rhizosphere It has been reported that low molecular weight organic acids, including acetic acid, malic acid, citric acid, and oxalic acid, promoted the adsorption of Pb2+ on the surface of NHAP [34] Therefore, the increase in tartaric acid could be considered as a beneficial aspect of NHAP for plant growth and reducing the bioavailabiliy of Pb. Tartaric acid had no inhibition effect on the remediation results of NHAP. Figure 4. Tartaric acid contents in ryegrass rhizosphere soil under different treatments. a–f: The different letters in the table represent significant differences between treatments at p < 0 05 Figure 4. Tartaric acid contents in ryegrass rhizosphere soil under different treatments. a–f: The different letters in the table represent significant differences between treatments at p < 0.05. Figure 4. Tartaric acid contents in ryegrass rhizosphere soil under different treatments. a–f: The different letters in the table represent significant differences between treatments at p < 0.05. Figure 4. Tartaric acid contents in ryegrass rhizosphere soil under different treatments. a–f: The Figure 4. Tartaric acid contents in ryegrass rhizosphere soil under different treatments. a–f: The different letters in the table represent significant differences between treatments at p < 0.05. Figure 4. Tartaric acid contents in ryegrass rhizosphere soil under different treatments. a–f: The different letters in the table represent significant differences between treatments at p < 0.05. Int. J. Environ. Res. Public Health 2017, 14, 532 8 of 9 4. Conclusions This study illustrated that NHAP could significantly reduce the mobility and bioavailability of Pb. The addition of NHAP effectively reduced the exchangeable and reducible fractions of Pb in soil, and transformed them into oxidizable and residual Pb, limiting its mobility and bioavailability. NHAP could play a very large role in controlling and mitigating the dangers of Pb pollution for organisms and the environment. The Pb contents of shoots and roots decreased and soil pH did not change significantly with the addition of NHAP; moreover, NHAP promoted the growth of ryegrass and the secretion of tartaric acid. This also indicated that the application of NHAP was beneficial to the growth of plants, and did not have negative impacts on the environment. The results in this study showed that NHAP could immobilize Pb in contaminated soil effectively, and can benefit the growth of ryegrass. Acknowledgments: This study was supported by the Natural Science Foundation of Hebei Province (B2014201175) and the Hebei Provincial Project of International Cooperation in Science and Technology (14394204D). Author Contributions: Shu-xuan Liang and Wei Liu conceived and designed the experiments; Ling Ding and Qingqing Zuo performed the experiments; Ling Ding analyzed the data; Ling Ding, Shu-xuan Liang and Jianbing Li wrote the paper. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Shrivastava, R.; Upreti, R.K.; Chaturvedi, U.C. Various cells of the immune system and intestine differ in their capacity to reduce hexavalent chromium. FEMS Immunol. Med. Microbiol. 2003, 38, 65–70. [CrossRef] 1. Shrivastava, R.; Upreti, R.K.; Chaturvedi, U.C. 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A novel model of urosepsis in rats developed by injection of Escherichia coli into the renal pelvis
Frontiers in immunology
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OPEN ACCESS OPEN ACCESS EDITED BY Alessandra Stasi, University of Bari Aldo Moro, Italy REVIEWED BY Vladimir M. Pisarev, Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitation, Russia Ming-Jun Shi, Department of Urology, Capital Medical University, China *CORRESPONDENCE Tongzu Liu liutongzu@163.com Xiaojie Bai 13476839931@163.com †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Inflammation, a section of the journal Frontiers in Immunology RECEIVED 19 October 2022 ACCEPTED 15 December 2022 PUBLISHED 05 January 2023 CITATION Cao Y, Bai C, Si P, Yan X, Zhang P, Yisha Z, Lu P, Tuoheti K, Guo L, Chen Z, Bai X and Liu T (2023) A novel model of urosepsis in rats developed by injection of Escherichia coli into the renal pelvis. Front. Immunol. 13:1074488. doi: 10.3389/fimmu.2022.1074488 Yuanfei Cao 1†, Can Bai 1†, Penghui Si 1†, Xin Yan 1, Peng Zhang 2, Zuhaer Yisha 1†, Peixiang Lu 1, Kuerban Tuoheti 1, Linfa Guo 1, Zhao Chen 1, Xiaojie Bai 1* and Tongzu Liu 1* Yuanfei Cao 1†, Can Bai 1†, Penghui Si 1†, Xin Yan 1, Peng Zhang 2, Zuhaer Yisha 1†, Peixiang Lu 1, Kuerban Tuoheti 1, Linfa Guo 1, Zhao Chen 1, Xiaojie Bai 1* and Tongzu Liu 1* 1Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan, China, 2Institute of Hepatobiliary Diseases , Zhongnan Hospital of Wuhan University, Wuhan, China Despite extensive research, urosepsis remains a life-threatening, high- mortality disease. Currently, animal models of urosepsis widely accepted by investigators are very scarce. This study aimed to establish a standardized and reproducible model of urosepsis in rats. Forty adult Wistar rats were randomly divided into four groups according to the concentration of injected E. coli suspensions: Sham, Sep 3×, Sep 6×, and Sep 12×. Because the ureter is so thin and fragile, no conventional needle can be inserted into the ureter, which is probably why rats are rarely used to develop models of urosepsis. To solve this problem, the left ureter was ligated in the first procedure. After 24 hours, the left ureter above the ligation was significantly dilated, then saline or different concentrations of E. coli at 3 ml/kg were injected into the left renal pelvis using a 30G needle. The left ureter was subsequently ligated again at a distance of 1 cm from the renal hilum to maintain high pressure in the renal pelvis. Following injection of E. TYPE Original Research PUBLISHED 05 January 2023 DOI 10.3389/fimmu.2022.1074488 TYPE Original Research PUBLISHED 05 January 2023 DOI 10.3389/fimmu.2022.1074488 urosepsis, rats, animal model, Escherichia coli, upper urinary tract obstruction, pathophysiology COPYRIGHT © 2023 Cao, Bai, Si, Yan, Zhang, Yisha, Lu, Tuoheti, Guo, Chen, Bai and Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original authors(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. KEYWORDS urosepsis, rats, animal model, Escherichia coli, upper urinary tract obstruction, pathophysiology Experimental procedures Forty adult Wistar rats (250-300g) were randomly divided into four groups according to the concentration of injected E. coli suspensions: Sham (injected with saline), Sep 3× (injected with 3×108 cfu/mL E. coli), Sep 6× (injected with 6×108 cfu/mL E. coli), Sep 12× (injected with 12×108 cfu/mL E. coli). Before the experiment, Wistar rats were fasted overnight but allowed to drink freely. All rats were anesthetized with intraperitoneally injected 30 mg/kg of 1% sodium pentobarbital. After anesthesia, the abdomen of the rats was shaved, and a 3 cm-long incision was performed on the left side of the abdomen. The abdominal cavity of the rats was opened to expose the left kidney, and the left ureter was carefully isolated. At a distance of 2 cm from the left renal hilus, we ligated the left ureter using 4-0 silk, placing the left kidney and intestine back into the abdominal cavity, closing the abdominal cavity, and suturing the skin. 24 h later, the rats were reanesthetized, and the abdominal cavity was reopened, showing that the left renal pelvis and left ureter were significantly dilated compared with those before ligation. Groups Sep3×, 6×, and 12× were injected with 3 ml/kg E. coli solution in the left ureter above the ligation at a concentration of 3×108, 6×108, and 12×108 cfu/ml, respectively. Saline was also injected into the left ureter of the sham group at 3 ml/kg. Subsequently, the left ureter was ligated again at a distance of 1 cm from the renal hilum to maintain a state of pelvic hypertension. The rats were then sutured and received postoperative analgesic meloxicam (1 mg/kg, s.c.). Injection of endotoxin or bacteria, cecum ligation and puncture (CLP), and colonic ascending stent peritonitis (CASP) are the commonly used models of sepsis (7–9). Among these, the rodent cecum ligation and puncture (CLP) model of experimental sepsis has grown to be the most popular and is currently regarded as the gold standard for sepsis research (10–12). However, widely applied and standardized animal models of urosepsis are relatively rare. Some scholars have found that rabbits can be used to develop models of urosepsis by injecting E. coli into the renal pelvis (13–16). Rodents, the most widely used for experimental research, are rarely used to make models of urosepsis. Therefore, the establishment of a standardized rat model of urosepsis may rapidly advance the study of the pathophysiological mechanisms of urosepsis. OPEN ACCESS coli or saline for 24 h, three rats from each group were sacrificed and their organs (lung, liver, and right kidney) were collected. In contrast, the remaining seven rats continued to be observed for survival. At 10 days after E. coli injection, rats in the sep12× group had a higher mortality rate (100%) compared to the sep3× group (28.6%) or the sep6× group (71.4%). The significant changes in peripheral blood WBC count, serum IL-6 and TNF-a levels were also in the sep12× group. In addition, rats in the sepsis group showed multi-organ dysfunction, including damage to the lungs, liver, and kidneys. The establishment of a standardized rat model of urosepsis may be of great value for studying the pathophysiological of urosepsis. CITATION Cao Y, Bai C, Si P, Yan X, Zhang P, Yisha Z, Lu P, Tuoheti K, Guo L, Chen Z, Bai X and Liu T (2023) A novel model of urosepsis in rats developed by injection of Escherichia coli into the renal pelvis. Front. Immunol. 13:1074488. doi: 10.3389/fimmu.2022.1074488 COPYRIGHT © 2023 Cao, Bai, Si, Yan, Zhang, Yisha, Lu, Tuoheti, Guo, Chen, Bai and Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original authors(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. frontiersin.org Frontiers in Immunology 01 Cao et al. 10.3389/fimmu.2022.1074488 Experimental procedures Blood samples were collected at four postoperative time points (0h, 2h, 24h, and 48h). A portion of the fresh samples was utilized for routine blood testing (WBC count). Additional blood samples were processed at 3000 rpm for 20 minutes using a cryogenic centrifuge, and the supernatant was stored in a -80°C refrigerator for TNF-a and IL-6 assay. In this study, we attempted to utilize rats to produce a standardized and reproducible model of urosepsis by injecting E. coli into the renal pelvis. We evaluated the effectiveness of a rat model of urosepsis by observing survival rates and blood cultures, detecting changes in WBC and inflammatory factors, and verifying multi-organ damage to the lungs, liver, and kidneys. The establishment of a standardized rat model of urosepsis may be of great value for studying the pathophysiological of urosepsis. Introduction (45-55%) with a 12-hour light/dark cycle for 7 days to acclimate to the environment. Sepsis, a life-threatening organ dysfunction with rapid progression and high mortality (17-26%), is the leading cause of death in critically ill patients worldwide (1–3). Depending on the site of infection, infections originating in the urinary tract and/or male genital tract are referred to as urosepsis (4, 5). It is estimated that approximately 20-30% of all sepsis cases are urosepsis. In total, there are an estimated 31.5 million sepsis cases each year, representing a potential of up to 9.45 million cases of urosepsis (6). Therefore, sepsis and urosepsis have been recognized as very concerning problems by many hospitals and are made a global health priority by the World Health Organization. Despite many new research results in recent years, the pathophysiology of sepsis is still incompletely understood. Several animal models have been created that all seek to mimic the typical pathophysiological changes in septic patients to study the pathophysiological causes of sepsis. Materials and methods Escherichia coli (E. coli) (ATCC 25922) was purchased from Guangdong Huankai Microbial Technology Co. and cultured on McConkey Agar (Solarbio Life Science Technology Co., Beijing, China) for 24h at 37°C to form individual colonies. Afterward, a single colony of bacteria was picked and inoculated in LB medium at 37°C with shaking at 200 rpm for 18-24h. The bacteria medium was precipitated by centrifugation at 2000g for 10 min and then resuspended in saline to a concentration of 3×108,6×108,12×108 cfu/mL. To ensure accurate concentration, bacterial suspensions were tested using a bacterial turbidimeter (Thermo Fisher Scientific, USA). Results Under the manufacturer agreement, total RNA was extracted from bladder cancer cells using RaPure Total RNA Micro Kit (Magen, China). The RNA NanoPhotometer spectrophotometer (IMPLEN, Westlake Village, CA, USA) quantified the RNA at 260 nm/280 nm. Following the package recommendations, 2 mg of total RNA was reverse transcribed to cDNA utilizing ABScript II RT Master Mix (ABclonal, Wuhan, China). Using a Bio-Rad (Hercules, CA, USA) CFX96 system, qRT-PCR was used to ascertain the mRNA level of an interesting gene predicated upon SYBR green. The primer sequence is shown in Table S1. Each target gene’s relative mRNA expression level was estimated using the 2−DDCT method in conjunction with ACTB as an internal loading control. Immunohistochemical (IHC) Formalin-fixed, paraffin-embedded tissue sections were first deparaffinized. And then, endogenous peroxidase activity was inhibited using H2O2. The indicated primary antibody (Table S2) and secondary antibody (Table S3) were added to the sections according to the suggested methods offered by the manufacturer. All the slides were examined under an inverted microscope at 200× magnification. Measurement of WBC, Cytokines(IL-6, TNF-a), Serum CRE, BUN, AST, and ALT Statistical analysis All experimental data were represented as the means ± standard error. Student t-tests or one-way ANOVA were employed to assess the statistical analyses, with P < 0.05 regarded as statistically significant. Surgical methods and critical points of the rat urosepsis model To better investigate the mechanism of urosepsis, we tried to establish an animal model of urosepsis in rats. Briefly, we first ligated the left ureter, injected E. coli suspension into the dilated ureter after 24 h, and then ligated the left ureter again at 1 cm from the renal hilum to prevent the flow of E. coli out of the renal pelvis. Three rats in each group were killed at 24 h postoperatively, and the organs were collected, while the remaining seven rats continued to be observed for survival (Figure 1A). We found that the ureters of rats were very slim, not even more than 1 mm in diameter (Figure 1B), which made it extremely difficult to establish a model of urosepsis. After 24 h of ureteral ligation, the original slender ureter became significantly dilated, and its diameter could reach 3-4 mm (Figure 1C). We could easily inject E. coli into the dilated ureter using a syringe with a 30G needle (Figure 1D). Measurement of WBC, Cytokines(IL-6, TNF-a), Serum CRE, BUN, AST, and ALT White blood cell (WBC) counts were determined using an automatic hematology analyzer (Nihon Kohden, Japan). Rat serum concentrations of interleukin IL-6 and tumor necrosis factor-alpha (TNF-a) were determined by the Ellisa enzyme immunoassay kit (Wuhan antigene Biotechnology Co., Ltd. Wuhan, China) according to the manufacturer’s protocol. Determination of markers of kidney function by BUN and CRE kits and changes in liver function by AST and ALT kits according to the manufacturer’s instructions (Nanjing Jiancheng Institute of Biotechnology, Nanjing, China) Animal Adult Wistar rats of either sex (weight 250–300 g) were purchased from Beijing Speford Biotechnology Co. (Beijing, China). All experiments were performed at the Animal Research Institute of Zhongnan Hospital at Wuhan University. The animal study was evaluated and approved by the ethics committee of the Zhongnan Hospital at Wuhan University. Rats were raised at controlled temperature (21-25°C) and humidity frontiersin.org Frontiers in Immunology 02 Cao et al. 10.3389/fimmu.2022.1074488 After injection of E. coli for 24h, blood samples were collected and inoculated onto MacConkey agar for 24h at 37°C. If pink colonies were found, E. coli in the blood was proven. Louis, MO). 40 mg total protein was separated by 10–12.5% SDS- PAGE electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, cat# IPVH00010). After blocking with 5% skim milk at room temperature for 2 h, the membrane was first treated with the primary antibody (Table S2) at 4°C for an overnight period, followed by incubation with the secondary antibody—goat anti-rabbit IgG (Table S3)—at room temperature for an additional two hours. The bands on the membrane were monitored on a Tanon-5200 ECL imager (Tanon, Shanghai, China) and visualized by an enhanced chemiluminescence kit (Thermo Scientifisher, Waltham, MA, USA). Louis, MO). 40 mg total protein was separated by 10–12.5% SDS- PAGE electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, cat# IPVH00010). After blocking with 5% skim milk at room temperature for 2 h, the membrane was first treated with the primary antibody (Table S2) at 4°C for an overnight period, followed by incubation with the secondary antibody—goat anti-rabbit IgG (Table S3)—at room temperature for an additional two hours. The bands on the membrane were monitored on a Tanon-5200 ECL imager (Tanon, Shanghai, China) and visualized by an enhanced chemiluminescence kit (Thermo Scientifisher, Waltham, MA, USA). Clinical observations After intrarenal pelvis injection of E. coli, rats were examined for general responses such as consciousness, activity, weakness, and mortality. Every 4-6 hours, rectal body temperature was measured (Thermometer type T15SGF; Panasonic, Japan), as well as respiratory rate, heart rate, and body weight. Rats were euthanized for humanitarian reasons when they reached a behavior score of 1, where 1 represented Moribund [adapted from Yang et (17)]. Western blotting analysis Cells were lysed sufficiently in RIPA that contained 1% protease inhibitor and 1% PMSF (all from Sigma-Aldrich, St. frontiersin.org Frontiers in Immunology 03 Cao et al. Cao et al. 10.3389/fimmu.2022.1074488 A B C D FIGURE 1 The experimental procedure and critical steps in the process of urinary sepsis model in rats.(A) Schematic diagram of the experimental procedure. (B) The ureter prior to ligation. (C) Dilated ureter after ligation for 24h. (D) 30G syringe needle. A B D D B C C FIGURE 1 The experimental procedure and critical steps in the process of urinary sepsis model in rats.(A) Schematic diagram of the experimen procedure. (B) The ureter prior to ligation. (C) Dilated ureter after ligation for 24h. (D) 30G syringe needle. Characterization of urosepsis in rats. showed that blood cultures were positive in the three sepsis groups while negative in the sham group. In addition, we collected blood samples at four-time points (0h, 2h, 24h, and 48h postoperatively) for WBC counts. And the data performed statistically significant changes in WBC of all rats 2h after renal pelvis injection of E. coli solution compared to preoperative values except for the control group. These changes were most We detected that greater concentrations of E. coli led to higher mortality in rats at 10 days, with 28.6% mortality in the sep 3× group, 71.4% mortality in the sep 6× group, and 100% mortality in the sep 12× group (Figure 2A). After 24 h of E. coli or saline injection, all rats were tested by blood culture. Figure 2B A B FIGURE 2 Survival analysis and Blood cultures after ureter ligation and inoculation of E coli. (A) Survival analysis after ureter ligation and inoculation of E coli. (B) Blood specimens were incubated on MacConkey agar at 37°C after 24h inoculation with E coli. A analysis and Blood cultures after ureter ligation and inoculation of E coli. (A) Survival analysis after ureter ligation and inoculation of E Blood specimens were incubated on MacConkey agar at 37°C after 24h inoculation with E coli. frontiersin.org Frontiers in Immunology 04 10.3389/fimmu.2022.1074488 Cao et al. evident in the sep12× group with a mean ± SD WBC of 1.24 ± 0.47× 109/L (Figure 3A). Similarly, the changes in IL-6 and TNF-a levels were most remarkable in the sep12× group with mean± SD values of 114.78 ± 7.18 pg/mL and 531.46± 61.99 ng/ L, respectively. It is important to note that the increase in IL6 and TNF-a concentrations occurred at 24h postoperatively instead of 2h (Figures 3B, C). levels of IL-6 and TNF-a were significantlyhigher than those of the sham group (P<0.01). Immunohistochemistry and Western blotting analysis of liver tissue also revealed that the levels of IL-6 and TNF-a protein expression were considerably greater in the sepsis group (Figures 5A–G). Lung injury in the rat model of urosepsis We performed HE staining of the right kidney tissue and discovered that renal tissue sections from the sepsis group had apparent features of renal injury, such as vacuolar degeneration of renaltubularepithelialcells,separationofrenaltubularepithelialcells, and inflammatory cell infiltration (Figure 6A). Using measurements ofserumbiochemicalparametersinrats,wedemonstratedthatserum Cre and BUN were significantly increased in the sepsis group 24 h afterE.coliinjection (Figures6B,C).The mostsignificant elevation of serum Cr and BUN was examined in the sep 12× group compared to theshamgroup.Inaddition,RT-PCRanalysisofratlivers(Figure6D) revealed that the relative mRNA levels of IL-6 and TNF-a were considerably greater than those in the sham group. Finally, we identified that IL-6 and TNF-a protein expression levels were significantly elevated in the sepsis group by immunohistochemistry and Western blotting analysis of renal tissues compared to the sham group (Figures 6A, 6E–G). The sep 6× group was selected as a representative of the sepsis groups compared with the sham group. HE staining examination (Figure 4A) showed that the alveolar wall was widened, and the alveolar lumen collapsed in most areas due to edema in the sepsis group. At the same time, the alveolar wall and alveolar lumen of the sepsis group also had a large number of inflammatory cell infiltrates and erythrocyte exudates. Immunohistochemical (IHC) analysis of paraffin-embedded lungs illustrated that the levels of IL-6 and TNF-a were considerably greater in the sepsis group than in the sham group (Figure 4A). As shown in Figure 4B, there was a statistically significant difference in the relative mRNA levels of IL-6 and TNF-a in the lung tissue of the sepsis group vs the sham group (P<0.05). Furthermore, compared with the sham group, Western blotting analysis of the collected lung tissues revealed that the expression levels of IL-6 and TNF-a proteins were increased in the model sepsis group (Figures 4C–E). Liver injury in the rat model of urosepsis (A) Representative images of hematoxylin and eosin (H&E) staining of lung tissue from the sham and sepsis groups, immunohistochemical analysis of IL-6 and TNF-a in lung tissue. (B) The relative mRNA levels of IL-6 and TNF-a in the lung tissue. (C) Expressions of IL-6 and TNF-a in lung tissue were tested by western blot, and b-actin was used as a loading control. (D, E) Quantitative data of the levels of IL-6 and TNF-a. The sep 6× group was selected as a representative of the sepsis groups. Values were shown as mean ± SD. *P < 0.05, vs sham group. 22), which confirmed our hypothesis. After several attempts, we finally chose to inject 3ml/kg of E. coli into the renal pelvis. Autopsy of the dead rats revealed that the left kidney was enlarged while renal parenchyma was thinned, and the renal capsule was intact without rupture, indicating that the injection dose of 3 ml/kg was safe. Therefore, we believe that maintaining renal pelvic hypertension is necessary for this urosepsis model. pelvic pressure leading to bacterial entry into the bloodstream is also a factor contributing to the development of urosepsis (19). Nguyen et al. (20) demonstrated that when the renal pelvic pressure exceeds 40 cm H2O, the urine can reflux into the bloodstream carrying bacteria and metabolic waste products. Wu et al. (13) discovered that E. coli was injected into the renal pelvis at 2 ml/kg to maintain intrapelvic hypertension, leading to the development of a New Zealand rabbit model of urosepsis. In a preliminary pre-experiment, we observed low mortality in rats when 2 ml/kg of E. coli was injected into the renal pelvis. We hypothesized that intrapelvic injection of a small number of bacteria leads to a pyelonephritis model rather than a urosepsis model because of insufficient pressure in the renal pelvis. Gupta K et al. established pyelonephritis models by injecting small amounts of bacteria into the renal pelvis of rats or rabbits (21, CLP is similar to the clinical development of human sepsis, which is why it has been called the gold standard for sepsis research (23). The model features a pathogen from the host interior and mimics the pathogenesis of peritonitis (24). However, the severity of sepsis is not well standardized due to the difficulty of quantifying surgical operations, such as the percentage of cecum ligation and the dose of bacteria that enter the peritoneum after the puncture (25). Liver injury in the rat model of urosepsis This research depicts a standardized rat model of urosepsis with ligation of one ureter and consequent injection of Escherichia coli. The experimental results demonstrate our success in developing a standardized model of urosepsis in rats and confirm a significant correlation between the severity of urosepsis and the concentration of inoculated E. coli. Histopathological examination proved that liver sections from sepsis rats had features of liver injury, such as inflammatory infiltration, disorganized cell arrangement, vacuolated necrosis, and ductal hyperplasia(Figure 5A). And serum levels of ALT and AST were elevated (compared to sham surgery) due to sepsis- induced liver injury (Figures 5B, C). In addition, we also performed a reverse transcription-polymerase chain reaction (RT-PCR) analysis on the livers of rats (Figure 5D). The relative mRNA The incidence of urosepsis is increasing approximately 8.7% per year, which is closely linked to the widespread availability of upper urinary tract endoscopic procedures (18). In addition to stone co-infection and prolonged surgery, high intraoperative A B C FIGURE 3 Changes in WBC (A), serum IL-6 (B), and serum TNF-a (C) in rats at different E coli concentrations and intervals. Values were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P <0.001, and ****P < 0.0001 vs sham group. B FIGURE 3 Changes in WBC (A), serum IL-6 (B), and serum TNF-a (C) in rats at different E coli concentrations and intervals. Values were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P <0.001, and ****P < 0.0001 vs sham group. 05 Frontiers in Immunology frontiersin.org Cao et al. 10.3389/fimmu.2022.1074488 A B D C E FIGURE 4 Lung injury in the rat model of urinary sepsis. (A) Representative images of hematoxylin and eosin (H&E) staining of lung tissue from the sham and sepsis groups, immunohistochemical analysis of IL-6 and TNF-a in lung tissue. (B) The relative mRNA levels of IL-6 and TNF-a in the lung tissue. (C) Expressions of IL-6 and TNF-a in lung tissue were tested by western blot, and b-actin was used as a loading control. (D, E) Quantitative data of the levels of IL-6 and TNF-a. The sep 6× group was selected as a representative of the sepsis groups. Values were shown as mean ± SD. *P < 0.05, vs sham group. A C B B C D E E D FIGURE 4 Lung injury in the rat model of urinary sepsis. Liver injury in the rat model of urosepsis Therefore, Rittirsch et al. renormalized the details of the CLP Frontiers in Immunology frontiersin.org 06 Cao et al. 10.3389/fimmu.2022.1074488 A B C D E F G FIGURE 5 Liver injury in the rat model of urinary sepsis (A) Representative images of hematoxylin and eosin (H&E) staining of liver tissue from the sham and sepsis groups, immunohistochemical analysis of IL-6 and TNF-a in liver tissue (magnification, 200×; bar). (B, C) Serum AST and ALT levels in rats 24 hours after E. coli or saline inoculation of the renal pelvis. (D) The relative mRNA levels of IL-6 and TNF-a in the lung tissue. (E) Expressions of IL-6 and TNF-a in liver tissue were tested by western blot, and b-actin was used as a loading control. (F, G) Quantitative data of the levels of IL-6 and TNF-a. The sep 6× group was selected as a representative of the sepsis groups. Values were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P <0.001, and ****P < 0.0001 vs sham group. "ns" means "not signifcant". A A B C D B C C D D B C E F G E F F G FIGURE 5 Liver injury in the rat model of urinary sepsis (A) Representative images of hematoxylin and eosin (H&E) staining of liver tissue from the sham and sepsis groups, immunohistochemical analysis of IL-6 and TNF-a in liver tissue (magnification, 200×; bar). (B, C) Serum AST and ALT levels in rats 24 hours after E. coli or saline inoculation of the renal pelvis. (D) The relative mRNA levels of IL-6 and TNF-a in the lung tissue. (E) Expressions of IL-6 and TNF-a in liver tissue were tested by western blot, and b-actin was used as a loading control. (F, G) Quantitative data of the levels of IL-6 and TNF-a. The sep 6× group was selected as a representative of the sepsis groups. Values were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P <0.001, and ****P < 0.0001 vs sham group. "ns" means "not signifcant". and mortality predictions (32). As the concentration of inoculated E. coli increased, we observed a significant increase in serum IL-6 and TNF-a levels, indicating that adjusting the concentration of E. coli could control the severity of sepsis in our study. techniquetocontroltheseverityofsepsisbythelengthoftheligated cecum and the size and/or the number of punctures (26). Liver injury in the rat model of urosepsis In our study, we obtained a similar mortality rate using ligation of the ureter 1 cm from the renal hilum to standardize the surgical procedure. Compared to the CLP technique of regulating the ligation site of the cecum, it is undoubtedly much simpler to adjust different concentrations of E. coli to achieve control of sepsis severity in our experiments. In contrast, peripheral blood WBC counts were significantly decreased in the Sep 12× group at 2 h postoperatively after E. coli inoculation. Wu et al. (13) revealed a decrease in WBC counts 2 h after formation in a New Zealand rabbit model of urosepsis, which is consistent with our results. The host inflammatory response might be seen as a balanced response between pro-inflammatory and anti-inflammatory mediators (27). In the early stages of sepsis, activation of the host’s innate immune system leads to a massive release of pro- inflammatorymediators, the main ones includingIL-6 andTNF-a, as well as chemokines (24, 28). In various animal models of sepsis, IL-6andTNF-aexpressionweresignificantlyelevatedcomparedto the normal group (29–31). Besides, plasma IL-6 levels have been found to be potential indicators of the intensity of inflammation In 2016, sepsis was redefined as life-threatening organ dysfunction resulting from dysregulated host responses to infection (33). The severity of organ dysfunction is quantified using the Sequential Organ Failure Assessment (SOFA) score (34). Compared to the previous definition, the new version of the definition of sepsis places more emphasis on multi-organ damage. Our data suggested that the relative mRNA levels and protein levels of inflammatory factors (IL-6 and TNF-a) Frontiers in Immunology frontiersin.org 07 Cao et al. 10.3389/fimmu.2022.1074488 A B C D E F G FIGURE 6 Kidney injury in the rat model of urinary sepsis (A) Representative images of hematoxylin and eosin (H&E) staining of kidney tissue from the sham and sepsis groups, immunohistochemical analysis of IL-6 and TNF-a in kidney tissue (magnification, 200×; bar, 50 mm). (B, C) Serum BUN and Cre levels in rats 24 hours after E. coli or saline inoculation of the renal pelvis. (D) The relative mRNA levels of IL-6 and TNF-a in kidney tissue. (E) Expressions of IL-6 and TNF-a in kidney tissue were tested by western blot, and b-actin was used as a loading control. (F, G) Quantitative data of the levels of IL-6 and TNF-a. The sep 6× group was selected as a representative of the sepsis groups. Values were shown as mean ± SD. Data availability statement extracted from three organs—the lung, liver, and kidney—were considerably greater in the sepsis group than in the sham group. Our rat urosepsis model confirmed three organ damages by HE staining, immunohistochemistry, RT-PCR, and Western blotting. The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors. Ethics statement Current animal sepsis models do not fully replicate the pathophysiological process of human sepsis. In this study, our novel rat sepsis model simulates human urosepsis due to upper urinary tract obstruction combined with urinary tract infection. In addition, this model can manipulate the severity of sepsis by adjusting the concentration of E. coli suspensions. Therefore, this rat model may be an essential tool for studying the pathophysiological mechanisms of sepsis or urosepsis. The animal study was reviewed and approved by Zhongnan Hospital of Wuhan University’s Ethics Committee. Liver injury in the rat model of urosepsis *P < 0.05, **P < 0.01, ***P <0.001, and ****P < 0.0001 vs sham group. "ns" means "not signifcant". A B C B C D C D D B C F G E F G F E G FIGURE 6 Kidney injury in the rat model of urinary sepsis (A) Representative images of hematoxylin and eosin (H&E) staining of kidney tissue from the sham and sepsis groups, immunohistochemical analysis of IL-6 and TNF-a in kidney tissue (magnification, 200×; bar, 50 mm). (B, C) Serum BUN and Cre levels in rats 24 hours after E. coli or saline inoculation of the renal pelvis. (D) The relative mRNA levels of IL-6 and TNF-a in kidney tissue. (E) Expressions of IL-6 and TNF-a in kidney tissue were tested by western blot, and b-actin was used as a loading control. 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Auton Neurosci (2016) 200:1–10. doi: 10.1016/j.autneu.2015.07.425 6. Rhodes A, Evans LE, Alhazzani W, Levy MM, Antonelli M, Ferrer R, et al. Conflict of interest The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ fimmu.2022.1074488/full#supplementary-material The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Author contributions YC, CB, and PS designed the study, analyzed the data, and prepared the original draft. YC, XY, and PZ collected the data and conductedthestatisticalanalysis.ZY,PL,andKTcollectedthedata. Frontiers in Immunology frontiersin.org 08 Cao et al. 10.3389/fimmu.2022.1074488 Publisher’s note LGandZCraisedrats.YCwrotethepaper.XBandTLdesignedand monitored the study, together with significant revisions to the manuscript. All authors have approved the final draft submitted. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Acknowledgments We are very grateful to the laboratory staff at the Zhongnan HospitalofWuhanUniversityfortheirhelpincompletingthisstudy. References Surviving sepsis campaign: International guidelines for management of sepsis and septic shock: 2016. Intensive Care Med (2017) 43(3):304–77. doi: 10.1007/s00134- 017-4683-6 21. Gupta K, Donnola SB, Sadeghi Z, Lu L, Erokwu BO, Kavran M, et al. Intrarenal injection of escherichia coli in a rat model of pyelonephritis. J Vis Exp (2017) 125:54649. doi: 10.3791/54649 22. Giamarellos-Bourboulis EJ, Antonopoulou A, Raftogiannis M, Koutoukas P, Tsaganos T, Tziortzioti V, et al. 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Endogenous monocyte chemoattractant protein-1 (MCP-1) protects mice in a model of acute septic peritonitis: cross-talk between MCP-1 and leukotriene B4. J Immunol (1999) 163(11):6148–54. 15. Qiu H, Chen X, Luo Z, Zhao L, Zhang T, Yang N, et al. Inhibition of endogenous hydrogen sulfide production exacerbates the inflammatory response Frontiers in Immunology frontiersin.org 09 Cao et al. 10.3389/fimmu.2022.1074488 Frontiers in Immunology frontiersin.org 10
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IS THERE GENETIC VARIABILITY IN DWARF COCONUT ACCESSIONS PRESERVED IN BRAZIL?
Revista Caatinga
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Rev. Caatinga, Mossoró, v. 32, n. 1, p. 52 – 61, jan. – mar., 2019 52 _______________________________ *Corresponding author 1Received for publication in 02/17/2017; accepted in 09/06/2018. Paper extracted from the doctoral thesis of the first author. 2Department of Biological Sciences, Universidade Estadual de Feira de Santana, Feira de Santana, BA, Brazil; milambrito@hotmail.com – ORCID: 0000-0002-2005-6445. 3Department of Technology and Social Sciences, Universidade do Estado da Bahia, Juazeiro, BA, Brazil; manoelabiliomaq@gmail.com – ORCID: 0000-0001-9501-2343. 4Center for Agrarian Sciences Universidade Federal do Vale do São Francisco, Petrolina, PE, Brazil; izaias.limaneto@univasf.edu.br – ORCID: 0000-0002-7557-1102. 5Instituto Federal de Educação, Ciência e Tecnologia Baiano, Xique-Xique-BA, Brasil; ronaldosimaorso@gmail.com – ORCID: 0000-0003- 0996-9144. 6Embrapa Tabuleiros Costeiros, Aracaju, SE, Brazil; semiramis.ramos@embrapa.br – ORCID: 0000-0003-1289-1341. KAMILA MARCELINO BRITO SOBRAL2, MANOEL ABILIO DE QUEIROZ3, IZAIAS DA SILVA LIMA NETO4, RONALDO SIMÃO DE OLIVEIRA5, SEMÍRAMIS RABELO RAMALHO RAMOS6* ABSTRACT - Dwarf coconut tree is the main variety for commercial use in Brazil, which ranks fourth in world coconut production. However, the genotypes used still have limitations and genetic variability is required. The aim of this study was to estimate the genetic variability in dwarf coconut accessions preserved at the Germplasm Bank of Brazil at different harvesting times and using agronomic descriptors of plant and fruits. The accessions Brazilian Green Dwarf-Jiqui, Cameroon Red Dwarf, Malayan Red Dwarf, Brazilian Red Dwarf -Gramame, Brazilian Yellow Dwarf-Gramame, and Malayan Yellow Dwarf were assessed by means of 30 descriptors Variance analysis was performed and the genetic diversity was quantified by using the Mahalanobis’ generalized distance and expressed by means of UPGMA clusters, Tocher optimization, and canonical variables. The maximum likelihood analysis was used to estimate the components of variance with the data of each plant in a sample of 11 descriptors of great importance for the genetic improvement of the coconut tree. A phenotypic divergence was found among the accessions using the UPGMA clusters, Tocher optimization and graphic dispersion obtained with canonical variables. The use of the maximum likelihood analysis confirms the existence of genetic variability in the accessions for the descriptors fruit polar and equatorial diameter, nut polar diameter, total fruit weight, and epicarp thickness, which presented a heritability varying from 0.17 to 0.39. There is a possibility of genetic gains with the selection of these traits for use of accessions in breeding programs. Keywords: Cocos nucifera L. Phenotypic traits. Multivariate analysis. Germplasm. Plant genetic resources. Keywords: Cocos nucifera L. Phenotypic traits. Multivariate analysis. Germplasm. Plant genetic resources ISSN 0100-316X (impresso) ISSN 1983-2125 (online) 590/1983 21252019 32 106 ISSN 0100-316X (impresso) ISSN 1983-2125 (online) 590/1983 21252019 32 106 ISSN 0100-316X (impresso) ISSN 1983-2125 (online) http://dx.doi.org/10.1590/1983-21252019v32n106rc Universidade Federal Rural do Semi-Árido Pró-Reitoria de Pesquisa e Pós-Graduação q ç https://periodicos.ufersa.edu.br/index.php/caatinga http://dx.doi.org/10.1590/1983-21252019v32n106rc 1Received for publication in 02/17/2017; accepted in 09/06/2018 MATERIAL AND METHODS Six 15-year-old dwarf coconut accessions were assessed at the International Coconut Genebank for Latin America and the Caribbean (ICG–LAC). The accessions were planted in 2003 at the Experimental Field of Itaporanga belonging to Embrapa Coastal Tablelands, located in Itaporanga d’Ajuda, SE, on the SE 100, km 3 (11°07′ S and 37° 11′ W), 28 km from Aracaju. Indonesia is the world’s largest producer (17,722,429 tons), followed by the Philippines (13,825,080 tons), and India (11,127,898 tons) (FAOSTAT, 2016). Brazil occupies the fourth position, with a production of over 2,649,246 tons in a planted area of 234,012 ha (FAOSTAT, 2016), and the green dwarf is the cultivar mainly used for commercial production. The accessions Brazilian Green Dwarf-Jiqui (BGDJ), Cameroon Red Dwarf (CRD), Malayan Red Dwarf (MRD), Brazilian Red Dwarf-Gramame (BRDG), Brazilian Yellow Dwarf-Gramame (BYDG), and Malayan Yellow Dwarf (MYD) were assessed during three cultivation cycles from 2014 to 2016, resulting in three assessments indicated as Year 1 (2014), Year 2 (2015), and Year 3 (2016). In recent years, an increase of areas used for cultivation and production has been seen in different parts of the world. In Brazil, the advance of the crop occurs not only by the evolution in production levels, giving it a prominent place among the world’s largest coconut producers, but also by the expansion into regions not traditionally used for cultivation. Coconut cultivation in Brazil traditionally occurs in the Northeast region, but in the last 30 years, the cultivation areas have spread into other regions of Brazil, mainly in the Southeast, Midwest, and North (MARTINS; JESUS JUNIOR, 2014). The climate of the Itaporanga d’Ajuda region is classified as A′s, i.e., a rainy tropical climate with a dry summer, according to the Köppen classification. The soil of the experimental area is classified as an arenosols (Quartzipsamments) of low natural fertility (MELO-FILHO; SILVA; JACOMINE, 1982). The average temperature of the region is 25.6 °C and the average monthly precipitation for 2014, 2015, and 2016 were 98, 180, and 99.2 mm, respectively. The cultivation practices and phytosanitary treatments were carried out as normally recommended for the crop (FONTES; FERREIRA, 2016). In Brazil, dwarf coconut accessions are preserved in the International Coconut Genebank for Latin America and the Caribbean (ICG–LAC), unique in the country and located at Embrapa Coastal Tablelands, in Aracaju, SE. INTRODUCTION conservation and for breeding programs. In this sense, the dwarf coconut is an autogamous variety and its germplasm has a high degree of homozygosity. Thus, knowledge about the variability among the accessions preserved in the germplasm bank will also allow the identification of parental potentials and exploration of the heterosis. The aim of this study was to estimate the genetic variability in accessions of dwarf coconut preserved in the ICG– LAC at different harvest times and using agronomic fruit and plant descriptors. The coconut tree is a monospecific palm composed of three botanical varieties: Cocos nucifera L. var. typica (tall coconut), C. nucifera L. var. nana (dwarf coconut), and C. nucifera L. var. aurantiaca (intermediate coconut) (LIYANAGE, 1958). The dwarf variety is small in size, reaching up to 12 m when fully grown, an early cycle when compared to the tall coconut, and depending on the environment, it can start flowering around two years and six months after planting, producing a high number of small fruits (150 to 200 fruits/plant/year) (MENON; PADALAI, 1958; ARAGÃO et al., 2002). This variety is composed of yellow, green, and red cultivars (OHLER, 1984). In Asian, African, and some Latin American countries, dwarf coconuts are usually used for ornamental purposes and in breeding programs, especially in the intervarietal hybridization process with tall coconut (ARAGÃO et al., 2002). K. M. B. SOBRAL et al. K. M. B. SOBRAL et al. HÁ VARIABILIDE GENÉTICA EM ACESSOS DE COQUEIRO-ANÃO CONSERVADOS NO BRASIL? RESUMO - O coqueiro anão é a principal variedade para uso comercial no Brasil, que ocupa atualmente a quarta posição na produção mundial. No entanto, os genótipos utilizados no país ainda apresentam limitações e há necessidade de variabilidade genética. Este trabalho teve por objetivo estimar a variabilidade genética em acessos de coqueiro-anão conservados no Banco de Germoplasma existente no Brasil, em diferentes épocas de colheita, utilizando descritores agronômicos de planta e frutos. Os acessos anão-verde-do-Brasil-de-Jiqui; anão- vermelho-de-Camarões; anão-vermelho-da-Malásia; anão-vermelho-de-Gramame; anão-amarelo-de-Gramame e anão-amarelo-da-Malásia foram avaliados por meio de 30 descritores. Análise de variância foi realizada e a diversidade genética foi quantificada utilizando a distância generalizada de Mahalanobis e expressa por meio de agrupamentos UPGMA, otimização de Tocher e variáveis canônicas. A análise de máxima verossimilhança foi utilizada para estimar os componentes de variância com os dados de cada planta em uma amostra de 11 descritores de maior importância para o melhoramento genético do coqueiro. Foi encontrada divergência fenotípica entre os acessos usando os agrupamentos UPGMA, Tocher e a dispersão gráfica obtida com variáveis canônicas. O emprego da análise de máxima verossimilhança confirma a existência de variabilidade genética nos acessos para os descritores diâmetro polar e equatorial do fruto, diâmetro polar da noz, peso total do fruto e espessura de epicarpo que apresentaram herdabilidade variando de 0,17 a 0,39. Há possibilidade de ganhos genéticos com a seleção desses caracteres para uso dos acessos em programas de melhoramento genético. Palavras-chave: Cocos nucifera L. Características fenotípicas. Análise multivariada. Germoplasma. Recursos genéticos vegetais. 52 RESULTS AND DISCUSSION The descriptors related to fruits and leaf distinguished the accessions of dwarf coconut. A total of 23, 26, and 26 descriptors were significant, respectively, in the first, second, and third year, and most of them significant at 1% level (Table 1). In the joint analysis, considering both the fruit and leaf descriptors, only three descriptors did not present a significant difference (Table 1). Considering the three years and the joint assessment, low coefficients of variation (CV) were observed for the great majority of the descriptors, but with some exceptions, indicating good experimental precision. In order to quantify the genetic diversity among the accessions, the Mahalanobis’ generalized distance (CRUZ; FERREIRA; PESSONI, 2011) was used. Five individual analyses were carried out with six variables each and the matrices of each analysis corresponding to each year were summed, obtaining a single matrix. For the joint analysis, the matrices of the three years were summed. The hierarchical clustering was obtained from the genetic distance matrix using the UPGMA (unweighted pair group method with arithmetic mean) method (SNEATH; SOKAL, 1973) and the Tocher optimization method. The assessment of the relative importance of traits was measured by the Singh (1981) method and the method of the canonical variables (CRUZ; FERREIRA; PESSONI, 2011). All the analyses were performed using the software GENES (CRUZ, 2016). The clustering consistency was determined by the cophenetic correlation coefficient according to Sokal and Rohlf (1962). The significance of cophenetic correlation coefficients was calculated by the Mantel test with 1000 permutations (MANTEL, 1967). The cut-off point was defined by the Mojena (1977) method and the dendrograms were built using the software R (R DEVELOPMENT CORE TEAM, 2012). For the study of the variance components, considering that the data of accessions were available in one place and with three production p g g p p The UPGMA cluster method showed the formation of two groups and some subgroups in all years and in the joint analysis (Figure 1). The accession CRD formed an isolated group in years 1 and 2. However, in the joint analysis of year 3, this accession was placed in a subgroup next to other accessions and the first group was formed by the BGDJ and BRDG accessions (Figure 1C). It is important to note that the cophenetic correlation coefficient in this year was 0.70, i.e. the lowest among the four analyses. Statistical analysis The data were analyzed individually (each year) and then a joint analysis was performed using the mean obtained for each descriptor over the three years. The data were tested for ANOVA assumptions, analysis of variance homogeneity (BARTLETT, 1937), and normality (SHAPIRO; WILK, 1965). Descriptors that did not meet the assumptions were transformed and then the ANOVA was performed to observe phenotypic variability among the accessions. K. M. B. SOBRAL et al. K. M. B. SOBRAL et al. cycles, the methodology of mixed linear models (RESENDE, 2002) was used by the restricted maximum likelihood method (REML procedure) and the estimate of the best linear unbiased prediction (BLUP) by the statistical model 9: y = Xm + Zg + Wp + Ts + e, where y is the data vector, m is the vector of effects of measurement-repeating combinations (assumed to be fixed) added to the overall mean, g is the vector of genotypic effects (assumed to be random), “p” is the vector of plot effects (random), “s” is the vector of permanent environmental effects (random), and “e” is the vector of errors or residuals (random). Uppercase letters represent the incidence matrices for the respective effects. The parameters were estimated using the genetic-statistical software Selegen-Reml/Blup (RESENDE, 2002). For this analysis, 11 important agronomic descriptors (fruit polar diameter, fruit equatorial diameter, nut polar diameter, nut equatorial diameter, quantity of liquid endosperm, soluble solids content of endosperm, pH of the liquid endosperm, total fruit weight, epicarp weight, epicarp thickness, and number of fruits) were selected for the dwarf coconut. equatorial circumference (FEC, cm), fruit equatorial diameter (FED, cm), fruit polar diameter (FPD, cm), nut polar diameter (NPD, mm), nut equatorial diameter (NED, mm), total fruit weight (TFW, kg), fruit weight without liquid endosperm (FWWLE, kg), endocarp weight (shell) (EDW, kg), epicarp weight (husk) (EPW, kg), nut weight (NW, kg), solid endosperm weight (solid albumen) (SAW, kg), liquid endosperm weight (LAW, kg), solid endosperm thickness (AWT, mm), endocarp thickness (EDT, mm), epicarp thickness (EPT, mm), and number of fruits per plant (NF). The leaf number 14 in each plant was used for vegetative assessments. Three fruits/plant/accession/ replication had their inflorescences previously marked, being harvested seven months after formation. After harvesting, the fruits were transported, washed, and identified for recording the various fruit descriptors. MATERIAL AND METHODS Since their implantation, the accessions have been assessed and characterized and most of the studies were carried out using an official descriptive list for the species (IPGRI, 1995). However, some assessments were carried out in a preliminary scope and with data only measured from a single production cycle. In order to assess the preserved accessions in depth and to access as much information as possible, it is necessary to carry out studies of different production cycles, in sequential years, in which the maximum number of descriptors can be used. The results obtained from these studies will provide information about the genetic variability, which is essential for future decision-making, both for accession The accessions were arranged in a completely randomized block design with five replications with up to 16 useful plants per plot and a spacing of 7.5 × 7.5 × 7.5 m in an equilateral triangle. We used 30 quantitative descriptors adapted from the IPGRI (1995) list, being 10 vegetative and 20 of fruits. The vegetative descriptors were the number of live leaves (NLL), number of dead leaves (NDL), number of emitted leaves (NEL), rachis length (RL, m), petiole length (PL, cm), petiole thickness (PT, mm), petiole width (PW, cm), number of leaflets (NL), leaflet length (LL, cm), and leaflet width (LW, cm). The fruit descriptors were the soluble solids content of endosperm (water) (SSC, °Brix), pH of the liquid endosperm (pH), quantity of liquid endosperm (VLE, mL), fruit polar circumference (FPC, cm), fruit 53 Rev. Caatinga, Mossoró, v. 32, n. 1, p. 52 – 61, jan. – mar., 2019 IS THERE GENETIC VARIABILITY IN DWARF COCONUT ACCESSIONS PRESERVED IN BRAZIL? K. M. B. SOBRAL et al. Table 1. Summary of the variance analysis for the 30 descriptors assessed in six accessions of dwarf coconut preserved at the International Coconut Genebank for Latin America and the Caribbean (ICG–LAC). IS THERE GENETIC VARIABILITY IN DWARF COCONUT ACCESSIONS PRESERVED IN BRAZIL? 1 Descriptors1 Year1 (2014) Year 2 (2015) Year 3 (2016) Joint analysis MS CV Mean MS CV Mean MS CV Mean MS CV Mean FDP 0.70** 4.06 21.07 0.30** 2.84 20.19 0.09** 1.53 20.22 0.10** 1.99 20.50 FED 1.10** 6.90 15.22 0.40* 4.70 14.92 0.10** 2.10 15.27 0.30** 3.67 15.14 NPD 23.80* 4.55 107.36 25.80** 4.93 103.12 5.30** 2.20 104.36 10.90** 3.15 104.95 NED 38.30ns 6.42 95.17 36.30ns 5.95 101.26 8.10** 2.65 107.34 17.50ns 4.13 101.26 FPC 5.80** 4.38 55.10 2.90** 3.15 54.95 0.80** 1.66 54.56 1.70** 2.40 54.87 FEC 8.20** 6.12 46.98 3.90** 4.12 47.88 0.70** 1.79 47.84 2.50** 3.36 47.57 VLE 1992.90ns 17.28 258.32 3283.60ns 18.05 317.37 934.6** 8.11 376.87 1160.1* 10.27 317.52 SSC 0.04** 3.16 6.54 0.06** 3.87 6.37 0.08ns 4.68 6.06 0.20** 7.54 6.40 pH 0.01ns 1.62 6.31 0.01** 2.24 5.26 0.00** 1.48 5.21 0.00** 1.06 5.60 TFW 0.05** 8.95 1.69 0.05** 12.56 1.83 0.00** 5.17 1.84 0.02** 8.45 1.790 FWWLE 0.03** 13.23 1.46 0.02** 10.75 1.50 0.00** 5.89 1.43 0.01** 7.82 1.450 LAW 0.00ns 15.21 0.28 0.00ns 21.67 0.329 0.00** 6.63 0.41 0.01* 12.11 0.34 SAW 0.00ns 16.56 0.15 0.00** 15.02 0.172 0.00ns 9.34 0.17 0.00** 11.27 0.17 EDW 0.00* 10.83 0.14 0.00** 15.39 0.150 0.00** 6.03 0.15 0.00** 9.60 0.15 NW 0.00ns 13.21 0.44 0.01* 17.78 0.652 0.00** 5.20 0.73 0.00** 10.52 0.65 EPW 0.02** 13.76 1.12 0.01** 10.30 1.184 0.00** 8.31 1.09 0.00** 7.88 1.13 AWT 0.39* 10.40 6.06 0.06** 4.60 5.67 0.10** 7.70 5.33 0.06** 4.43 5.69 EDT 0.08** 7.19 4.05 0.05** 6.70 3.64 0.04** 6.13 3.62 0.02** 3.97 3.78 EPT 2.71** 7.38 22.31 0.72** 4.24 20.06 1.10** 6.03 17.60 0.70** 4.34 19.99 NF 291.4** 27.25 62.62 208.2** 11.79 70.70 108.30** 17.83 58.37 144.8** 19.06 63.16 NLL 5.70** 9.27 24.79 4.70** 8.02 27.15 4.00** 5.04 27.63 4.40** 7.98 26.29 NEL 1.97** 11.89 11.84 1.90** 8.31 16.82 2.00** 9.30 15.28 1.05** 7.01 14.64 NDL 1.59** 11.74 10.75 1.40ns 18.13 6.52 2.40** 12.67 12.39 0.90** 9.95 9.89 PL 53.2ns 6.95 104.84 55.50* 6.59 113.16 24.30ns 4.14 119.13 32.80ns 5.10 112.38 PW 0.11** 4.87 6.94 0.09** 4.48 6.96 0.04** 3.11 6.47 0.05** 3.38 6.80 PT 0.64** 3.26 24.63 1.00* 4.44 23.53 0.40** 2.70 24.23 0.40** 2.83 24.14 RL 0.07* 6.89 4.09 0.04* 4.46 4.30 0.01** 2.75 4.37 2.80ns 4.47 37.97 NL 20.5** 2.42 187.28 113.50** 1.89 184.67 9.60ns 1.67 186.57 17.80** 2.28 185.19 LL 59.4** 6.66 115.75 146.60** 5.07 117.05 18.10** 3.44 123.65 43.30** 5.29 119.80 LW 0.07** 5.40 5.20 0.04** 4.10 5.23 0.02** 3.14 4.84 0.02** 2.91 5.09 **and *significant to 1 and 5%, respectively, by the teste of F; ns not significant. IS THERE GENETIC VARIABILITY IN DWARF COCONUT ACCESSIONS PRESERVED IN BRAZIL? Descriptors: FPD = fruit polar diameter (cm), FED = fruit equatorial diameter (cm), NPD = nut polar diameter (mm), NED = nut equatorial diameter (mm), FPC = fruit polar circumference (cm), FEC = fruit equatorial circumference (cm), VLE = quantity of liquid endosperm (mL), SSC = soluble solids content of endosperm (water) (°Brix), pH = of the liquid endosperm (pH), TFW = total fruit weight (kg), FWWLE = fruit weight without liquid endosperm (kg), LAW = liquid endosperm weight ( kg), SAW = solid endosperm weight (solid albumen) (kg), EDW = endocarp weight (shell) ( kg), NW = nut weight (kg), EPW = epicarp weight (husk) (kg), AWT = solid endosperm thickness (mm), EDT = endocarp thickness (mm), EPT = epicarp thickness (mm), NF = number of fruits per plant (unit), NLL = number of live leaves (unit), NEL = number of emitted leaves (unit), NDL = number of dead leaves (unit), PL = petiole length (cm), PW = petiole width (cm), PT = petiole thickness (mm), RL = rachis length (m), NL = number of leaflets (unit), LL = leaflet length (cm), and LW = leaflet width (cm). CV= coefficient of variation, MS= mean squares. Table 1. Summary of the variance analysis for the 30 descriptors assessed in six accessions of dwarf coconut preserved at the International Coconut Genebank for Latin America and the Caribbean (ICG–LAC). Thus, the results indicate that the accession CRD was different from the others (Figures 1A, 1B, 1C, and 1D). The other accessions maintained a constant position in the second group but forming different subgroups. In particular, the MRD and BRDG accessions were very close in the same subgroup in years 1, 2, and 3 and in the joint analysis, as well as the BYDG and MYD accessions, which were also shown to be very close when considering all years and the joint assessment. The accession BGDJ was isolated in a division within the subgroup (Figures 1A, 1B, 1C, and 1D). These results indicate a diversity between the accessions, especially for CRD in relation to the others, and show a great similarity between the pairs of accessions BYDG and MYD in one subgroup and MRD and BRDG in another subgroup. The molecular data of a study performed with all dwarf coconut accessions showed the accessions in a single cluster, with BGDJ and MYD as the most distant (DAHER et al., 2002). RESULTS AND DISCUSSION The minimum desirable value for this coefficient is 0.80 (ROHLF; FISHER, 1968) and hence the groups formed in this year are less precise and the data may have been influenced by some environmental factors such as precipitation indices, which presented a variation of 98, 180, and 99.2 mm in years 1, 2, and 3, respectively. However, the coefficients of joint analysis for years 1, 2, and 3 were 0.90, 0.82, and 0.92, respectively, indicating an accurate separation of the groups. 54 Rev. Caatinga, Mossoró, v. 32, n. 1, p. 52 – 61, jan. – mar., 2019 IS THERE GENETIC VARIABILITY IN DWARF COCONUT ACCESSIONS PRESERVED IN BRAZIL? It is important to note that these data were not consistent with the present study since the formation of at least two groups was observed in the joint analysis (Figures 1A, 1B, 1C, and 1D). This difference found among the results is probably because molecular markers, especially those used by the authors (RAPD), use tags that anneal to random regions of the genome of plants and are not associated with the numerous assessed traits. Rev. Caatinga, Mossoró, v. 32, n. 1, p. 52 – 61, jan. – mar., 2019 55 IS THERE GENETIC VARIABILITY IN DWARF COCONUT ACCESSIONS PRESERVED IN BRAZIL? Table 2. Clustering by the Tocher method in six dwarf coconut accessions based on the dissimilarity expressed by the Mahalanobis’ generalized distance estimated from 30 quantitative descriptors in years 1, 2, and 3 and in the joint analysis. Group Accessions in each group with fruit format* 1 MRD, BRDG, BYDG, MYD, BGDJ MRD BRDG BYDG MYD BGDJ 2 CRD CRD *Accessions: MRD - Malayan Red Dwarf; BRDG - Brazilian Red Dwarf-Gramame; BYDG - Brazilian Yellow Dwarf- Gramame; MYD - Malayan Yellow Dwarf; BGDJ - Brazilian Green Dwarf-Jiqui; CRD - Cameroon Red Dwarf. 1 MRD, BRDG, BYDG, MYD, BGDJ MRD *Accessions: MRD - Malayan Red Dwarf; BRDG - Brazilian Red Dwarf-Gramame; BYDG - Brazilian Yellow Dwarf- Gramame; MYD - Malayan Yellow Dwarf; BGDJ - Brazilian Green Dwarf-Jiqui; CRD - Cameroon Red Dwarf. (Table 3). Most of the descriptors (50%) presented a contribution below 1% (Table 3). Thirty-three percent of the descriptors showed a good contribution ranging from 10.47 to 21.60% in the different years. Only the descriptor FPC was present in at least three analyses (Table 3). These data did not indicate a greater consistency in the discrimination capacity of the descriptor over the years, showing the need to continue the accession assessments, as established for the crop (SANTOS et al., 1996). In this case, more assessment cycles would be required for some descriptors, which would possibly allow a greater stability and consistency of the data of the assessed plants. It is important to deepen these observations by analyzing different aspects of the descriptors, such as the correlation between them, in order to have a selection of descriptors that may be more appropriate for the understanding of dwarf coconut accessions. The Tocher optimization clustering method revealed the formation of two groups for years 1, 2, and 3 and joint analysis (Table 2). The results are consistent with those observed in the UPGMA cluster analysis. When considering the individual (years 1, 2, and 3) and joint analyses, the clustering formed by the Tocher optimization method showed the same pattern in the grouping formation, with accessions BGDJ, MRD, BRDG, BYDG and MYD in one group and the accession CRD in another group. In general, when observing the clustering formed by UPGMA and Tocher optimization analyses, the accession CRD is the most dissimilar among those assessed since it is not grouped to any other accession. K. M. B. SOBRAL et al. K. M. B. SOBRAL et al. Figure 1. Dendrogram based on the Mahalanobis distance and UPGMA cluster method for six dwarf coconut accessions estimated from 30 quantitative descriptors referring to years 1 (A), 2 (B), and 3 (C) and the joint analysis of these years (D). A B C D D C Figure 1. Dendrogram based on the Mahalanobis distance and UPGMA cluster method for six dwarf coconut accessions estimated from 30 quantitative descriptors referring to years 1 (A), 2 (B), and 3 (C) and the joint analysis of these years (D). A difference was also observed between the data found in this study when compared to those found by Sobral et al. (2012). These authors assessed, in a single cycle, dwarf coconut accessions by means of vegetative and reproductive descriptors. The differences between the accessions in both studies are credited to the difference in the ages of the assessed plants (6 and 11 years, respectively), the number of descriptors used (49 and 30, respectively), as well as the number of assessed production cycles (1 and 3, respectively). In addition, the quantitative phenotypic descriptors are usually of low heritability and the recommendation is for them to be assessed for more production cycles, which was considered in this study, as recommended by Santos et al. (1996). According to these authors, five years is considered a good period for the assessment of vegetative data, four years for fruit components, and at least 10 production cycles for flowering and yield and production stability data. 56 Rev. Caatinga, Mossoró, v. 32, n. 1, p. 52 – 61, jan. – mar., 2019 56 K. M. B. SOBRAL et al. important for breeding programs. important for breeding programs. fruit weight and epicarp thickness presented heritability values of 0.39 and 0.33, respectively, indicating the possibility of genetic gains through selection. Thus, considering the current demand scenario, the germplasm bank of dwarf coconut preserves and provides satisfactory genetic variability to be worked on breeding programs that consider both advances in intravarietal (dwarf X dwarf) and intervarietal (tall X dwarf) crosses conducted in Brazil. The analyses using the maximum likelihood (RESENDE, 2002) show that, in fact, there is a genetic variance for some descriptors and these variances in relation to the environmental variances (Table 4) allow heritability even in a broad sense, which indicates the possibility of genetic gains in breeding. Thus, the individual broad sense heritability (h2G) estimated for the descriptors fruit polar and equatorial diameter and nut polar diameter presented values from 0.17 to 0.21, while the total Table 3. Relative importance of the 30 descriptors for the assessment of the genetic diversity in dwarf coconut accessions in years 1, 2, and 3 and in the joint analysis between the years using the Singh (1981) method. e 3. Relative importance of the 30 descriptors for the assessment of the genetic diversity in dwarf coconut accession ars 1, 2, and 3 and in the joint analysis between the years using the Singh (1981) method. K. M. B. SOBRAL et al. Year I Year II Year III Joint analysis Descriptors1 % TAV % TAV % TAV % TAV FDP 9.09 9.09 0.72 0.72 3.45 3.45 16.63 16.63 FED 11.86 20.95 4.52 5.24 1.03 4.47 1.46 18.09 NPD 2.06 23.01 8.72 13.96 2.16 6.64 11.60 29.69 NED 0.98 23.99 7.81 21.76 1.76 8.39 3.51 33.21 FPC 10.47 34.47 0.94 22.70 7.74 16.13 21.66 54.87 FEC 2.57 37.03 4.61 27.31 12.28 28.41 1.19 56.06 VLE 1.48 38.51 1.16 28.46 4.67 33.08 4.78 60.84 SSC 0.05 38.57 1.19 29.65 0.43 33.51 0.38 61.22 pH 0.16 38.73 0.21 29.87 0.74 34.25 0.91 62.13 TFW 0.47 39.20 0.78 30.65 1.37 35.62 1.90 64.03 FWWLE 0.08 39.28 0.35 30.99 0.32 35.94 0.49 64.52 LAW 0.06 39.34 0.02 31.02 0.29 36.22 0.35 64.87 SAW 0.01 39.35 0.08 31.10 0.05 36.27 0.46 65.33 EDW 0.01 39.36 0.03 31.12 0.04 36.31 0.40 65.73 NW 0.01 39.38 0.23 31.36 0.93 37.24 1.16 66.89 EPW 4.90 44.28 0.93 32.29 2.16 39.40 0.48 67.37 AWT 0.87 45.15 7.82 40.11 2.07 41.47 0.74 68.11 EDT 1.60 46.75 0.24 40.35 0.20 41.67 1.64 69.74 EPT 6.91 53.66 18.22 58.56 4.70 46.37 2.22 71.96 NF 0.68 54.34 8.81 67.38 4.40 50.77 9.15 81.11 NLL 7.70 62.04 4.23 71.61 14.68 65.44 0.64 81.76 NEL 0.96 62.99 0.75 72.35 7.31 72.75 6.55 88.31 NDL 0.72 63.71 0.51 72.86 4.79 77.54 2.40 90.71 PL 0.26 63.98 1.62 74.48 2.11 79.65 2.14 92.85 PW 1.62 65.60 0.97 75.46 1.81 81.46 1.61 94.46 PT 13.41 79.01 0.59 76.04 8.81 90.27 0.90 95.36 RL 0.68 79.70 1.70 77.74 2.07 92.34 0.01 95.37 NL 4.59 84.29 11.58 89.32 0.90 93.24 2.09 97.46 LL 12.59 96.88 9.57 98.89 4.43 97.67 1.67 99.13 LW 3.12 100.00 1.11 100.00 2.33 100.00 0.87 100.00 1Descriptors: FPD = fruit polar diameter (cm), FED = fruit equatorial diameter (cm), NPD = nut polar diameter (mm), NED = nut equatorial diameter (mm), FPC = fruit polar circumference ( cm), FEC = fruit equatorial circumference (cm), VLE = quantity of liquid endosperm (mL), SSC = soluble solids content of endosperm (water) (°Brix), pH = of the liquid endosperm (pH), TFW = total fruit weight (kg), FWWLE = fruit weight without liquid endosperm (kg), LAW = liquid endosperm weight ( kg), SAW = solid endosperm weight (solid albumen) (kg), EDW = endocarp weight (shell) ( kg), NW = nut weight (kg), EPW = epicarp weight (husk) (kg), AWT = solid endosperm thickness (mm), EDT = endocarp thickness (mm), EPT = epicarp thickness (mm), NF = number of fruits per plant (unit), NLL = number of live leaves (unit), NEL = number of emitted leaves (unit), NDL = number of dead leaves (unit), PL = petiole length ( cm), PW = petiole width (cm), PT = petiole thickness (mm), RL = rachis length (m), NL = number of leaflets (unit), LL = leaflet length ( cm), and LW = leaflet width (cm). IS THERE GENETIC VARIABILITY IN DWARF COCONUT ACCESSIONS PRESERVED IN BRAZIL? This accession presents interesting characteristics that can facilitate the harvest such as leaf arrangement and low plant height. The fruits have physical attributes similar to BGDJ, which is the most used in commercial production (MACIEL et al., 2009). However, traits such as lower endocarp thickness make the fruit more susceptible to a higher nut breakage, causing losses during transportation (RIBEIRO et al., 1999). To confirm the variability shown in the previous analyses, the data were plotted in scatter plots. The first two canonical variables explained more than 80% of the variation in each year and in the joint assessment (Figure 2). In fact, it is desirable that the percentage of accumulated variation in the first two canonical variables be higher than 80% (CRUZ; FERREIRA; PESSONI, 2011). The analyses showed a consistent genetic divergence between dwarf coconut accessions from ICG–LAC since the same groupings were formed in years 1, 2, and 3 and in the joint analysis of the data. However, different results were obtained by Cambuí, Aragão and Leal, (2007), which were probably because both studies were carried out in different environments and periods, with plants of different ages, sample size (number of plants considered in the analysis), number and state of descriptors (according to the period, work objective, and plant age), and choice of estimated genetic distance for the analysis of the obtained results. The scatter plots for the three assessed years and joint analysis were in accordance with the results for UPGMA clusters and Tocher optimization, which are similar to the results obtained from the genetic diversity between accessions and their respective groups and subgroups (Figure 1 and Table 2). The results presented and discussed to date consistently indicate that the dwarf coconut accessions present a genetic divergence as attested by the univariate and multivariate analyses. However, since it is an autogamous crop (PASSOS, 1998), it is very important to examine the components of variance in order to have reliable information about the existence of the genetic variance for the descriptors, especially those most The use of the Singh (1981) method showed that about 10 to 11 descriptors in years 1, 2, and 3 and in the joint analysis presented an importance level above 3% and, in a few cases, reaching up to 21.66% for the descriptor FPC in the joint analysis 57 Rev. Caatinga, Mossoró, v. 32, n. 1, p. 52 – 61, jan. – mar., 2019 K. M. B. SOBRAL et al. TAV= Total Accumulated Variance. Rev. Caatinga, Mossoró, v. 32, n. 1, p. 52 – 61, jan. – mar., 2019 58 Rev. Caatinga, Mossoró, v. 32, n. 1, p. 52 – 61, jan. – mar., 2019 58 IS THERE GENETIC VARIABILITY IN DWARF COCONUT ACCESSIONS PRESERVED IN BRAZIL? IS THERE GENETIC VARIABILITY IN DWARF COCONUT ACCESSIONS PRESERVED IN BRAZIL? K. M. B. SOBRAL et al. Figure 2. Dispersion of scores of six dwarf coconut accessions in relation to the first two canonical variables (CV1 and CV2) and accumulated variance (%) based on agronomic traits measured in years 1 (A), 2 (B), and 3 (C) and in the joint analysis for these years (D). A B C D CONCLUSIONS anão (Cocos nucifera, L. var. nana). Revista Brasileira de Biociências, v. 5, Sup., p. 165-167, 2007. There is a genetic variability for fruit traits in dwarf coconut accessions preserved in Brazil. CRUZ, C. D.; FERREIRA, F. M; PESSONI, L. A. Biometria Aplicada ao Estudo da Diversidade Genética. 1 ed. Viçosa, MG: UFV, 2011. 620 p. The coconut variety Cameroon Red Dwarf is the most divergent accession among those considered in this study. The descriptors used in this study are efficient in estimating the genetic variability among accessions. CRUZ, C. D. Genes Software – extended and integrated with the R, Matlab and Selegen. Acta Scientiarum, v. 38, n. 4, p. 547-552, 2016. The descriptors fruit polar and equatorial diameter, fruit nut polar diameter, total fruit weight, and epicarp thickness are liable to genetic gain through selection. DAHER, R. F. et al. Assessment of coconut tree genetic divergence by compound sample RAPD marker analysis. Crop Breeding and Applied Biotechnology, v. 2, n. 3, p. 431-438, 2002. IS THERE GENETIC VARIABILITY IN DWARF COCONUT ACCESSIONS PRESERVED IN BRAZIL? K. M. B. SOBRAL et al. Table 4. Estimates of genotypic variance (Vg), environmental variance between plots (Vplot), residual variance (Ve), individual phenotypic variance (Vf), individual broad sense heritability (h2G), and coefficient of determination of plot effects (c2plot) using 11 descriptors of economic importance in the International Coconut Genebank for Latin America and the Caribbean (ICG–LAC). 1Descriptors: FPD = fruit polar diameter (cm), FED = fruit equatorial diameter (cm), NPD = nut polar diameter (mm), NED = nut equatorial diameter (mm), VLE = quantity of liquid endosperm (mL), SSC = soluble solids content of endosperm (water) (°Brix), pH = of the liquid endosperm (pH), TFW = total fruit weight (kg), EPW = epicarp weight (husk) (kg), EPT = epicarp thickness (mm), NF = number of fruits per plant (unit). Descriptors 1 Vg Vparc Ve Vf h2G c2parc FDP 0.62 0.77 2.19 3.60 0.17 ± 0.04 0.21 FED 0.58 0.52 1.62 2.74 0.21 ± 0.05 0.19 NPD 13.08 19.18 44.13 76.82 0.17 ± 0.04 0.24 NED 2.18 47.56 69.80 120.00 0.01 ± 0.01 0.39 VLE 602.26 4554.18 5476.74 10676.53 0.05 ± 0.02 0.42 SSC 0.02 0.02 0.37 0.42 0.05 ± 0.02 0.05 pH 0.00 0.28 0.05 0.34 0.00 ± 0.00 0.84 TFW 0.08 0.03 0.08 0.20 0.39 ± 0.07 0.17 EPW 0.07 0.01 0.04 0.13 0.57 ± 0.08 0.11 EPT 8.15 6.00 10.04 24.31 0.33 ± 0.06 0.24 NF 108.48 392.89 867.36 1378.79 0.07 ± 0.03 0.28 1Descriptors: FPD = fruit polar diameter (cm), FED = fruit equatorial diameter (cm), NPD = nut polar diameter (mm), NED = nut equatorial diameter (mm), VLE = quantity of liquid endosperm (mL), SSC = soluble solids content of endosperm (water) (°Brix), pH = of the liquid endosperm (pH), TFW = total fruit weight (kg), EPW = epicarp weight (husk) (kg), EPT = epicarp thickness (mm), NF = number of fruits per plant (unit). ACKNOWLEDGMENTS FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS - FAOSTAT. Culturas ano 2016. Disponível em: <http:// www.fao.org/faostat/en/#data/QC> Acesso em:19 abr. 2018. The authors thank Mariana N. R. Lima for the statistical support. To the MAPA/SNPC for the financial support and to the Embrapa Coastal Tablelands for the facilities to carry out work in the field and in the laboratory. FONTES, H. R.; FERREIRA, J. M. S. A cultura do coqueiro. 2.ed. Aracaju: Embrapa Tabuleiros Costeiros; Brasília: Embrapa Informação Tecnológica, 2016. (Sistemas de Produção, 1). K. M. B. SOBRAL et al. K. M. B. SOBRAL et al. A B A B D C C D Figure 2. Dispersion of scores of six dwarf coconut accessions in relation to the first two canonical variables (CV1 and CV2) and accumulated variance (%) based on agronomic traits measured in years 1 (A), 2 (B), and 3 (C) and in the joint analysis for these years (D). environmental homogeneity within blocks remained for these traits. According to Guerra et al. (2009), the coefficients of determination of plot effects (c2plot) quantify the variability within blocks. The nut equatorial diameter, quantity of the liquid endosperm, pH, and the number of fruits had a very low genetic variance (0.01 to 0.07), higher plot effects (Table 4), and low heritability. The other descriptors presented a low magnitude (0.05 to 0.24), indicating a low environmental variation between plots within the block, in addition to indicating that the experimental design was adequate since the The results of the maximum likelihood analyses are important because they corroborate the results obtained with the univariate and multivariate analyses and indicate the existence of genetic variability in the germplasm of dwarf coconut preserved in the ICG–LAC. From them, we will be able to select superior individuals for different coconut production environments based on the main descriptors which are of economic importance. 59 Rev. Caatinga, Mossoró, v. 32, n. 1, p. 52 – 61, jan. – mar., 2019 59 K. M. B. SOBRAL et al. ROHLF, F. S.; FISHER, D. L. Test for hierarchical structure in random sets. Systematic Zoology, v. 17, n. 4, p. 407-412. 1968. ROHLF, F. S.; FISHER, D. L. Test for hierarchical structure in random sets. Systematic Zoology, v. 17, n. 4, p. 407-412. 1968. LIYANAGE, D. V. Varieties and forms of the coconut palm grown in Ceylon. Ceylon Coconut Quarterly, v. 9, s/n, p. 1-10, 1958. MACIEL, V. T. et al. Caracterização física dos frutos de seis cultivares de coqueiro-anão em diferentes estádios de desenvolvimento. Revista Brasileira de Ciências Agrárias, v. 4, n. 4, p. 395- 398, 2009. SANTOS, G. A. et al. Manual on standardized research techniques in coconut breeding. International Plant Genetic Resources Institute, Rome, Italy, 1996. 46 p. SHAPIRO, S. S.; WILK, M. B. An analysis of variance teste for normality. Biometrika, v. 52, n. 3- 4, p. 591-611, 1965. MANTEL, N. The detection of disease clustering and generalized regression approach. Cancer Research, v. 27, n. 2, p. 209-220, 1967. SINGH, D. The relative importance of characters affecting genetic divergence. The Indian Journal of Genetic and Plant Breeding, v. 41, n. 2, p. 237-245, 1981. MARTINS, C. R.; JESUS JUNIOR, L. A. Produção e comercialização de coco no Brasil frente ao comércio internacional: panorama 2014. 1. ed. Aracaju: Embrapa Tabuleiros Costeiros, 51 p. 2014 (Documentos, 184). SNEATH, P. H.; SOKAL, R. R. Numerical taxonomy: The principles and practice of numerical classification. San Francisco: W. H. Freeman, 1973. 573 p. MELO FILHO, H. F. R.; SILVA, F. B. R.; JACOMINE, P. K. T. Levantamento detalhado dos solos da Fazenda, UEPAE/ARACAJU. Rio de Janeiro: Empresa Brasileira de Pesquisa Agropecuária; Serviço Nacional de Levantamento e Conservação de Solos, 1982, 59 p. (Boletim Técnico,78). SOBRAL, K.M.B. 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Interval Estimation of Motion Intensity Variation Using the Improved Inception-V3 Model
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Research Article Posted Date: March 24th, 2021 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Abstract In the process of acute resistance exercise, repeated variation in motion intensity can lead to muscle fatigue and heart failure. Therefore, acquiring the interval of motion intensity variation in time the training pattern and effect can be improved by acquiring the interval of motion intensity variation. In order to achieve this goal, an improved Inception-V3 model is proposed for motion intensity variation interval estimation. The MIVIE(Motion Intensity Variation Interval Estimation) dataset consisting of Strong, Moderate, Weak groups achieve centralized and uninterrupted collection. Then, the multi-modal fusion vectors of time-frequency eigenvalues are stacked up to 227 × 227 grayscale images fed into improved inception-CNN. Finally, the manipulator's trajectory optimization is completed under the guidance of ATO-DQN (Adaptive Trajectory Optimization-Deep Q Network) algorithm based on the motion intensity interval estimation. This work can improve the non-stationary effect of motor speed caused by changes in motion intensity during rehabilitation, which can better guarantee the safety of patients. Keywords: Improved inception-v3 model, Motion intensity variation interval estimation, DQN-based trajectory optimization. Keywords: Improved inception-v3 model, Motion intensity variation interval estimation, DQN-based trajectory optimization. ed inception-v3 model, Motion intensity variation interval estimation, DQN-based trajectory optimization to solve the existing time-series classification problems. In the literature[15-17], the researchers used conditional and gated restricted Boltzmann machines, deep belief networks, recurrent neural networks, auto encoders, HMMs, and also extended form of CNN to improve the details of various problems. 1. Introduction With the development of medical technology, many more diseases can be cured. However, for patients with hemiplegia, the current main medical treatments are still nursing and rehabilitation training[1]. Through targeted treatments that depend on the appropriate diagnosis and therapy measures, the movement ability of patients can be partially and even completely restored[2]. Thus, rehabilitation training with the assistance of an exoskeleton arm is proposed to alleviate this practical problem[3]. Patients can gain the ability to conduct rehabilitation exercises independently through the exoskeleton rehabilitation robot, which will reduce the treatment cost and obtain more effective treatment[4]. In the process of acute resistance exercise, repeated variation in motion intensity can lead to muscle fatigue and heart failure. In order to optimize the training mode and improve the training effect, the reasonable use of this information can effectively avoid the motor out-of-step in the trajectory control of the manipulator[5]. p Specifically, the technical contributions of this paper can be concluded as follows: MIVIE dataset consisting of Strong, Moderate, Weak groups realizes the data collection of setting specific situation according to the individual’s condition. We build an improved Inception-V3 model is proposed for motion intensity variation interval estimation. the manipulator's trajectory optimization is completed under the guidance of ATO-DQN algorithm based on the motion intensity interval estimation. In the field of technological innovation, use physiological information obtained by BMD101 ECG sensor and angular acceleration sensor to comprehensively judge and divide the boundary of rehabilitation motion intensity through the improved model achieve complementary advantages and mitigate the negative effects. The existing method[6, 7], which uses the heart rate as a detection mechanism, has been mainly used in medical diagnosis. Compared with EMG signals or EEG signals, the heart rate could alleviate individual differences and would be more easily detected by wearable devices[8, 9]. In terms of the existing research results[10, 11], collecting accurate feedback signals and developing accurate and effective control strategies could improve the efficiency and quality of rehabilitation training. 3.1.3 Data Acquisition Methods. Subjects must be trained before they formally participate in the data collection project. The collected data includes ECG at 512 Hz sampling frequency and a joint line acceleration and angular velocity signal at 60 Hz sampling frequency. The dataset named MIVIE is divided into three groups: SI (Strong Intensity), MI (Moderate Intensity), WI (Weak Intensity). Specific Standards and rules for each group are shown in Table 1. Group SI exercise protocol included 30 seconds of warming up, 120 seconds of running at 9~12 km/h, 30 seconds of rest, 120 seconds of joint flexion/extension at 2 times/s, and 30 seconds of walking to relax. Group MI exercise protocol included 30 seconds of warming up, 100 seconds of running at 6~8 km/h,30 seconds of rest, 90 seconds of arm lifting at 1 times/s, and 50 seconds of walking to relax. Group WI exercise protocol included 30 seconds of warming up, 80 seconds of running at 3~4 km/h, 45 seconds of rest, 40 seconds of arm lifting at 0.6 times/s, and 50 seconds of walking to relax. 2. Related Work The difference of rehabilitation motion intensity results in the difference of rehabilitation effect for various diseases among different populations. Weak and moderate intensity motion is associated with protection against chronic diseases, especially cardiovascular disease[18, 19]. High intensity interval training (HIIT) may be an incredibly effective way to increase VO2peak and improve cardiorespiratory fitness compared to moderate intensity continuous training[18]. Therefore, the setting of rehabilitation motion intensity should be personalized and self-adaptive. Traditional methods of processing kinematic signals (obtained by multiple sensors) or bioelectrical signals (EMG, ECG) seem to be hard to couple the nonlinear relationship. To achieve rapid responses from rehabilitation robots, the control system needs to respond in real-time. Deep learning provides more possibilities for this response[20, 21]. Combined with medical and kinesiology theories, motion intensity refers to the degree of force exerted by the human body when performing actions and the degree of tension of the body. The motion intensity directly affects the stimulation effect of the current motion on the human body[12]. Existing research has put forward the algorithms and framework for heart rate monitoring during intensive exercise[13, 14]. Given the strong feature learning capabilities of CNNs, the researchers have been active in applying deep neural networks （b） Online ECG waveform display interface Figure.1 BMD101 Bluetooth ECG acquisition module （b） Online ECG waveform display interface Figure.1 BMD101 Bluetooth ECG acquisition module Some scholars have proposed a transfer learning (TL) algorithm[22] that obtains rich data among users by mixing models of multiple topics. This mapping relationship can realize online data analysis for new objects. The Sensor-Wise method[23] achieves a high-precision and lightweight structure with only two hidden layers, but this method has limitations in the depth and accuracy of the motion intensity classification. On the whole, the use of physiological signals may be hampered by individual differences that are not easy to model, and there is a problem of hysteresis in kinematic signals[24]. From another perspective, the kinematic signals were used to sense the motion state and detect the deviation of the motion trajectory to achieve trajectory tracking[25]. Therefore, the deep learning algorithm is used to construct the multi-mode vector composed of physiological signals and kinematic signals to build the nonlinear coupling relationship among the multi-mode information[26, 27]. Figure.1 BMD101 Bluetooth ECG acquisition module Figure.1 BMD101 Bluetooth ECG acquisition module 3.1.2 Time-window Length. In the processing of bioelectrical signal sequences, the performance of the classifier should take priority over the speed[28]. Recent studies[29] have shown that the Qi (Quality index) can measure classification performance. Furthermore, when there are fewer eigenvalues, a high Qi value can be obtained by using the Kaiser window function and the window length of 512ms[29]. In order to improve the classification efficiency[30], we choose the Kaiser window function with a maximum delay window of 256ms to reduce the delay without affecting the classification performance. The 256 samples (256ms time window) separated by sliding window with the 32ms sliding window step length and the 224ms overlap allow approximately 15s~20s for window data pre-processing. 3.1 Datasets The ECG signals of 24 able-bodied subjects were collected at a sampling rate of 512Hz using the BMD101 Bluetooth ECG acquisition module. At the same time, the angular velocity and linear acceleration of the joints are measured by an integrated industrial MEMS attitude sensor installed at the joints of elbow and shoulder of rehabilitation exoskeleton robotic arm. The MIVIE(Motion Intensity Variation Interval Estimation) dataset we set up was divided into two sub- datasets, in which the data of 18 able-bodied subjects were used as training datasets to build models and optimize parameters, another 6 able-bodied subjects’ data were used as test datasets for algorithm validation and trajectory optimization. 3.1.1 ECG Recording Hardware. Frequency band distribution of main information and noise of ECG Baseline wander P-T wave QRS complex wave Power-line interference EMG interference 0~0.5 3~10 3~40 50 30~300 (2).VO2max(Maximum oxygen uptake) reaches 80%/65%/50%. (2).VO2max(Maximum oxygen uptake) reaches 80%/65%/50%. For the nonlinear and non-stationary weak signal with strong randomness and noise, the traditional method has poor effect. Considering the characteristics of HR signal and the speed of operation, this paper chooses wavelet threshold denoising method and sets threshold function: Figure.2 Rehabilitation scenario The sensor placement on the body is shown in Figure 2. For the classification of the weak motion intensity, the test subject hardly takes the initiative to exercise his or her limbs during the entire rehabilitation exercise process and can complete the entire exercise track. For the classification of the moderate motion intensity, the maximum output torque of the actuator is limited, and the exercise can just barely be undertaken under no-load conditions. For the classification of the strong motion intensity, the output torque of the actuator on the rehabilitation robot arm is further restricted, and it can hardly drive itself to perform the rehabilitation exercises. In this case, the subject needs to actively exert force to complete the entire rehabilitation exercise trajectory. 1 3 1 3 0.01 2ln = ln( 1) med( ) = W W W W W W N j d E          − −    = +          +     (1 (1) where W is primitive wavelet coefficient, W is the adaptive modified wavelet coefficient, is the correction factor which is set to 0.4, is the threshold setting value, N is the amount of ECG sampling points, is the noise intensity estimation under the wavelet coefficient, med( ) d is the median of the absolute value of the wavelet coefficients at each scale, E is the correction constant 0.65. where W is primitive wavelet coefficient, W is the adaptive modified wavelet coefficient, is the correction factor which is set to 0.4, is the threshold setting value, N is the amount of ECG sampling points, is the noise intensity estimation under the wavelet coefficient, med( ) d is the median of the absolute value of the wavelet coefficients at each scale, E is the correction constant 0.65. 3.1.1 ECG Recording Hardware. BMD101 is the 3rd generation bio-signal system-on-chip (SoC) of NeuroSky. Because of the BMD101’s extremely low system noise and programmable gain, it can detect bio-signals and convert them into digital words using a 16-bit high resolution ADC. As shown in Figure.1(a), BMD101 Bluetooth ECG sensor’s sampling frequency is set at 512 Hz with the heart rate monitoring range of 24~200bpm that measurement error can be controlled within ±1bpm. In order to better observe the quality of signal collection, a specific online ECG waveform display interface was designed shown in Figure.1(b). To avoid the noise and baseline deviation caused by wire connection during the intense motion, the subjects will perform the muscle contraction and joint flexion and extension using a single-lead remote Bluetooth transmission protocol which can effectively solve the problems. In order to prevent muscle fatigue caused by excessive exercise during one day, each subject completed 15 sets of motion intensity training at intervals in a 3-week period in hi h 24 ti i t d d which 24 participants were recorded. Table 1. Standard and Rules of Different Training Motion Intensity Group which 24 participants were recorded. Table 1. Standard and Rules of Different Training Motion Intensity Group p p Table 1. Standard and Rules of Different Training Motion Intensity Group Gr oup War ming up(s) Running (s) Re st (s) Joint flexion/e xtension (s) W alk ing (s) Effective indicators SI 30 120(9~12 km/h) 30 120(2 times/s) 30 85%+HRmax/80 %VO2max MI 30 100(6~8k m/h) 30 90(1 times/s) 50 60%~70%HRma x/65%+VO2max WI 30 80(3~4k m/h) 45 40(0.6tim es/s) 50 45%~60%HRma x/50%+VO2max p （a）BMD101 Bluetooth ECG sensor （a）BMD101 Bluetooth ECG sensor 2 Effective indicators must meet any of the following requirements. Otherwi it will be classified as invalid data: (1).The average heart rate during exercise reaches the maximum heart ra of 85%/60-75%/45%-60%. (2).VO2max(Maximum oxygen uptake) reaches 80%/65%/50%. 3~40Hz, and the frequency band of P-T wave is 3~10 Hz, so the interval frequency is filtered to reduce the noise. Table 2. Frequency band distribution of main information and noise of ECG Effective indicators must meet any of the following requirements. Otherwise, it will be classified as invalid data: (1).The average heart rate during exercise reaches the maximum heart rate of 85%/60-75%/45%-60%. (1).The average heart rate during exercise reaches the maximum heart rate of 85%/60-75%/45%-60%. q y Table 2. 3.2.1 Time Domain and Frequency Domain Analysis of ECG and Kinematics Signal Like other physiological signals, ECG is a kind of low- frequency weak signal under strong noise background and has the following characteristics: The general sampling value of normal ECG fluctuates from 0.05 mv to 5 mv, the frequency range is from 0.05 Hz to 100 Hz, and 90% of ECG spectrum energy is concentrated from 0.25 Hz to 35 Hz[31]. g g Figure.3 Comparison of denoising effect of improved Coif4 wavelet threshold algorithm and original signal The process of collecting ECG signal will be disturbed by various kinds of noise, the noise sources are usually as follows: (1) Power-line interference: 50Hz Power-line interference is caused by the electromagnetic field formed by the lead device which collects HR and the loop circuit of human body. (1) Power-line interference: 50Hz Power-line interference is caused by the electromagnetic field formed by the lead device which collects HR and the loop circuit of human body. (2) EMG interference: The EMG interference can be regarded as the instantaneous zero mean band limited noise, and the main energy is concentrated in the range of 30 ~ 300Hz. Figure.3 Comparison of denoising effect of improved Coif4 wavelet threshold algorithm and original signal (3) Baseline wander: Baseline wander in ECG amplitudes is generally caused by low frequency interference[32], such as aspiration and electrode movement, and the frequency is less than 5 HZ; the change can be regarded as a sine component added to HR at the same frequency as the aspiration frequency, at 0.015 ~ 0.03 Hz, the amplitude of baseline change was 15% of the ECG peak value. 3.1.1 ECG Recording Hardware. All methods were carried out in accordance with relevant guidelines and regulations. All subjects’ informed consent were confirmed by themselves. The data acquisition protocol was approved by Medical and Experimental Animal Ethics Committee of Northwestern Polytechnical University, Xi'an, China (approbation number: 6101030222595-202001001). ECG signals with noise was decomposed into 8 levels of wavelet by Coif4 wavelets, then baseline wander was removed by wavelet decomposition and reconstruction. EMG and power frequency interference were removed by improved threshold algorithm, the specific effect of noise removal is shown in Figure.3. Grayscale Pixel Matrix Considering the real-time nature of the control accuracy, the frequency domain signal was obtained by a fast Fourier transform (FFT). We mainly used the root mean square frequency (RMSF) and the root variance frequency (RVF) as the frequency domain eigenvalues. These two parameters reflect the characteristics of the motion-related aspects more effectively. The specific algorithm is as follows. If the original one-dimensional time series data matrix is directly used as network input, the network input dimension is too large resulting in expensive network computation. After extracting the eigenvalues of the original signal, the original Matrix of the input data is normalized and aligned. In this paper, a method is proposed to convert batch one- dimensional time series to two-dimensional gray-scale images, that is, the original time-domain signal is the format of the one-dimensional data matrix, which is transformed into an m×n grayscale pixel matrix. 2 0 0 ( ) ( ) f S f df RMSF S f df + + =   (6) 0 0 ( ) ( ) fS f df FC S f df + + =   (7) 2 0 0 ( ) ( ) ( ) f FC S f df RVF S f df + + − =   (8) 2 0 0 ( ) ( ) f S f df RMSF S f df + + =   (6) 0 0 ( ) ( ) fS f df FC S f df + + =   (7) 2 0 0 ( ) ( ) ( ) f FC S f df RVF S f df + + − =   (8) (6) In order to keep the pixel matrix stable and reduce the computation time of the procedure of the transformation, the random principle of N choosing 3 is adopted before the stack of the eigenvalues. On the other hand, too much preprocessing will lose the small information of the eigenvalues such as the wave crests of the time-frequency series in the QRS complex, so only the grayscale, the open operation and the bicubic interpolation operations are used after the pixel matrix is generated. (7) (8) 0 ( ) S f df +  where S is the corresponding frequency domain amplitude, f is the corresponding frequency, RMSF is the Root Mean Square Frequency, FC is the frequency centre, and RVF is the Root Variance Frequency. 3.2.2 Extraction of ECG Eigenvalues The following eigenvalues were extracted as the input layer of the subsequent deep learning model. ⬧ The standard deviation of the time domain (SD): 2 1 N i i x SD N  = − =  (2) As shown in Table 2, the information of motion state mainly concentrates on P-T wave and QRS complex wave of ECG[33]. The frequency band of QRS complex wave band is (2) 3 where xi is the signal value corresponding to the time series, N is the total number of sampling points of the time series, and μ is the average value of the segment signal. rehabilitation exercise, the joint angular velocity was also changed under different motions. Therefore, it was difficult to determine the specific motion intensity by directly determining the angular acceleration signal. Hence, we calculated the motion velocity of the collected data set based on the collected signal points in this paper. ⬧ Approximate Entropy (ApEn): The approximate entropy is a nonlinear dynamic parameter used to quantify the regularity and unpredictability of time series fluctuations. ( ) [ ( ), ( ( 1)),..., ( )] ( ) [ ( ), ( ( 1)),..., ( )] m m R i r i m r i m r i Y i y i m y i m y i = − − −   = − − −  (9) (9) For a one-dimensional series such as a heart rate, the extraction method was designed to reconstruct the vector and calculate the vector distance. Then, we counted the number of conformances and expressed series in the form of non-negative numbers. The algorithm hyper parameter r=0.2*SD, which represents the parameter measure of "similarity", was used in this paper to reconstruct the vector dimension. The specific algorithm for the approximate entropy is expressed as follows. The one-dimensional heart rate discrete signal obtained by sampling at equal intervals is a(1), a(2) …, a(N). we reconstituted the signal into a 3-dimensional vector, namely, A(1), A(2) …, A(N-m+1). When 1≤i≤N-m+1, the number of reconstruction vectors satisfying the following conditions is: For a one-dimensional series such as a heart rate, the extraction method was designed to reconstruct the vector and calculate the vector distance. Then, we counted the number of conformances and expressed series in the form of non-negative numbers. ⬧ Frequency domain eigenvalues (FDE): ⬧ Frequency domain eigenvalues (FDE): 3.2.2 Extraction of ECG Eigenvalues The algorithm hyper parameter r=0.2*SD, which represents the parameter measure of "similarity", was used in this paper to reconstruct the vector dimension. The specific algorithm for the approximate entropy is expressed as follows. m 0 R ( )-Y ( ) = N m i i i AVD N = (10) (10) where ( ) m R i is the one-dimensional vector composed of the measured angular velocity values in the previous m seconds, ( ) m Y i is the one-dimensional vector of the angular velocity value of the expected motion trajectory in the time period and N is the total number of the vectors. The one-dimensional heart rate discrete signal obtained by sampling at equal intervals is a(1), a(2) …, a(N). we reconstituted the signal into a 3-dimensional vector, namely, A(1), A(2) …, A(N-m+1). When 1≤i≤N-m+1, the number of reconstruction vectors satisfying the following conditions is: The angular velocity deviation (AVD) was obtained by the collected data set and the actual feedback signal. In summary, the process block diagram of building a multi-mode vector is described in Figure.4. ( ) ( ) / ( 1) [ ( ), ( )] m i number of A j such that C r N m d A i A j r   = − +      (3) Figure.4 Multimode vector diagram where d[A(i), A(j)] is the vector distance. The vector distance is defined as the value of the largest absolute difference of each dimension in the two reconstructed vectors, where the range of j is [1, N-m+1], including j=i. 1 1 1 ( ) ( 1) log( ( )) m N m m r i i r N m C r  − − + = = − +  (4) (4) The state quantity of the current reconstructed dimension was defined by the above equation. The approximate entropy (ApEn) equals the difference value between the state quantity of the strong reconstruction dimension and the current dimension state quantity. Figure.4 Multimode vector diagram 1 ( ) ( ) m m ApEn r r   + = − (5) (5) 3.3 Resampling of the Rehabilitation Motion Trajectory The rehabilitation motion trajectory collected from volunteers through the teaching reply mode could not be directly used to simulate the patient's experiment. The sampled trajectory points needed to be resampled to facilitate the adjustment of the motion rate during the experiment[34]. 2 2 2 1 = ( 1) kp fs bs fs bs  − − + (15) (15) where kp represents the probability of a unit remaining in the traditional dropout (between 0.75 and 0.95, with a final value of 0.9), fs represents the size of the feature map, bs represents block size. The Lanczos resampling method is widely used in the two- dimensional vector resampling process in the field of image processing[35]. Considering the function mentioned above, the effect of smooth interpolation was available for the one- dimensional data such as the rehabilitation motion trajectory data. The purpose of the resampling is to double the number of points in the rehabilitation motion DropBlock has two main parameters, bs is set to 7,theis 3.373×10-3 caculated from equation 15. After the first two convolution layers, using the advantage of the Inception structure to reduce the number of parameters, Inception-sim is proposed for the small amount of classification (in our work, we need 6 classifications) based on the InceptionV3 convolution network structure. Let the input point be x. Then, the weight of the Lanczos window function corresponding to each point is defined as sin( ) sin ( ) x c x x   = (12) sin ( )sin ( / ) ( ) 0 c x c x a if a x a L x otherwise −    =  (13) sin( ) sin ( ) x c x x   = (12) (12) Compared with traditional Inception-V3[37] module “one 5×5 convolution replaced by two 3×3 convolution” (“5th layer” module), Inception-Simin also added “N×1 and 1×N” module (“6th layer” module) to reduce the network over- fitting and speed up the network , the specific acceleration fusion module is shown in Figure.6 sin ( )sin ( / ) ( ) 0 c x c x a if a x a L x otherwise −    =  (13) (13) where a can be taken as 2 or 3, as it is the hyper parameter that corresponds to the adjustment and reduction interpolation or the amplification interpolation. model After using the ZCA whitening method to reduce the redundant information, the model takes the feature value stack map as input, and then sends it to the convolutional layer of 5×5, 3×3 convolution kernel, which is used to extract the fluctuation trend information of eigenvalues in the low frequency band. The DropBlock layer can be used to simulate noise and improve generalization ability[36]. The traditional dropout regularization technique is not used in the proposed network, because for the convolution layer, the feature map adjacent position elements share semantics in the spatial block area, and the structured DropBlock layer performs better in the convolution network where mentioned in the Discussions and Results section. Figure.5 The conversion process from eigenvalue data matrix to gray pixel matrix Grayscale Pixel Matrix Firstly, we need to extract a R-R sequence from a continuous signal as a sample, and the grayscale pixel matrix is transformed by the data matrix: ⬧ Extraction of kinematic eigenvalues: The angle signal and the angular velocity signals of this article were obtained by the encoder of the INNFOS disc motor. Due to the diversification of the motion of the ( , ) min( ) ( , ) 255 max( ) min( ) A i j A C i j A A − =  − (11) (11) 4 where i is the ordinal number of eigenvalues, j is the ordinal number of the sampling points of R-R time series, max/min (A) represents the maximum and minimum values of the pixel matrix, and C matrix is the normalized matrix. where i is the ordinal number of eigenvalues, j is the ordinal number of the sampling points of R-R time series, max/min (A) represents the maximum and minimum values of the pixel matrix, and C matrix is the normalized matrix. The input layer of the motion-intensity perception model consists of multi-modal information fusion data, and the expected output is the specific exercise intensity classification result. The deep neural network (DNN) method is used to implement the model. Figure.5 shows the conversion process from eigenvalue data matrix to gray pixel matrix. After the normalization of the Matrix from 0 to 255, the Pixel Matrix with dimension n×m is obtained. Secondly, the “square” structure element performs an open operation on the grayscale image to remove the process noise caused by the low signal-to-noise ratio. Finally, the time-frequency image with the size of 800 × 800 is adjusted to 227 × 227 by using the bicubic interpolation algorithm. 3.4.1 Neural Network Environment The programming uses the TensorFlow framework and the Keras library, trains the model in the Python environment, uses Nvidia’s CUDA parallel computing architecture to achieve hardware acceleration, and uses the C++ language API interface in the control software to connect the control program of the actuator. g Figure.5 The conversion process from eigenvalue data matrix to gray pixel matrix 3.3 Resampling of the Rehabilitation Motion Trajectory Because the purpose of this paper is to up sample, a is set to 3. Then, the set of reconstructed points corresponding to the specific reconstruction function is defined as follows: 1 ( ) ( ) x a i i x a S x s L x i + = −+ = −  (14) (14) where S(x) is the resampled value at the position, which is the sampled value of the original position. 3.4 Motion Intensity Mutation Perception Model 5 Figure.6 Inception-Simin module: From left to right, the third sub-column represents mini module of the “5th layer” module, the fourth sub-column represents mini module of “6th layer”. intensity mutation mode, the specific network model is shown in the Figure.8 and Table 3. intensity mutation mode, the specific network model is shown in the Figure.8 and Table 3. Figure.8 Motion intensity mutation perception model’s network structure Figure.8 Motion intensity mutation perception model’s network structure Figure.8 Motion intensity mutation perception model’s network structure TABLE 3. Summary of Motion Intensity Mutation Perception Model Layer Output layer dimension Kernel dimension Parameter Feature-in 227×227×3 Conv 1 113×113×64 5×5 Leaky_relu Maxpool 1 57×57×64 2×2 Stride=2 Conv 2 57×57×128 3×3 Leaky_relu Maxpool 2 27×27×128 2×2 Stride=2 DropBlock 27×27×128 7 =3.373e-3 Inception-Simin 27×27×256 1×1/3×3 /7×1/1×7 Relu Inception- Simdeep 19×19×384 Relu Inception- Simdeep 7×7×512 Relu AvgPool 3×3×512 2×2 Stride=2 DropBlock 3×3×512 1 =3.373e-3 FC 1 1×1×4608 Dropout=0.9 FC 2 1×1×512 Dropout=0.7 Softmax 1×1×6 3.5 Adaptive trajectory optimization algorithm based on DQN TABLE 3. Summary of Motion Intensity Mutation Perception Model Figure.6 Inception-Simin module: From left to right, the third sub-column represents mini module of the “5th layer” module, the fourth sub-column represents mini module of “6th layer”. Inception-Simdeep shown in Figure.7 is used in deep layers and reduces the feature map size to increase network depth by combining of “6th layer” and “7th layer”. An example is given to illustrate the meaning of “split or connect” indicated by the arrow. The 19×19×64 layer of conv2d is connected to the 27×27×96 Maxpool layer, and finally the 19×19×160 layer of Maxpool layer is generated. Its advantage is that it realizes the function of inception to reduce the parameters, and can save the calculation time and improve the accuracy in part, which is discussed in the Result and Discussion section. Figure.7 Inception-Simdeep module：”3×3” represents the size of kernel,”/2” represents stride of 2,”64” represents the amount of kernel 3.5 Adaptive trajectory optimization algorithm based on DQN 3.5 Adaptive trajectory optimization algorithm based on DQN Impedance control allows only the actual trajectory of the robot to be adjusted, rather than associated with the desired trajectory of the robot[38], which leads to the out of step of motor in the face of motion intensity mutation. 4.1 Demonstration teaching mode The zero position of the joint angle corresponds to the initial state, the elbow joint has degrees of freedom in flexion/extension, and the shoulder joints M1, M2 and M3 correspond to abduction/adduction, flexion/extension, and internal/external rotation degrees of freedom, respectively. The motion trajectory is shown in Figure.9. p y Table 4. Corresponding Meaning of the Value of m Value of m 1,2 3,4 5,6 Motion intensity mutation types M→S W→M W→S M→W S→M S→W M(Moderate), W(Weak), S(Strong) represents the type of motion intensity mutation. The reward function rewards the maintenance of “moderate” intensity actions, and punishes motion intensity mutation actions. Moreover, the penalty of intensity mutation across two grades (m=5,6) is much greater than that of across one grade (m = 1,2) and its concrete effect is shown in the reward function. p y Table 4. Corresponding Meaning of the Value of m Value of m 1,2 3,4 5,6 Motion intensity mutation types M→S W→M W→S M→W S→M S→W M(Moderate), W(Weak), S(Strong) represents the type of motion intensity mutation. The reward function rewards the maintenance of “moderate” intensity actions, and punishes motion intensity mutation actions. Moreover, the penalty of intensity mutation across two grades (m=5,6) is much greater than that of across one grade (m = 1,2) and its concrete effect is shown in the reward function. Table 4. Corresponding Meaning of the Value of m Value of m 1,2 3,4 5,6 Motion intensity mutation types M→S W→M W→S M→W S→M S→W j y g Figure.9 Motion trajectory of the shoulder and elbow joint angle 4.2 Design of the Rehabilitation motion decomposition Figure 9 Motion trajectory of the shoulder and elbow joint angle M(Moderate), W(Weak), S(Strong) represents the type of motion intensity mutation. The reward function rewards the maintenance of “moderate” intensity actions, and punishes motion intensity mutation actions. Moreover, the penalty of intensity mutation across two grades (m=5,6) is much greater than that of across one grade (m = 1,2) and its concrete effect is shown in the reward function. M(Moderate), W(Weak), S(Strong) represents the type of motion intensity mutation. The reward function rewards the maintenance of “moderate” intensity actions, and punishes motion intensity mutation actions. Moreover, the penalty of intensity mutation across two grades (m=5,6) is much greater than that of across one grade (m = 1,2) and its concrete effect is shown in the reward function. 4.1 Demonstration teaching mode * * * * ' * * ' 3 5 =1,2 ( ) 1 ( ) ( ) 50 =3,4 ( ) 1 ( ) ( ) ( ) 10 =5,6 ( ) 1 ( ) ( ) -5 ( ) 0.75, ( ) ( 3) s e s e a ss s e i a ss j i m D i U i U i m D i U i U i r i m D i U i U i r j D i D i = − −     +  +          +  +     =  −     +  +         −   (18) To realize the demonstration teaching mode, it is necessary to collect the motion track position signal through motion capture. When collecting motion track position signals, the INNFOS joint actuator is set to the “current” mode. In this state, the current in the actuator is zero, there is no holding torque, the upper limbs of a healthy person can directly drive the INNFOS joint actuator, and the integrated encoder will record the current movement track position data. (18) The motion trajectory is collected for the rehabilitation. The rehabilitation motion requires the coordination of the four main joints of the upper limbs of the human body. The encoder data has been converted into joint motion angle data. The motion trajectory is collected for the rehabilitation. The rehabilitation motion requires the coordination of the four main joints of the upper limbs of the human body. The encoder data has been converted into joint motion angle data. The zero position of the joint angle corresponds to the initial state, the elbow joint has degrees of freedom in flexion/extension, and the shoulder joints M1, M2 and M3 correspond to abduction/adduction, flexion/extension, and internal/external rotation degrees of freedom, respectively. The motion trajectory is shown in Figure.9. where m represents the mutation type of motion intensity prediction（Table 4 shows the corresponding meaning of m）, * ( ), ( ) s e U i U i   are differentials between the current position and the desired position of shoulder joint, elbow joint, respectively. 3.4 Motion Intensity Mutation Perception Model 7: Store the(Si,ai,r,Si+1) data in experience replay 8: 1 1 1 ( ) 0.01 Set max ( , , ') i i i i i i a r D i y r q S a Otherwise   + + +   = +  9: Gradient descent method to update network layer parameters for evaluating Q network： ( ) ( ) 2 , , i i i L y q S a  = − 7: Store the(Si,ai,r,Si+1) data in experience replay 8: 1 1 1 ( ) 0.01 Set max ( , , ') i i i i i i a r D i y r q S a Otherwise   + + +   = +  9: Gradient descent method to update network layer parameters for evaluating Q network： ( ) ( ) 2 , , i i i L y q S a  = − 7: Store the(Si,ai,r,Si+1) data in experience replay 8: 1 1 1 ( ) 0.01 Set max ( , , ') i i i i i i a r D i y r q S a Otherwise   + + +   = +  9: Gradient descent method to update network layer parameters for evaluating Q network： ( ) ( ) 2 , , i i i L y q S a  = − ⬧ Action Space: ⬧ Action Space: 10: Copy the network layer parameters of the evaluated network to the target network per completion of a training branch 10: Copy the network layer parameters of the evaluated network to the target network per completion of a training branch 11 d f ( ( ), ( )) t s e A t t   = (17) (17) where ( ) s t  , ( ) e t  are the angular velocity of shoulder joint and elbow joint servo motor respectively, through the control of motor angular velocity to achieve state transfer. ⬧ Reward Function: 4.1 Demonstration teaching mode The ε-greedy algorithm is used to select the action of the actual Q value: Figure.9 Motion trajectory of the shoulder and elbow joint angle actual Q value: 4.2 Design of the Rehabilitation motion decomposition y 3: For i=1, do 2: Read the initial value of exoskeleton manipulator joint position and motion intensity state 5: Execute corresponding trajectory optimization action At and calculate the reward function r(i) 3.4 Motion Intensity Mutation Perception Model Finally, through the whole connection layer Softmax output six kinds of motion (16) ( ( ), ( ), ( ))T i s e t S U i U i D i = ( ( ), ( ), ( ))T i s e t S U i U i D i = 6 7: Store the(Si,ai,r,Si+1) data in experience replay 8: 1 1 1 ( ) 0.01 Set max ( , , ') i i i i i i a r D i y r q S a Otherwise   + + +   = +  9: Gradient descent method to update network layer parameters for evaluating Q network： ( ) ( ) 2 , , i i i L y q S a  = − 10: Copy the network layer parameters of the evaluated network to the target network per completion of a training branch 11: end for 12:end for where Us(i) is the angle signal value converted by shoulder encoder, Ue(i) is the angle signal value converted by the elbow encoder, De(i) is the distance value between the target point and the current position in the Cartesian space coordinate system. 3.4 Motion Intensity Mutation Perception Model The reason is that when the output of the joint motor does not reach the desired position, a new position control signal is received, and the original position control signal is overwritten, resulting in the deformation of the motion trajectory. Faced with such problems, this paper proposes a trajectory optimization algorithm aiming at stabilizing motion intensity. The strategy is to optimize the dynamic trajectory in each time window, and adjust the joint drive motor speed based on DQN (Deep Q Network) algorithm to keep the motion intensity in the “moderate” state. With the iteration of the algorithm, the deviation between the actual trajectory and the desired trajectory gradually tends to 0. Figure.7 Inception-Simdeep module：”3×3” represents the size of kernel,”/2” represents stride of 2,”64” represents the amount of kernel Markov decision process is generally described using Multi- tuple (S, A, P, R, γ), S is finite state set, A is action set, P is finite state transition conditional probability, r is reward function, γ is discount factor of reward function, used to measure long-term reward and penalty. ⬧ State Space: Through the collaboration of Inception-Sim layer and DropBlock layer to achieve acceleration, the implementation of “first Inception-Sim and then “Avgpooling” mode is conducive to maintaining feature stability, but it increases the complexity of calculation by three times. Finally, through the whole connection layer Softmax output six kinds of motion Through the collaboration of Inception-Sim layer and DropBlock layer to achieve acceleration, the implementation of “first Inception-Sim and then “Avgpooling” mode is conducive to maintaining feature stability, but it increases the complexity of calculation by three times. Table 5. Algorithm：Adaptive Trajectory Optimization Algorithm based on DQN 1:For episode=1, do 2: Read the initial value of exoskeleton manipulator joint position and motion intensity state 3: For i=1, do 4: With ε probability random logic True: Randomly selected action: Ai False: Select the action corresponding to the maximum Q value 5: Execute corresponding trajectory optimization action At and calculate the reward function r(i) 6: Record the next time slice of the manipulator system：Si+1 4.2 Design of the Rehabilitation motion decomposition 1- + a=arg max ( , ) ( ) a arg max ( , ) a a if q s a k a s if q s a k             (19) The motion intensity mutation perception model has achieved good results in offline testing, but the use of the model in actual scenarios requires the online model to achieve real-time interaction. The designed rehabilitation experiment is conducted on the two basic degrees of flexion/extension freedom of the shoulder and elbow joint. The specific action decomposition diagram we adopted is shown in Figure.10. The motion trajectory of the actuator is collected through the demonstration teaching mode as shown in Figure.11. (19) where  is the hyper-parameter of the algorithm; k(set k=4) represents the amount of action types that the algorithm can choose. In the specific implementation algorithm of trajectory optimization strategy based on DQN, the pseudo code is shown in Table 5. 7 Figure.10 Rehabilitation motion decomposition Table 5. Algorithm：Adaptive Trajectory Optimization Algorithm based on DQN Table 5. Algorithm：Adaptive Trajectory Optimization Algorithm based on DQN 1:For episode=1, do 2: Read the initial value of exoskeleton manipulator joint position and motion intensity state 3: For i=1, do 4: With ε probability random logic True: Randomly selected action: Ai False: Select the action corresponding to the maximum Q value 5: Execute corresponding trajectory optimization action At and calculate the reward function r(i) 6: Record the next time slice of the manipulator system：Si+1 4: With ε probability random logic True: Randomly selected action: Ai False: Select the action correspondin p g 5: Execute corresponding trajectory optimization action At and calculate the reward function r(i) Figure.10 Rehabilitation motion decomposition 7 Figure.11 Rehabilitation motion trajectory through the demonstration teaching mode The data obtained by the online motion intensity mutation perception model are shown in Table 6. The results show that in the first three rehabilitation cycles, the motion intensity of the subjects is mainly concentrated in the mutual transformation between “strong” and “moderate”. The main reason is that the subjects are full of energy at the beginning of the experiment, producing a strong interactive force between the muscles and the manipulator. 4.2 Design of the Rehabilitation motion decomposition During the last two cycles, the subjects’ motion intensity gradually changed from “moderate” to “weak”, accompanied by a decrease in motion velocity and deformation of the motion trajectory, which was caused by the subject's muscle fatigue and a decrease of upper limb strength. Figure.11 Rehabilitation motion trajectory through the demonstration teaching mode As shown in Figure.12, the exoskeleton arm was fixed on the aluminium alloy steel frame. Additionally, the upper arm and the forearm were loaded with extra 1.0 kg weights. The simulated patient wore the exoskeleton arm by grasping the grip of this arm with the right hand. The nylon bolts were tightened to adjust the overall length to a comfortable location. The heart rate sensor was worn on the patient’s left-hand finger. After confirmation that the setup was correct, we turned on the power for the rehabilitation experiment. This experiment was conducted in five consecutive rehabilitation cycles with a pause of 5 seconds after each cycle. In summary, the data obtained by the online perception model are basically consistent with the motion trajectory measured in the experiment. The classification accuracy of the perception model has been verified, laying a foundation for the adaptive trajectory optimization algorithm based on DQN. p j y p g Table 6. Data of Online Motion Intensity Perception Model Intensity The first The second The third The fourth The fifth Strong 25.2% 24.3% 6.6% 0.0% 0.0% Moderate 74.8% 75.9% 65.3% 13.4% 18.8% Weak 0.0% 0.0% 28.1% 86.6% 81.2% y p y Figure.12 Rehabilitation motion experimental platform 5.1 Multi-mode information fusion The multi-mode feature vector constructed in this paper was composed of ECG eigenvalues and kinematic eigenvalues. A parallel coordinate plot adopting the standardized pre- processing is shown in Figure.14(a). For the intensity label, the solid line indicates that the prediction vector of the model matches the actual label, while the dashed line is opposite. From the perspective of feature value dimension, the feature vectors of four-dimensional heart rate signal are densely distributed with high accuracy, while the feature vectors of two-dimensional kinematic signal are distributed dispersedly, which is roughly due to the relative hysteresis and anisotropy of the kinematic signal. Figure.12 Rehabilitation motion experimental platform 4.3 Verification and optimization of the motion- intensity perception model1.1 Subsection heading Model Evaluation On MIT-BIH ECG Compression Test Database(CDB) Intensit y W to S Precisi on/Rec all M to S Precisi on/Rec all Overall Precisi on Overall Recall F1 ACR （1/s ） MLP- CNN 0.6145/ 0.6568 0.7985/ 0.6125 0.6342 0.5852 0.6087 4.032e- 3 ECG- CNN 0.7652/ 0.4823 0.9456/ 0.6935 0.8654 0.6561 0.7463 2.932e- 3 VGGN ET 0.8211/ 0.7520 0.6425/ 0.5354 0.7524 0.5628 0.6439 3.932e- 3 34- layer CNN 0.8452/ 0.6245 0.8012/ 0.6723 0.8124 0.6724 0.7358 4.932e- 3 Propos ed method 0.9223/ 0.6123 0.8145/ 0.2453 0.8721 0.6242 0.7276 5.132e- 3 Figure.15 Radar chart of classification effect of CDB dataset Table 7. Model Evaluation On MIT-BIH ECG Compression Test Database(CDB) Intensit y W to S Precisi on/Rec all M to S Precisi on/Rec all Overall Precisi on Overall Recall F1 ACR （1/s ） MLP- CNN 0.6145/ 0.6568 0.7985/ 0.6125 0.6342 0.5852 0.6087 4.032e- 3 ECG- CNN 0.7652/ 0.4823 0.9456/ 0.6935 0.8654 0.6561 0.7463 2.932e- 3 VGGN ET 0.8211/ 0.7520 0.6425/ 0.5354 0.7524 0.5628 0.6439 3.932e- 3 34- layer CNN 0.8452/ 0.6245 0.8012/ 0.6723 0.8124 0.6724 0.7358 4.932e- 3 Propos ed method 0.9223/ 0.6123 0.8145/ 0.2453 0.8721 0.6242 0.7276 5.132e- 3 nates plot with scaling of Normalization Table 7. Model Evaluation On MIT-BIH ECG Compression Test Database(CDB) l Evaluation On MIT-BIH ECG Compression Test Database(CDB) (b). Parallel coordinates plot with scaling of Normalization Figure.14 Parallel coordinates plot with scaling of Standardization and Normalization Figure.14 Parallel coordinates plot with scaling of Standardization and Normalization 5.2.2 Evaluation index Precision, Recall, ACR (average convergence rate) and F1 are used as network performance evaluation indexes, which are expressed as follows: 1 1 2 average convergence rate epoch time TP Recall TP FN Recall F Recall TP Precision=TP FP Precision Precision +  + = = + =  (20) 1 1 2 average convergence rate epoch time TP Recall TP FN Recall F Recall TP Precision=TP FP Precision Precision +  + = = + =  (20) TP Precision=TP FP + (20) where epoch time represents the overall time when the loss function value stabilizes, TP FP TN FN represents for True Positives, False Positives, True Negatives and False Negatives, respectively. 5.2.1 Baselines In order to verify the performance of the proposed model in individual motion intensity mutation monitoring, the multiple state-of-the-art methods are listed below for comparison: MLP-CNN[39]: The model includes two algorithms which were integrated in a concise and effective way using a rule- based decision fusion approach. ECG-CNN[40]: The deep two-dimensional CNN for ECG arrhythmia classification using the Xavier initialization and exponential linear units. p VGGNET[41]:The model uses very small (3x3) convolution filters thorough evaluation of networks with the depth to 16-19 weight layers. p g y 34-layer CNN[42]: The network contains 33 layers of convolution followed by a fully connected layer and a softmax. Besides the above networks, we also compare with widely- used image classification frameworks, such as VGG16, Resnet50 and Inception-V3. Figure.15 Radar chart of classification effect of CDB dataset Table 8. Model Evaluation On MIVIE Database Figure.15 Radar chart of classification effect of CDB dataset Table 8. Model Evaluation On MIVIE Database Table 8. Model Evaluation On MIVIE Database Intensi ty W to S Precisi on/Rec all M to S Precisi on/Rec all Overall Precisi on Overall Recall F1 ACR （1/s ） MLP- CNN 0.8522/ 0.8268 0.7565/ 0.7245 0.8245 0.7852 0.8043 4.724e- 2 ECG- CNN 0.8678/ 0.8521 0.8785/ 0.6732 0.8425 0.7541 0.7958 5.584e- 2 VGGN ET 0.8842/ 0.8289 0.9752/ 0.8354 0.9124 0.5628 0.6961 6.4263 2e-2 34- layer CNN 0.8452/ 0.6245 0.8012/ 0.6723 0.8124 0.6842 0.7428 7.546e- 2 VGG1 6 0.8183/ 0.6423 0.8523/ 0.6247 0.8345 0.6423 0.7258 5.542e- 2 Resnet 50 0.8745/ 0.8523 0.9123/ 0.7825 0.8915 0.8354 0.8625 7.678e- 2 Incepti on-V3 0.8824/ 0.8524 0.9254/ 0.7852 0.9041 0.8123 0.8557 6.852e- 2 Propos ed metho d 0.8952/ 0.8123 0.9345/ 0.7845 0.9081 0.7563 0.8252 9.132e- 2 4.3 Verification and optimization of the motion- intensity perception model1.1 Subsection heading The transformation from an offline model to an online model requires optimization of the program packaging interface and running time. The engineering optimization is mainly to use the CPU multi-threading feature, which can encapsulate the perception model and translate it into the Visual Studio 2017 platform. The control program uses the reserved input layer and output layer program interfaces. After the test, a thread is established in the main program for data collection, and multi-mode fusion and standardization are performed. The running time of identification using the perception model is approximately 1.8 s, the thread resource occupancy rate is in line with expectations, and the heart rate signal acquisition cycle is 3 seconds. In the designed experiment, the motion intensity will be initially set to the medium intensity, and the online perception model will correct the real-time exercise intensity every 5 seconds. The encoder records five consecutive cycles of motion trajectory, which are shown in Figure.13. The vectors preprocessed by standardization are more densely distributed under the same label and more dispersed under different labels, which is convenient for classification and identification of the model. In contrast, the vectors shown in Figure.14(b) that are preprocessed by normalization are scattered under the same label, mainly because normalization does not change the relative order of the data, but it does not keep the space between the dimensions of the initial vector. Finally, the gradient identification of each dimension of the initial vector disappears. (a). Parallel coordinates plot with scaling of Standardization g Figure.13 Experimental motion trajectory of five consecutive cycles (a). Parallel coordinates plot with scaling of Standardization (a). Parallel coordinates plot with scaling of Standardization Figure.13 Experimental motion trajectory of five consecutive cycles 8 (b). Parallel coordinates plot with scaling of Normalization Figure.14 Parallel coordinates plot with scaling of Standardization and Normalization Table 7. 5.2.3 Comparison with the state-of-the-art methods We compared the proposed method with the latest method, and tested on MIT-BIH ECG Compression Test database (CDB) and MIVIE database(collected by experiments), respectively. To save space, we show two typical classification labels (W to S, to S) precision and recall (left of the table), overall (right of the table) precision, recall, F1 scores and ACR. In all experiments and evaluations of the proposed method is based on the results of 5-fold cross-validation. 9 Figure 16 Radar Chart of Classification Effect of MIVIE Dataset Figure.17 t-SNE embeddings of multimode feature vectors from different subjects Figure.17 t-SNE embeddings of multimode feature vectors from different subjects j The different colours of W(Weak), M(Moderate), and S(Strong) in the figure represent the multi-mode vector of weak motion intensity, moderate motion intensity and strong motion intensity, respectively. The position of the letter is determined by the value of the original multi-mode fusion vector data through the dimensionality reduction. The first three graphs separately represent the experimental data of each subject, and the fourth graph combines the data of three subjects and then draws them by the intensity classification. Analysing the position of the multi-mode vector data after the dimensionality reduction shows that the distribution of the individual multi-mode fusion vectors has a high degree of discrimination. The same letters are clustered together, and different letters are far apart. The result of the dimensionality reduction for the classification is more extreme. Figure.16 Radar Chart of Classification Effect of MIVIE Dataset Figure.16 Radar Chart of Classification Effect of MIVIE Dataset Results are demonstrated in Table 7, Table 8, respectively. In order to observe the results more intuitively, we visualize the model performance evaluation into Radar Chart shown in Figure 15 and Figure 16 through data visualization. On the whole, poor performance of classification methods based on time series (ECG-CNN and 34-layer CNN) can be observed in both datasets. It is worth noting that the general image classification framework (VGG16, inception-v3, Resnet50) achieves better performance than these digital-based approaches. This situation is in line with our original intention of transformation from eigenvalue data matrix to grayscale pixel matrix, that is, the application of time series data to image classification framework will be limited. 5.2.3 Comparison with the state-of-the-art methods When the proposed method comparing with these conventional image classification frameworks, index of Precision, F1, and Recall remains at a high level but the most prominent performance index is ACR, the structure optimization of our improved inception-CNN network plays a key role in reducing redundant network parameters and improving network running time. In the first two graphs, the classifications of the letter S and the letter M is more cross-over, which can also support the source of error between strong motion intensity and medium motion intensity in the confusion matrix. Compared with the first three figures, the mixed fourth figure shows that the distribution of the different letters is relatively scattered. The same letter logos converge into clusters, and more logos converge into multiple coherent blocks. On the whole, the model still shows a certain degree of discrimination for data with large individual differences. The results verify that the method of adding kinematic eigenvalues to the multi-mode fusion vector can alleviate the negative impact of the ECG signal due to individual differences to a certain extent. 5.4 Analysis of adaptive trajectory optimization algorithm ATO(adaptive trajectory optimization)-DQN algorithm emphasizes on dynamic state interaction. In order to improve the processing speed of the algorithm, the offline data pre- training of Evaluating Q Network is carried out. The ratio of offline and online samples with motion intensity mutation labels is 1:5, and the feedback angle information recorded in the pre-training process is put into experience replay. 6. Conclusions In this paper, the time domain, the frequency domain eigenvalue of ECG and the angular velocity deviation of the encoder were extracted as the input layer of the motion intensity variation interval estimation model, which was constructed for classification and identification. In the experiment, the results verify that the method of adding kinematic eigenvalues to the multi-mode fusion vector can alleviate the negative impact of the ECG signal due to individual differences to a certain extent. When the proposed method comparing with those conventional image classification frameworks, the structure optimization of our improved inception-CNN network plays a crucial role in reducing redundant network parameters and improving network running time. After the optimization of ATO-DQN algorithm, the tracking effect of motion trajectory on real-time environmental changes enhance, which can better guarantee the motion safety of patients. Figure.19 Performance of reward value and convergence rate under different learning rates. When the learning rate e is 0.7, the model step length is too short and the convergence speed is too slow; When the learning rate is 0.98, the convergence speed is greatly improved but can not converge. Figure.19 shows the performance of reward value and convergence rate under different learning rates. For each motion cycle, 400 groups of samples can be obtained and the learning rate (e = 0.9) is used to accelerate the iteration. Then, the action output is randomly selected by the ε-greed algorithm, and the stability is high under multiple tests. Our work also has many limitations. Compared with other methods, the precision, recall and F1 index have not been significantly improved. In Analysis of adaptive trajectory optimization algorithm, the types and evaluation indicators of DQN methods are not comprehensive enough. Most importantly, the current technology does not have good versatility in the application of manipulators, only in the angular velocity sensor and ECG signal multimodal feature vector shows good result. In subsequent research, our work can refine the classification limit of motion intensity and improve the speed correction accuracy of trajectory. We hope the trajectory correction technology will develop towards the direction of generalization and diversification. The rehabilitation motion trajectory used in the perception model validation experiment is imported into ATO-DQN algorithm model as the test datasets, and the real-time motion intensity is set as the mixed state of “Strong” and “Weak”, and the trajectory control signal is outputted to the exoskeleton manipulator and executed without load. 5.3 Analysis of individual differences It is not sufficiently comprehensive to analyse the problem of individual differences based on the classification accuracy of the perception model. We randomly extract 100 sets of data from each measured exercise intensity label, and we use the t- distributed stochastic neighbour embedding (t-SNE) method to perform dimensionality reduction and visualization of the data. The t-SNE method is used to reduce the dimensionality of the multi-mode fusion vector to two dimensions. The two- dimensional distribution graphics are shown in Figure.17. 0 10 20 30 40 50 60 0 10 20 30 40 50 60 70 80 Cumulative delayed reward Episode MADQN DQN ATO-DQN Figure.18 Comparison of cumulative delayed reward for MADQN, traditional DQN and ATO—DQN Figure.18 Comparison of cumulative delayed reward for MADQN, traditional DQN and ATO—DQN The cumulative delayed reward for MADQN (Multi-agent DQN), traditional DQN and ATO-DQN under motion intensity mutation state as the episode increases is shown in Fig.18. Attributed to the pre-training operation, the convergence speed and stability of ATO-DQN algorithm are significantly higher than those of MADQN and DQN. More obviously, ATO-DQN algorithm obtains higher cumulative delayed reward. In contrast, MADQN is more suitable for multi-agent (at least more than two agents) cooperative control. 10 0 5 10 15 20 25 30 35 40 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 Cumulative reward value Episode e=0.9 e=0.7 e=0.98 Figure.19 Performance of reward value and convergence rate under different learning rates. When the learning rate e is 0.7, the model step length is too short and the convergence speed is too slow; When the learning rate is 0.98, the convergence speed is greatly improved but can not converge. 0 5 10 15 20 25 30 35 40 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 Cumulative reward value Episode e=0.9 e=0.7 e=0.98 is small, which can better guarantee the motion safety of patients. Abbreviations ECG: Electrocardiogram; MIVIE: Motion Intensity Variation Interval Estimation; ATO-DQN: Adaptive Trajectory Optimization-Deep Q Network; MADQN: Multi-agent DQN; CNN: Convolutional Neural Network; ACR: average convergence rate (a). Rehabilitation motion trajectory without ATO-DQN algorithm Rehabilitation motion trajectory with ATO-DQN algorithm 6. Conclusions The actual motion trajectory is shown in Figure. 20(a), while the trajectory without algorithm optimization is shown in Figure. 20(b). (a). Rehabilitation motion trajectory without ATO-DQN algorithm (b). Rehabilitation motion trajectory with ATO-DQN algorithm Contributions Figure.20 Comparison of the trajectories of two states Figure.20 Comparison of the trajectories of two states W.W. implemented and performed the experiments, analyzed the data and wrote the manuscript. H.L. performed the experiments, analyzed the data and wrote the manuscript, C.Z. conceived the experiment, analyzed the data and critically revised the manuscript. D.K. performed the experiments and critically revised the manuscript, P.Z. conceived the experiment and critically revised the manuscript. It can be seen from Figure.20 that the output speed of the control method without ATO-DQN algorithm is relatively unstable in the face of continuous motion state mutation, resulting in the distortion of the actual output trajectory. The trajectory of elbow and shoulder joint begins to jitter and deform between 0~20% cycles (Corresponding to the first two stages in Table 6, namely the transition stage from “strong” to “moderate” state). Acknowledgements We thank the participants in this study for their willingness to devote energy and sweat to completing the research work of this project. (b). Rehabilitation motion trajectory with ATO-DQN algorithm otion trajectory with ATO-DQN algorithm References [1] C. Fisahn, M. Aach, O. Jansen, M. Moisi, A. Mayadev, K. T. Pagarigan, J. R. Dettori, and T. A. 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Encouraging data from a randomised-clinical-trial," Clinical Neurophysiology, vol. 130, pp. 767-780, 2019. [9] J. Sun, Z. Guo, D. Sun, S. He, and X. Xiao, "Design, modeling and control of a novel compact, energy-efficient, and rotational serial variable stiffness actuator (SVSA-II)," Mechanism and Machine Theory, vol. 130, pp. 123-136, 2018. [27] B. Peerdeman, D. Boere, H. Witteveen, H. Hermens, S. Stramigioli, H. Rietman, P. Veltink, and S. Misra, "Myoelectric forearm prostheses: state of the art from a user-centered perspective.," Journal of Rehabilitation Research & Development, vol. 48, 2011. [10] N. Yozbatiran, J. Berliner, M. O Malley, A. Pehlivan, Z. Kadivar, C. Boake, and G. Francisco, "Robotic training and clinical assessment of upper extremity movements after spinal cord injury: A single case report," Journal of Rehabilitation Medicine, vol. 44, p. 186, 2012. [28] Z. Comert, A. M. Boopathi, S. Velappan, Z. Yang, and A. F. Kocamaz, "The influences of different window functions and lengths on image-based time-frequency features of fetal heart rate signals," in 2018 26th Signal Processing and Communications Applications Conference (SIU), 2018, pp. 1-4. [11] F. Zyener, H. B. Rossiter, S. A. Ward, and B. J. Ethics approval and consent to participate The data acquisition protocol was approved by Medical and Experimental Animal Ethics Committee of Northwestern Polytechnical University, Xi'an, China (approbation number: 6101030222595-202001001). [16] W. Wang, A. Tan and L. Teow, "Semantic memory modeling and memory interaction in learning agents," IEEE Transactions on Systems, Man, and Cybernetics: Systems, vol. 47, pp. 2882-2895, 2016. [17] G. Petmezas, K. Haris, L. Stefanopoulos, V. Kilintzis, A. Tzavelis, J. A. Rogers, A. K. Katsaggelos, and N. Maglaveras, Funding In contrast, after the optimization of ATO-DQN algorithm, due to the action selection refinement and dynamic interaction ability, the tracking effect of motion trajectory on real-time environmental changes is enhanced, and the overall fluctuation This work is supported by the Natural Science Foundation of Shaanxi Province (Grant No. 2020JM-131 and 2020KW- 058), the Guangdong Basic and Applied Basic Research 11 Foundation (2019A1515111176), and the Guangdong Science and Technology Innovation Strategy Special Foundation (2019B090904007). Foundation (2019A1515111176), and the Guangdong Science and Technology Innovation Strategy Special Foundation (2019B090904007). [14] F. S. Lin and K. Dana, "Online Segmentation of Human Motion for Automated Rehabilitation Exercise Analysis," IEEE Transactions on Neural Systems and Rehabilitation Engineering, 2013. [15] M. Längkvist, L. Karlsson and A. Loutfi, "A review of unsupervised feature learning and deep learning for time-series modeling," Pattern Recognition Letters, vol. 42, pp. 11-24, 2014. Competing in interests "Automated Atrial Fibrillation Detection using a Hybrid CNN- LSTM Network on Imbalanced ECG Datasets," Biomedical Signal Processing and Control, vol. 63, p. 102194, 2021. [18] T. Karlsen, I. Aamot, M. Haykowsky, and Ø. Rognmo, "High Intensity Interval Training for Maximizing Health Outcomes " "Automated Atrial Fibrillation Detection using a Hybrid CNN- LSTM Network on Imbalanced ECG Datasets," Biomedical Signal Processing and Control, vol. 63, p. 102194, 2021. The author(s) declare(s) that they have no competing interests. The author(s) declare(s) that they have no competing interests. p [18] T. Karlsen, I. Aamot, M. Haykowsky, and Ø. Rognmo, "High Intensity Interval Training for Maximizing Health Outcomes," Progress in Cardiovascular Diseases, vol. 60, pp. 67-77, 2017. References Whipp, "Influence of exercise intensity on the on- and off-transient kinetics of pulmonary oxygen uptake in humans," Journal of Physiology, vol. 533, pp. 891-902, 2010. pp [29] M. Wewege, R. van den Berg, R. E. Ward, and A. Keech, "The effects of high-intensity interval training vs. moderate-intensity continuous training on body composition in overweight and obese adults: a systematic review and meta-analysis," Obesity Reviews, vol. 18, pp. 635-646, 2017. [12] Y. BIN and Q. JIANG, "Human Motion Intensity Detection Embedded in Holter," Chinese Journal of Medical Physics, p. 18, 2012. [13] H. Lee, H. Chung, H. Ko, and J. Lee, "Wearable Multichannel Photoplethysmography Framework for Heart Rate Monitoring During Intensive Exercise," IEEE Sensors Journal, vol. 18, pp. 2983-2993, 2018. [30] K. Ranjeet, A. Kuamr and R. K. Pandey, "ECG signal compression using optimum wavelet filter bank based on Kaiser window," Procedia engineering, vol. 38, pp. 2889-2902, 2012. 12 [31] S. Cuomo, G. De Pietro, R. Farina, A. Galletti, and G. Sannino, [31] S. Cuomo, G. De Pietro, R. Farina, A. Galletti, and G. Sannino, "A revised scheme for real time ecg signal denoising based on recursive filtering," Biomedical Signal Processing and Control, vol. 27, pp. 134-144, 2016. [32] R. R. Sharma and R. B. Pachori, "Baseline wander and power line interference removal from ECG signals using eigenvalue decomposition," Biomedical Signal Processing and Control, vol. 45, pp. 33-49, 2018. [33] Y. Xiang, Z. Lin and J. Meng, "Automatic QRS complex detection using two-level convolutional neural network," Biomedical engineering online, vol. 17, pp. 1-17, 2018. [34] S. A. Murray, K. H. Ha, C. Hartigan, and M. Goldfarb, "An assistive control approach for a lower-limb exoskeleton to facilitate recovery of walking following stroke," IEEE Transactions on Neural Systems and Rehabilitation Engineering, vol. 23, pp. 441- 449, 2014. [35] G. Budzik and P. Turek, "Improved accuracy of mandible geometry reconstruction at the stage of data processing and modeling," AUSTRALASIAN PHYSICAL \& ENGINEERING SCIENCES IN MEDICINE, vol. 41, pp. 687-695, 2018. [36] N. Srivastava, G. Hinton, A. Krizhevsky, I. Sutskever, and R. Salakhutdinov, "Dropout: a simple way to prevent neural networks from overfitting," The journal of machine learning research, vol. 15, pp. 1929-1958, 2014. [37] C. Szegedy, V. Vanhoucke, S. Ioffe, J. Shlens, and Z. Wojna, "Rethinking the Inception Architecture for Computer Vision,", 2016, pp. 2818-2826. [38] X. Wu, Z. Li, Z. Kan, and H. References Gao, "Reference Trajectory Reshaping Optimization and Control of Robotic Exoskeletons for Human–Robot Co-Manipulation," IEEE Transactions on Cybernetics, vol. 50, pp. 3740-3751, 2020. [39] C. Zhang, X. Pan, H. Li, A. Gardiner, I. Sargent, J. Hare, and P. M. Atkinson, "A hybrid MLP-CNN classifier for very fine resolution remotely sensed image classification," ISPRS Journal of Photogrammetry and Remote Sensing, vol. 140, pp. 133-144, 2018. [40] T. J. Jun, H. M. Nguyen, D. Kang, D. Kim, D. Kim, and Y. Kim, "ECG arrhythmia classification using a 2-D convolutional neural network," arXiv preprint arXiv:1804.06812, 2018. [41] K. Simonyan and A. Zisserman, "Very Deep Convolutional Networks for Large-Scale Image Recognition," arXiv preprint arXiv:1409.1556, 2014. [42] P. Rajpurkar, A. Y. Hannun, M. Haghpanahi, C. Bourn, and A. Y. Ng, "Cardiologist-Level Arrhythmia Detection with Convolutional Neural Networks," arXiv preprint arXiv:1707.01836, 2017. 13 Figures Figures Figures Figure 1 BMD101 Bluetooth ECG acquisition module Figure 1 Figure 4 Figure 4 Multimode vector diagram Multimode vector diagram Figure 5 The conversion process from eigenvalue data matrix to gray pixel matrix Figure 1 BMD101 Bluetooth ECG acquisition module BMD101 Bluetooth ECG acquisition module Figure 2 Figure 2 Figure 2 Rehabilitation scenario Rehabilitation scenario Figure 3 Comparison of denoising effect of improved Coif4 wavelet threshold algorithm and original signal Figure 3 Comparison of denoising effect of improved Coif4 wavelet threshold algorithm and original signal Figure 3 Comparison of denoising effect of improved Coif4 wavelet threshold algorithm and original signal Comparison of denoising effect of improved Coif4 wavelet threshold algorithm and original signal Figure 4 Multimode vector diagram Figure 5 Figure 5 The conversion process from eigenvalue data matrix to gray pixel matrix ure 6 eption-Simin module: From left to right, the third sub-column represents mini module of the “5th laye odule, the fourth sub-column represents mini module of “6th layer”. Figure 6 Inception-Simin module: From left to right, the third sub-column represents mini module of the “5th layer” module, the fourth sub-column represents mini module of “6th layer”. Figure 7 nception-Simdeep module฀”3×3” represents the size of kernel,”/2” represents stride of 2,”64” represent the amount of kernel Figure 7 Figure 7 Inception-Simdeep module฀”3×3” represents the size of kernel,”/2” represents stride of 2,”64” represents the amount of kernel Figure 8 Motion intensity mutation perception model’s network structure Figure 9 Motion trajectory of the shoulder and elbow joint angle Figure 8 Motion intensity mutation perception model’s network structure Figure 9 Motion trajectory of the shoulder and elbow joint angle Motion intensity mutation perception model’s network structure Motion intensity mutation perception models network structure Figure 9 Motion trajectory of the shoulder and elbow joint angle Figure 10 Rehabilitation motion decomposition Figure 10 Figure 10 Rehabilitation motion decomposition Figure 11 Rehabilitation motion trajectory through the demonstration teaching mode Figure 12 Rehabilitation motion experimental platform Figure 13 Experimental motion trajectory of ¦ve consecutive cycles Figure 11 Figure 11 Rehabilitation motion trajectory through the demonstration teaching mode Rehabilitation motion trajectory through the demonstration teaching mode Rehabilitation motion trajectory through the demonstration teaching mode Figure 12 Rehabilitation motion experimental platform p p Figure 13 Experimental motion trajectory of ¦ve consecutive cycles Experimental motion trajectory of ¦ve consecutive cycles arallel coordinates plot with scaling of Standardization and Normalization Figure 14 Parallel coordinates plot with scaling of Standardization and Normalization Figure 15 Radar chart of classi¦cation effect of CDB dataset Figure 16 Radar Chart of Classi¦cation Effect of MIVIE Dataset Radar Chart of Classi¦cation Effect of MIVIE Dataset Figure 17 t-SNE embeddings of multimode feature vectors from different subjects Figure 17 t-SNE embeddings of multimode feature vectors from different subjects t-SNE embeddings of multimode feature vectors from different subjects Figure 18 Comparison of cumulative delayed reward for MADQN, traditional DQN and ATO—DQN Figure 18 Comparison of cumulative delayed reward for MADQN, traditional DQN and ATO—DQN Figure 19 Performance of reward value and convergence rate under different learning rates. When the learning rate e is 0.7, the model step length is too short and the convergence speed is too slow; When the learning rate is 0.98, the convergence speed is greatly improved but can not converge. Figure 19 Figure 19 Performance of reward value and convergence rate under different learning rates. When the learning rate e is 0.7, the model step length is too short and the convergence speed is too slow; When the learning rate is 0.98, the convergence speed is greatly improved but can not converge. Performance of reward value and convergence rate under different learning rates. When the learning rate e is 0.7, the model step length is too short and the convergence speed is too slow; When the learning rate is 0.98, the convergence speed is greatly improved but can not converge. Fi 20 Figure 20 Comparison of the trajectories of two states Comparison of the trajectories of two states
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Catalan; Valencian
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La poesía catalana contemporània: Un breu itinerari a la recerca d1una tradició
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1. Totes les literatures del món occidental pateixen la síndrome del moviment. Quan hi ha un corrent que predomina, els autors en voga utilitzen tots els mitjans perquè el lector oblidi que hi ha altres manifestacions literàries que o bé són diferents, o bé havien estat interessants immediatament abans d’ells. Cauen en la vella rutina de voler matar els pa- res. Aquest fet és cada cop més evident en el món modern, on els mitjans de comunicació s’han convertit de vegades en els grans aliats de les aparicions agosarades, arriscades, trencadores dels nous autors. Això no vol pas dir, però, que els autors nous puguin acon- seguir el seu objectiu. La pròpia literatura, o el conjunt de lectors, la societat o el que la nova teoria de torn afirmi s’encarrega de fer filtrar els autors que persisteixen en unes contingències determinades, les obres que representen fites en el moviment literari ante- rior. S’assisteix, aleshores, a una activitat de balanceig en què trobar l’equilibri equival a tenir la capacitat per poder destriar quins seran els objectius de la nova fornada d’autors, sense oblidar els vells creadors que malden per mantenir les seves tendències. És un balanceig de vegades dramàtic en què es veu involucrada tota la màquina que la societat contemporània ha format al voltant de la literatura, que ens guardarem prou de titllar com a necessària o innecessària per al seu creixement. Cauen alguns autors, plens d’ira per la impotència de la seva veu que ja no se sent als mitjans de comunicació, i pugen com l’escuma els creadors novells, omplint tots els espais de què són capaços, amb una golafre- ria abusiva per aconseguir els estratagemes del poder. La situació descrita és necessària per a la renovació d’una cultura, d’una literatura, sempre i quan aquesta gaudeixi de bona salut. Però pot arribar a tenir conseqüències força negatives, encara que no sempre ha de ser d’aquesta manera, en una altra que o bé és molt més fràgil, o bé és d’un àmbit molt reduït. És, efectivament, aquest darrer element el que entra en joc en la literatura catalana. Fins fa no gaire, encara havíem de tenir en compte també el primer. Si, a més, el terreny no és en la prosa, sinó en la poesia, un gènere que, com se sap, és només de minories, la situació ja és dramàtica fins a un extrem. Xavier Farré Universitat de Ljubljana Xavier Farré Universitat de Ljubljana 1. L’any 2004, fa només un parell de mesos, un fenomen ha trasbalsat el panorama de la poesia catalana. Es presentà, enmig d’un desplegament de mitjans de comunicació que fins ara no s’havia vist mai, l’antologia Els imparables. Nou autors que obrien el llibre amb un manifest bel·ligerant, descarat, però sobretot anacrònic (potser a les avantguardes aquests manifests tenien algun sentit, principalment perquè anaven acompanyats d’un ideari estètic clarament definit, però en ple segle XXI, després de les malalties del mo- dernisme i el postmodernisme, han perdut qualsevol tipus de credibilitat i de vigència). Es definiren, en el llibre, les línies de la nova i jove poesia catalana, deixaven arraconats els autors de la generació anterior, i proposaven la valoració d’uns autors que, evident- ment, fins aleshores havien estat bandejats pels poetes que plàcidament ja havien pres possessió dels pocs llocs que pot oferir el món poètic català. Alhora, ha posat la crítica davant d’un dilema que té un fons totalment maniqueista i que, en poc temps, ha fet re- duir la visió que fins fa molt poc es podia tenir de la poesia catalana. 87 87 2. Aproximadament cinquanta anys enrere, hi va haver una situació que podria tenir molts paral·lelismes amb l’actual. No pas en la forma, sense manifests explícits. Alesho- res es començava a congriar el camí que possibilitaria l’estat actual. En aquells anys, en plena dictadura del general Franco, els autors catalans no es podien permetre gairebé cap aparició. Dominaven els peus falsos d’impremta, les reunions i els canvis de llibres a les rebotigues, les trobades que esdevenien conspiracions (literàries, i no tan sols) contra el règim; uns fets que segurament tenen encara presents molts lectors polonesos. En aquella situació, el simbolisme o el postsimbolisme, amb la figura del poeta tancat en la seva torre de vori, aliè al que passava al seu voltant, no era la millor opció per a la literatura. I tan- mateix, era aquest moviment el que seguia dominant l’escena catalana, on al capdavant figurava una de les personalitats més importants de la literatura catalana moderna: Carles Riba (1893-1959), considerat com un dels últims grans humanistes. Home d’una vasta cultura, les traduccions que realitzà, sobretot dels clàssics grecs i de poetes alemanys, ja li asseguren un lloc d’honor en qualsevol literatura. La seva poesia representa un camí de coneixement on l’absolut i la recerca metafísica esdevenen centrals. 1. El punt àlgid de la seva obra és Elegies de Bierville (1943), on expressa el dolorós sotrac que esdevé l’exili per al poeta. Escrit al castell de Bierville, a França, Riba entronca els seus poemes amb les Elegies romanes de Goethe i, principalment, amb les Elegies de Duino de Rilke. D’aquest darrer fins i tot n’extreu una gènesi molt similar a l’hora de concebre el poema. Escrites en hexàmetres adaptats al català, tant el món grec com la imatge d’aquest a partir dels poetes esmentats i l’exili conflueixen en instants d’autèntica epifania. Sens dubte, és un dels millors llibres de poemes que ha donat la poesia catalana del segle XX. La figura de Carles Riba era tan important que la tendència poètica a què s’adscri- via fou la que predominà en la poesia catalana fins a la seva mort. Malgrat que hi havia intents de projectar els nous corrents poètics que ja feia temps s’havien implantat a Euro- pa, aquests no reeixiren. Els autors hagueren d’esperar el 1959 per poder donar a conèixer una estètica que era més “necessària” per als temps foscos de la dictadura. Una nova estèti- ca que aviat tingué molta acceptació, el públic lector es podia identificar per primera vegada des de feia molt de temps amb el poeta que representava la seva veu, perquè el discurs esdevingué més clar, i el tema del poema no se centrà únicament en la introspec- ció interior sinó que es mourà al voltant de l’experiència de l’home en una realitat con- tingent, amb una voluntat de canviar-ne els aspectes desagradables. S’imposà el realisme històric. 3. La identificació del públic amb el poeta, a qui consideren anostrat i a qui li atribueixen uns valors que personalitzen els valors del poble, i en conseqüència nacionals, és un fet pròpiament del Romanticisme. En la literatura catalana el Romanticisme (conegut com a Renaixença, i amb unes particularitats pròpies) té una doble importància, car no tan sols hi ha l’establiment dels valors nacionals, sinó que alhora esdevé la recuperació de la llen- gua per a la literatura culta, recuperació encarnada en la figura de Jacint Verdaguer (1845- 1902). Autor de dos poemes èpics, el Canigó i L’Atlàntida, la riquesa del seu llenguatge, i sobretot la varietat formal dels seus versos, en què explota totes les possibilitats rítmi- ques i mètriques, recuperen la dignitat per a la llengua. Altres aspectes, tant literaris com 88 no literaris, la temàtica religiosa i patriòtica dels seus poemes, fan d’ell el primer gran poeta popular. Després vindrà Maragall, poeta modernista de ressons nietszcheans i vita- listes. És un altre cop en una època d’enfrontament amb el poder establert que apareix el tercer poeta popular: Salvador Espriu (1913-1985). Aquest poeta esdevé la veu de la re- sistència davant de la dictadura franquista. A diferència dels poetes anteriors, la poesia d’Espriu és deliberadament difícil, amb un rigor i una dedicació per l’obra perfecta que no té equivalent en cap altre autor català contemporani. També, com tot poeta popular, ha estat fruit d’una sèrie de lectures parcials, esbiaixades, simplificades. I tanmateix, Espriu és el constructor d’un edifici poètic singular, propi, que té el seu eix en la reflexió al vol- tant de la mort. El coneixement de les cultures jueva i grega, el Llibre dels morts egipci, són les fonts en una poesia que s’aparta dels corrents imperants a Europa. Si abans po- dríem parlar d’un cert retard en la mort del simbolisme i la introducció del realisme històric, ara podríem parlar de la creació d’un món propi, mític, aliè a qualsevol estètica que hagi traspassat el segle XX. Només la visió mediterrània de la seva poesia podria te- nir algun nexe amb la poètica de Salvatore Quasimodo, però els cinc llibres de poemes que conformen el cicle de Sinera (espai mític que crea escrivint el nom del seu poble a l’inrevés, Arenys) no troben equivalent en la poesia contemporània. 3. Si haguéssim de bus- car-li més afinitats, es podria afirmar que de vegades sembla un predecessor del poeta anglès Geoffrey Hill. El cicle el formen Cementiri de Sinera (1946), Les hores (1952), Mrs. Death (1952), El caminant i el mur (1954) i Final del laberint(1955). L’any 1960 apareix el llibre que, d’una banda, representa la plena vigència de l’entrada del realisme històric, i de l’altra, estableix la figura de poeta popular del seu autor. La pell de brau és una paràbola per explicar les diferents cultures que conviuen a la Península Ibèrica, i un intent de voler conciliar-les. Alhora denuncia els fets que van conduir a la cruel guerra fratricida que fou la Guerra civil espanyola. Dins de les files del moviment que comença a despuntar com a realisme històric apa- reix un jove autor que es convertirà, anys a venir, en el poeta popular més recent: Miquel Martí i Pol (1929-2003). El seu decés, l’any passat, es convertí en un dels esdeveniments més importants de la cultura catalana. Es començà a reflexionar i a discutir sobre el valor de la figura de poeta popular o, si es vol, nacional; i també sobre la vàlua de la seva ex- tensa obra poètica. El lector es pot fer una idea de la gran acceptació de la seva poesia si pensa que era l’únic poeta present a totes les llibreries del país, la seva popularitat tren- cava les fronteres de la pròpia literatura, i s’endinsava en terrenys com la música (s’ha de valorar la col·laboració amb el cantautor Lluís Llach) i fins i tot l’esport. I tanmateix, la seva poesia no era tan accessible com aquests fets poden fer pensar. L’ús del llenguatge de Martí i Pol, sobri, que combinava amb un gran encert en crear imatges que esdevenien el correlat per a una reflexió de caràcter introspectiu, on predominaven la soledat, l’en- frontament a la buidor que comporta el pas del temps i la lenta desaparició i destrucció d’elements relacionats amb la natura (metàfora de la malaltia que patí el poeta) són les coordenades de la seva poesia. Fou un autor capaç de llegar a la poesia catalana un to gairebé de confessió, amb un ús molt hàbil del decasíl·lab blanc que fluïa en acurats en- cavallaments de versos. En resultava una aparent naturalitat, com una conversa. 4. Un dels moviments més perjudicats a causa de la impossibilitat de coexistència de diferents moviments literaris a la mateixa època ha estat les avantguardes. Potser no tan la primera, contemporània als grans moviments d’avantguarda que hi va haver arreu d’Europa, però que tingué una incidència mínima en la poesia catalana, ja que aquesta patia una absència de tradició que, evidentment, no es podia destruir. Enfront de quines manifestacions s’havia de declarar l’avantguarda, quan el que hi havia era tan fràgil que no necessitava ni un sol cop per esmicolar-se completament? Els autors de la segona avantguarda van haver d’esperar que uns autors joves, que es rebel·laven en contra del moviment anterior, els recuperessin i els atorguessin el lloc de mestres de la nova generació. Aquells autors formaven part d’un corrent que es formà a finals dels anys vuitanta i que reberen el nom d’una de les editorials independents més creatives i eficaces que ha donat el mercat català: les edicions del Mall. Van recuperar noms com Joan Brossa o Guillem Viladot, i alhora redescobrien autors maleïts però de gran importància com Josep Palau i Fabre (1917). Aquest darrer representa un lligam di- recte amb la poètica de Rimbaud, la creació del geni, el símbol dut fins a un extrem; i combinat amb la noció de la poesia com a alquímia en què l’alteritat és el motiu central de l’estranyesa en el llenguatge i en la pròpia existència. La seva obra magna Els poemes de l’alquimista (1972) representa una de les culminacions d’una estètica que ha estat obli- dada i marginada en el conjunt de la poesia catalana. Aquesta obra l’ha situat com una de les figures més interessants de la poesia contemporània en català, i això ja representa tot un triomf. És clar que ha hagut d’esperar un moment en què hi ha hagut una visió més oberta quant a l’actitud lectora. L’autor que ha esdevingut sense pal·liatius central en el moviment avantguardista ha estat J. V. Foix (1893-1987). Present tant en la primera com en la segona avantguardes, im- portant en totes dues, la radicalitat de la seva proposta s’ha de concebre en el context de la poesia catalana. Per a Foix, l’irracional, les imatges agosarades, les relacions inconne- xes que pot arribar a provocar la ment, el joc amb el llenguatge són aspectes de gran rellevància. 3. Un tipus de poesia que trobarà ressons en el corrent que rep el nom de poesia de l’experiència. L’espai de poeta popular quedà buit de sobte a la seva mort. Però aquesta no com- portà pas un canvi d’estètica, ni de la possibilitat de donar entrada a noves vies d’ex- pressió. Fou una figura ben diferent a la de Riba, i la seva vàlua ha de ser analitzada en 89 uns paràmetres completament diferents. L’existència de Martí i Pol no excloïa altres co- rrents, i la seva obra, literàriament, ha deixat traces poc profundes tant en els autors de la seva generació com en autors més joves. uns paràmetres completament diferents. L’existència de Martí i Pol no excloïa altres co- rrents, i la seva obra, literàriament, ha deixat traces poc profundes tant en els autors de la seva generació com en autors més joves. 4. Però per una altra banda trobem un profund coneixement dels clàssics de la poesia catalana, sobretot de la seva figura màxima i un dels poetes més imponents del se- gle XV: Ausiàs March. Un coneixement que es tradueix no tan sols en l’ús d’unes formes determinades (com per exemple, el sonet) sinó en un llenguatge ric que recollia i remetia constantment a fonts medievals (el segle XV és el segle d’or de la literatura catalana). Foix era conscient de la manca de tradició que tenia al darrere, i la seva proposta avantguardis- ta no és destructora en absolut, és constructora. Dinamita la tradició al mateix temps que la reelabora en unes coordenades completament noves, innovadores. Amb ell es pot dir que arrela un nou concepte de tradició poètica, un nou concepte del que pot arribar a ser un moviment literari. La seva és una anàlisi en profunditat del fet literari, a diferència d’al- tres autors que es queden exclusivament en la forma externa de les avantguardes. Molt lligada a la tradició avantguardista és la poètica de Pere Gimferrer (1945) que l’enllaça amb unes tendències postsimbolistes. Recull les dues vies que representen Josep Palau i Fabre i J. V: Foix, a les quals afegeix la concepció del poema de dos autors fona- 90 mentals: l’espanyol Vicente Aleixandre, i el mexicà Octavio Paz. El resultat és una de les creacions més consistents de la contemporaneïtat poètica, on actua com a nucli la reflexió sobre el procés d’escriptura poètica. És a dir, dóna entrada de ple a la metapoesia. Gim- ferrer és una dels poetes més originals que han aparegut en el panorama de tot l’estat es- panyol. Recordem que va començar a escriure en castellà, llengua en què va publicar tres llibres, tots amb una molt bona acollida per part de la crítica, i que no fou fins l’any 1970 que no comença a publicar en català, que esdevindrà la llengua ja de creació poètica. És interessant el motiu que addueix per poder realitzar aquest pas, molt en consonància amb la seva teoria poètica. Afirma que representa un joc de personalitats, com una espècie de miralls giratoris que són una de les imatges clau en els seus poemes. 4. L’obra de Gimferrer apareix en un moment en què hi ha una renovació en la poesia, just després del realisme històric, i els autors que formen part del moviment poètic corro- boren la tendència de destronar els poetes anteriors. És una poètica que gira la mirada a l’interior del llenguatge, que posa en dubte la capacitat del discurs de la realitat. És un fenomen que es produeix arreu d’Europa, a Itàlia per exemple, assoleix un gran ressò, o també a Polònia, que podria tenir alguns punts de referència amb la poesia lingüística dels autors de la Nowa Fala. Ara bé, posats a buscar concomitàncies en la poesia de Pere Gimferrer, potser l’autor amb què tindria més afinitats seria amb una de les veus europees més interessants de l’actualitat: el poeta portuguès Nuno Júdice. A Catalunya, la figura de Pere Gimferrer apareix com a aïllada, ja que la majoria d’autors que, per proximitat en l’aparició en la palestra literària i en les dates de naixe- ment, podrien formar un grup generacional afí, adopten altres coordenades per a la seva poesia. Aquest fet representa una de les tendències mes evidents en la poesia catalana, fruit de la seva recerca constant de la tradició poètica. No és fins a finals del segle XX que es pot mirar enrere i crear els propis moviments, tendències, poètiques dins el marge de la pròpia llengua, però durant el segle XX el que ha existit ha estat la creació d’unes personalitats que en el seu individualisme buscaven les deus de la poesia en altres litera- tures europees, principalment l’eix de la poesia francesa i italiana per una banda, i per una altra, la poesia espanyola. 5. La influència d’arrel anglosaxona, la predominant en tota la segona meitat del segle XX, entra a Catalunya a través d’una altra personalitat única: Gabriel Ferrater (1922- 1972). La seva poesia, breu i escrita ja en els anys de maduresa de l’autor, ha esdevingut una de les vies més fructíferes dels futurs creadors. Arran d’aquesta obra poètica s’ha creat tota una generació d’escriptors i, fins i tot podria afirmar, part d’una segona gene- ració. És l’autor de referència que trenca la tendència dels autors joves a reduir-lo al no res. Ferrater es manté com un poeta valorat i admirat sense interrupcions des de la seva mort fins a l’actualitat. Gabriel Ferrater fou en el seu moment una ruptura en les tendències dominants, però que no es va fer palesa fins anys més tard. En el seu moment, la crítica no s’adonà de la intencionalitat de la seva poètica, i adscriví l’autor en el mateix corrent que el realisme històric. Evidentment, hi havia alguns punts de contacte, com l’ús de la realitat, el de les experiències més immediates i palpables; en canvi, el to completament diferent, el jo poè- tic que jugava en la construcció del poema, i l’actitud moral entesa en relació amb l’in- dividu enfront d’unes contingències concretes l’allunyaven dels representants del realis- 91 91 me històric. La poesia de Ferrater entronca directament amb uns referents de la lírica an- glesa que ell mateix s’ocupa d’esmentar. Afirma que són, entre d’altres, W. H. Auden, Robert Frost o Thomas Hardy. La crítica ha entès aquesta afirmació i declaració de prin- cipis del mateix autor com un axioma i l’han repetit fins a la sacietat, sense plantejar-se que en el fons el poeta en recull alguns elements, d’aquests autors, però que el lligam directe no és tan evident ( a banda d’alguns poemes que tenen com a model poemes con- crets de la tradició anglesa, poemes que gairebé reelabora sense arribar al que podríem anomenar com a plagi). No es deté en la senzillesa aparent de Frost, ni en l’efusió tran- quil·la de Hardy. Tal vegada amb Auden té més punts de contacte, el seu discurs no és fàcil. També, a la manera del poeta anglo-americà en els seus poemes llargs, Ferrater crea en el Poema inacabat una llarga introspecció de l’actitud moral enfront d’un fet de con- seqüències desastroses com és la Guerra civil espanyola. 5. Més endavant, a Ferrater se’l va classificar dins un moviment anomenat la poesia de l’experiència, seguint-ne la classificació que en feia Langbaum en el seu llibre The Poetry of Experience, una poètica que es definia enfront del Romanticisme (principalment el Romanticisme anglès, el jo creat per Wordsworth en el seu The Prelude) i que, a partir del concepte de la màscara, la disfressa del poeta dins el mateix poema, com una altra veu, reflexionava sobre l’actitud moral de l’home. Com en el Romanticisme, un dels ele- ments que caracteritzaven aquesta poesia i que en conformaven un dels seus pilars era l’ús de la ironia. En aquest sentit, la poètica de Ferrater n’és un clar exemple, i l’ús d’aquesta figura retòrica amplia els horitzons de la poesia catalana. Les possibilitats que aporta Ferrater a la creació poètica en català són molt àmplies. Allibera la poesia de la rèmora del simbolisme i de la influència francesa, que havia estat la més important fins aleshores, i d’altra banda, demostra que l’ús d’un llenguatge quoti- dià no s’ha de tancar en les proclames de la poesia del realisme històric. No és necessària la identificació de la literatura, per bé que pot tenir en compte la realitat ingènua (en el sentit que afirmava Czes³aw Mi³osz tant en el seu discurs del Premi Nobel com en les conferències de Harvard), amb una funció que ha de complir en la societat. La poesia de Ferrater no vol canviar la realitat ni la societat, no vol convèncer ningú, tan sols vol donar compte de l’experiència humana, de les relacions personals i de la consciència del pas del temps. 6. En el context presentat, és lògic que la seva proposta hagi trobat nous seguidors, en el sentit que encarna l’evolució del simbolisme en un món dominat pel dubte i pel recel enfront de la pròpia realitat. I també és lògic que aquesta proposta s’aliï amb la de Ferrater, on la realitat subjectiva planteja el conflicte de la pròpia acceptació moral i aleshores aquesta realitat s’erigeix com el correlat directe amb el que es pot convertir com un símbol de la conducta humana. Entre els autors que han sabut conciliar ambdós vessants, sense caure en la simpli- ficació de la poesia de l’experiència, i han creat una obra de força qualitat amb caracte- rístiques pròpies hi ha principalment Francesc Parcerisas (1944) i Joan Margarit (1938). El primer és l’autor d’un dels llibres crucials de la poesia catalana recent: L’edat d’or (1983). La seva poesia, bastida a partir de reflexions entorn de l’experiència moral, amb el recurs d’alguns símbols i amb un ús habitual de referents culturals, es perfila com una de les línies més interessants. Han estat molts els poetes joves que s’han vist enlluernats per aquesta poesia discursiva, aparentment senzilla, que té la base de la construcció no pas en els metres tradicionals sinó en el to conversacional que enllaça la realitat amb la reflexió. L’edat d’or proposa un viatge de la mà de Marguerite Yourcenar i l’Hadrià que crea, i d’altra banda, el guia per excel·lència en la literatura: Virgili. Un viatge on es des- cobreixen els petits paradisos que un pot tenir encara després de les diferents pèrdues a què sotmet la vida. En un cert sentit, és una poesia de caire elegíac. En la mateixa direcció actua la poesia de Joan Margarit, en aquest moment un dels poetes més prestigiosos, no tan sols en el panorama català, sinó també en la literatura cas- tellana, on les traduccions dels seus llibres (recentment ha aparegut la seva poesia com- pleta en edició bilingüe) són acollits d’una manera molt favorable. Fins i tot, alguns poetes castellans no dubten a qualificar-lo com el millor poeta que escriu actualment a Espanya. De fet, la primera etapa de la seva creació era en llengua castellana, i no fou fins l’any 1981 que no va publicar el primer llibre de poemes en català. D’aleshores ençà, la seva abundant producció s’ha imposat any rere any. 6. És difícil de trobar en el món hispànic una etiqueta que hagi estat tan malinterpretada com la de poesia de l’experiència. És evident que moviments com el surrealisme, que tenia la seva gènesi a França, quan passava les fronteres adquiria unes tonalitats diferents, els autors d’un altre país l’impregnaven d’elements que en un principi potser no preveien els seu fundadors. I això s’esdevé en la majoria de moviments literaris. Però en la poesia de l’experiència assistim a un canvi de perspectiva tan gran que les noves generacions de poetes apliquen aquest terme a una praxi poètica quasi radicalment oposada a la que exis- tia en un principi amb el mateix nom. De la seriositat del plantejament moral que propo- sava l’estètica de Ferrater es passa a una relació minuciosa de les experiències que el poeta té, on el fet amorós passa a un primer pla, i la reflexió moral queda diluïda en una super- ficialitat d’anàlisi. Els autors que pertanyen a aquest corrent tenen la particularitat de combinar dues in- fluències que en un principi tenen pocs elements en comú. Una d’elles és la de Ferrater, 92 i l’altra representa un altre descobriment tardà: Joan Vinyoli (1914-1984). A partir de la dècada dels 70, la figura de Joan Vinyoli adquireix una aura de mestratge que creixerà fins a la seva mort i en anys posteriors. La primera producció d’aquest poeta s’emmarca en el postsimbolisme, i té com a principals mentors a Carles Riba i sobretot a Rainer Maria Rilke, de qui en va fer belles traduccions. Posteriorment, la seva producció deriva vers una reflexió sobre el pas del temps i la buidor del món contemporani. Sense aban- donar algunes reminiscències simbolistes, la seva poesia es despulla de qualsevol element innecessari, hi ha una fusió d’elements reals a través del símbols, o d’elements simbòlics a través de la realitat. La realitat té plena vigència a partir de la transformació que hi ope- ra l’element simbòlic, i aleshores s’entra en el símbol del poema per atènyer un tercer nivell que retorna a la realitat per poder-la analitzar. És la seva poesia un pas més enllà del postsimbolisme, un pas per conciliar les esferes en què podem percebre la realitat. 6. En ell conflueixen les dues vies abans ini- ciades, més un coneixement profund de la poesia europea del segle XX, on es pot trobar tant referències a poetes russos com Mandelxtam o Akhmàtova, com també italians, sen- se oblidar, lògicament els anglesos, i passant per la poesia espanyola. Així mateix, les al·lusions al món clàssic i a referents culturals i literaris són abundants en la seva poesia. Margarit assoleix una gran naturalitat sense menystenir els aspectes formals, el decasíl·lab és el metre que més utilitza i, sobretot en la primera producció, hi predomina la rima, tan consonant com assonant, aquesta darrera ben poc utilitzada en l’àmbit català. La seva po- esia expressa la recança pel món perdut, pel pas ineludible del temps, però que, com en 93 l’esplèndid poema de Cavafis, el camí, en aquest cas de la pèrdua, és el que ens aporta coneixement. O, parafrasejant l’esplèndid final de l’elegia II (Elegies de Bierville) de Carles Riba, el pas de l’experiència, la pèrdua, és rica del que ha donat i pura en la seva ruïna. Tanca la tríade d’autors que reben la influència anglosaxona el poeta Narcís Coma- dira (1942). Els primers llibres de Comadira han estat des de sempre considerats com una mena de provatures, en què l’autor recercava la seva pròpia veu. Aquesta és distingible a partir dels anys 80, en què publica alguns dels llibres més importants de les darreres dèca- des, entre els que destaquen Enigma (1985) i En quarantena (1990). La poesia de Coma- dira, filigranada i rica en la seva forma, on destaca l’ús habitual de la rima amb què acon- segueix una gran musicalitat, es descobreix en aquests llibres com una introspecció vers les pors humanes, una dimensió metafísica on l’existència es debat enfront el desig. En Comadira és absent la petja de Joan Vinyoli, perquè la seva filiació enllaça direc- tament amb Josep Carner (1884-1970), un dels poetes més perfectes quant a l’ús de la forma, defensor del classicisme i creador d’una obra compacta en què l’equilibri s’alia amb un ús ben perfilat de la ironia a l’hora de mostrar, des d’un punt de vista gens agre, els aspectes més nimis i inofensius de la quotidianeïtat. 7. L’esclat poètic dels anys setanta és fruit de diversos factors, entre els quals no hem d’oblidar la mort del dictador, i la consegüent obertura i possibilitat ja de publicació en català. Però els elements més importants són d’índole literària. Des de la perspectiva que el lector pot tenir tot partint del panorama que arriba fins a aquestes línies, no li serà di- fícil veure que el que a principis de segle representava una clara mancança, l’absència de tradició, a meitats dels setanta qualsevol poeta ja comença a tenir el pes de la tradició, aquesta ja ha esdevingut un fet, no sense haver passat per moltes dificultats, és cert, però al llarg del segle s’ha fixat ja la vàlua d’alguns autors, les poètiques s’han succeït com havia de ser, i cada nou rei ha destronat l’anterior per poder ocupar un espai poètic propi i establir les noves línies d’actuació. És la primera vegada que es pot parlar d’un funciona- ment més o menys normal del fet literari. Apareixen noves editorials, encara la qualitat d’una obra passa per davant dels criteris comercials, i la poesia no únicament no se’n ressent sinó que en surt afavorida; i, per damunt de tot, apareix un públic lector en català. Es donen totes les condicions favorables perquè es pugui assistir a un autèntic esclat, apareix un nombre força elevat de nous autors, i per primera vegada, encara que sigui molt temporalment, poden coexistir diferents tendències alhora. Hi ha la segona generació dels poetes de l’experiència, hi ha la recuperació de veus que havien estat marginades, hi ha un corrent neopopularista que podria enllaçar amb alguns postulats de la Generación del 27 castellana, hi ha propostes agosarades que deriven de les segones avantguardes i del surrealisme. Tot en un moment únic, en una situació irrepetible, en un esclat sense precedents. D’entre totes les propostes que aparegueren sobresurt, per la seva solidesa i per la innovació i renovació que representa per a la poesia catalana, l’obra de Maria Mercè Marçal (1952-1998). S’hi pot comprovar la consistència de la tradició, a l’ombra d’aques- ta l’obra de la poetessa es constitueix com una amalgama de referències, de lectures en un àmbit completament nou. 7. De vegades, crea a redós de la poesia popular combinada amb imatges clarament surrealistes; d’altres, s’endinsa en l’experimentació formal, jugant 94 amb els sonets i amb les sextines (en una certa relació amb la mateixa activitat que dugué a terme Joan Brossa); i fins i tot d’altres s’endinsa en el vers lliure. Si ha aportat un caire de renovació quant a la forma, aquest encara s’accentua molt més si parlem de la temàtica que és present en la seva poesia. El poema esdevé el lloc d’una introspecció personal, d’una recerca del propi jo a través de l’alteritat i a través de la visió de la dona. Temes com l’amor, com el sexe, la maternitat, la necessitat de mantenir uns lligams amb el propi jo a través de l’altre són els elements fonamentals de la seva poesia. Amb ella les referències de la poesia catalana s’amplien. En un primer moment el lector pot ser remès a la poesia d’Anne Sexton, i principalment a la d’Adrianne Rich, després s’introdueixen les poetesses russes Anna Akhmàtova o Marina Tsvietàieva (de qui ha fet sengles traduccions en col·la- boració amb Monika Zgustová). La seva poesia ha esdevingut un mirall per a la línia ence- tada que arrenca de la consciència d’examinar el món a través de la singularitat de l’ésser femení, una línia que absorbeix aquesta tradició amb tota naturalitat, malgrat que amb una mica de retard. La importància de Maria-Mercè Marçal en la poesia catalana és deguda també a un altre factor. Juntament amb Ramon Pinyol i Xavier Bru de Sala funden l’editorial Llibres del Mall (1973-1988) en què s’aixoplugava quasi tota la nova poesia jove. És un dels fenòmens literaris i editorials més importants de les darreres dècades. La nova poesia, que es destacava per l’eclecticisme, com ja he apuntat en paràgrafs anteriors, possibilita que hi hagi la lluita pel poder de l’espai, i curiosament sembla que l’espai s’ha eixam- plat. Tanmateix, de retruc aquest fet comporta una segona conseqüència, en no haver-hi un espai clarament definit, la crítica es troba desorientada i busca delerosament la sortida a aquesta situació a través del descobriment de noves veus, d’antologies que intenten fixar els poetes que es poden consolidar com a alternativa. En aquest context apareixen poetes d’esclat tardà que ràpidament esdevenen centrals, sigui per la qualitat de la seva obra, sigui pel plantejament radicalment diferent de les seves poètiques. 7. Els dos exem- ples més paradigmàtics d’aquesta situació són Antoni Puigverd (1954) (publica el primer llibre l’any 1990) i Enric Cassasses (1951) (que publica d’una manera oficial l’any 1991). Aquest darrer autor, que conjuga una gran destresa formal amb una voluntat de relatar els aspectes més quotidians de la vida humana, sense escatimar en elements subversius, escatològics o violents, ha estat seguit per nombrosos autors joves. Cassasses és conegut tant per la seva obra, que ha tingut força acceptació, com també per la imatge de poeta que recull d’una certa tradició maleïda, marginal, que es dedica a posar en escena perfor- mances de gran efectivitat. El fet de l’existència d’un itinerari poètic com el que representa Enric Cassasses és saludable per a qualsevol literatura, però ja no ho és tant quan s’intenta presentar aquesta línia com la imperant en la nova poesia. Aquest ha estat un dels errors que últimament s’han comès en les files de la crítica de poesia catalana. 8. La dècada dels 90 transcorre en una certa fluïdesa, apareixen llibres de poemes remarcables, d’autors que després deixen de publicar, si més no fins l’actualitat, com l’excel·lent El test de Rorschach (1992) de Xavier Lloveras. També s’assisteix a l’ascensió i caiguda d’empreses editorials, essent la que obté més ressò l’editorial Columna, que s’havia convertit potser en l’editorial més important de poesia. S’assisteix igualment a l’entrada de noves traduccions, que renoven el panorama literari aportant noves influències. 95 És paradoxal que després d’haver assolit un nivell comparable a la poesia que existeix arreu, l’entrada d’autors com John Ashbery o Frank O’Hara ha estat tardana, fa dos anys va aparèixer una traducció del primer poeta, i el segon encara no ha estat traduït al ca- talà. Cito aquests dos autors perquè és el corrent que s’ha imposat en la poesia moderna europea, només cal veure, per exemple, la repercussió que tenen dins l’àmbit polonès. Ara bé, el fet que no hi hagi traduccions no indica que les influències no existeixin. En els autors més joves es comença a veure una actitud vers la realitat, vers el tractament del llenguatge que deriva tant de les teories postmodernistes com dels poetes que s’han erigit com els seus estendards. És la revolució imparable en el món modern, els poetes que s’agrupen sota un deno- minador pretensiós que proposen l’alternativa, l’única alternativa a la poesia catalana. Són autors que saben moure’s en un món on la imatge és tan important com el producte que s’ofereix i que han sabut manipular els ressorts necessaris per poder aconseguir situ- ar-se en el centre d’atenció de l’actualitat literària. Tanmateix, dins la nòmina d’autors que s’hi presenten, hi ha una gran divergència quant als estils, les poètiques, les influèn- cies. No tot sembla tan subversiu com pot semblar a primera vista. La voluntat de ruptura no es tradueix en una praxi poètica radicalment diferent a la que hi havia hagut fins al moment de l’aparició d’aquests autors. Quant a l’actitud lingüística no hi ha tampoc un acord establert, no s’endinsen en la mateixa problemàtica, no hi ha una renovació profun- da. Així doncs, on rau la novetat dels imparables, per què tan rebombori en el moment que surt publicada l’antologia? El primer factor és els mitjans de comunicació, per pri- mera vegada l’antologia traspassa les fronteres del gènere, també les de la literatura, i es converteix en un fenomen social. 8. El factor de la novetat sí el podem trobar en la poesia d’alguns d’aquests autors, però no pas en tots. Es podria afirmar que és la primera gene- ració que afronta la poesia com un exercici davant la relativitat del món modern. El tercer factor és el fet d’haver gosat posar en dubte la tradició, una tradició que havia costat tant d’assolir i que alguns autors ja consideraven com intocable. El grup dels imparables, so- bretot el que es considera com el nucli dur (Sebastià Alzamora, Hèctor Bofill i Manuel Forcano), ocupa ja un lloc destacat dins la poesia catalana. Són poetes que ja gaudeixen d’una obra força sòlida i que han demostrat les capacitats del seu quefer poètic, de vegades de gran qualitat. Alguns autors, potser els més ressentits amb els imparables, fins i tot dirien que s’han fet un lloc en el terreny del libel. La figura que els imparables han ata- cat més és la de Josep Carner. Però no tan sols han atacat el que es considera com una de les màximes figures del segle XX català, sinó tots aquells autors que, d’una manera o al- tra, tenen una filiació amb la seva poesia. Es discuteix el fet de crear una obra bella però que no té cap incidència ni en el lector ni en la realitat. Una obra que és exclusivament per poder ser admirada en la seva bellesa formal i amb l’ús dels diferents recursos lingüís- tics. Els imparables demanen una poesia que pugui plantejar un desafiament al lector, se- gons les paraules dels propis poetes, i que la literatura no sigui un mer passatemps sinó una forma de prolongar la vida, que s’hi involucri. La seva ruptura potser no és tan estèti- ca com d’actitud. Una actitud que pot agradar o no, però que no pot deixar indiferent. Han aparegut en un moment en què és plausible ja poder passar comptes amb la pròpia tradició, en un moment en què fins i tot és saludable. Al cap i a la fi, la solidesa de les propostes que s’han succeït al llarg del segle, i els noms que han destacat per la seva obra asseguren un lloc prou segur a la poesia catalana contemporània. 96 SODOBNA KATALONSKA POEZIJA: KRATKO POPOTOVANJE V ISKANJU NEKEGA IZRO^ILA Izid antologije »Neustavljivih« je povzro~il pretres v panorami katalonske poezije. Antologijo mladih katalonskih pesnikov so mo~no kritizirali, a pri tem niso nikoli posku- {ali analizirati, kak{ne posledice je imela ta izdaja in iz kak{nih vgibov je sploh nastala. Pri~ujo~i ~lanek govori o katalonski poeziji dvajsetega stoletja, ki je katalonsko literaturo umestila nazaj na evropsko raven, saj po treh stoletjih teme znova poustvarja svojo lastno tradicijo. Antologijo »Neustavljivih« lahko razumemo in analiziramo samo v tem kon- tekstu. 97 97 97
https://openalex.org/W3046952504
https://hal.inrae.fr/hal-03036333/file/2020_Fardet_Sustainability.pdf
English
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Ultra-Processed Foods and Food System Sustainability: What Are the Links?
Sustainability
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To cite this version: Anthony Fardet, Edmond Rock. Ultra-Processed Foods and Food System Sustainability: What Are the Links?. Sustainability, 2020, 12 (15), pp.6280. ￿10.3390/su12156280￿. ￿hal-03036333￿ Distributed under a Creative Commons Attribution 4.0 International License Review Review Received: 27 June 2020; Accepted: 31 July 2020; Published: 4 August 2020 Received: 27 June 2020; Accepted: 31 July 2020; Published: 4 August 2020 Abstract: Global food systems are no longer sustainable for health, the environment, animal biodiversity and wellbeing, culinary traditions, socioeconomics, or small farmers. The increasing massive consumption of animal foods has been identified as a major determinant of unsustainability. However, today, the consumption of ultra-processed foods (UPFs) is also questioned. The main objective of this review is therefore to check the validity of this new hypothesis. We first identified the main ingredients/additives present in UPFs and the agricultural practices involved in their provision to agro-industrials. Overall, UPF production is analysed regarding its impacts on the environment, biodiversity, animal wellbeing, and cultural and socio-economic dimensions. Our main conclusion is that UPFs are associated with intensive agriculture/livestock and threaten all dimensions of food system sustainability due to the combination of low-cost ingredients at purchase and increased consumption worldwide. However, low-animal-calorie UPFs do not produce the highest greenhouse gas emissions (GHGEs) compared to conventional meat and dairy products. In addition, only reducing energy dense UPF intake, without substitution, might substantially reduce GHGEs. Therefore, significant improvement in food system sustainability requires urgently encouraging limiting UPF consumption to the benefit of mildly processed foods, preferably seasonal, organic, and local products. Keywords: ultra-processed foods; food systems; sustainability; environment; animal wellbeing; socioeconomics Ultra-Processed Foods and Food System Sustainability: What Are the Links? Anthony Fardet * and Edmond Rock Unité de Nutrition Humaine, INRAE, Université Clermont Auvergne, CRNH Auvergne, F-63000 Clermont-Ferrand, France; edmond.rock@inrae.fr * Correspondence: anthony.fardet@inrae.fr; Tel.: +33-(0)4-7362-4704 Anthony Fardet * and Edmond Rock   Sustainability 2020, 12, 6280; doi:10.3390/su12156280 www.mdpi.com/journal/sustainability HAL Id: hal-03036333 https://hal.inrae.fr/hal-03036333v1 Submitted on 31 Mar 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License sustainability sustainability 1. Introduction In other words, UPFs are artificial foods with organoleptic and sensory properties modified by the addition of ‘cosmetic’ additives and/or highly processed ingredients. Therefore, UPFs are supplying to human organism new unstructured and recombined food matrices, but also new ultra-processed ingredients and additives [12], and whose health effects still needs to be studied on a long term. They are also the reflection of the last nutritional transition that occurred as a major event in the 1980s in Western countries. Since 2011, at least 260 peer-reviewed papers have used the UPF concept, as defined in the NOVA classification [14] (searched for “UPF” in the article title in the ISI Web of Science on 9 May 2020). A main finding is that the high and/or regular consumption of UPFs has been consistently associated with a higher prevalence of the main chronic diseases and metabolic deregulations in more than thirty-five ecological, epidemiological, and interventional human studies [9] from several different countries, indicating a globalization of UPF consumption. These studies focused on deleterious links with health, while the Brazilian Dietary Guidelines suggested that the massive consumption of UPFs may also be associated with an increased degradation of culinary traditions, social life, and the environment [11], thus affecting several dimensions of the sustainability of the food system itself. However, these suggestions need to be checked further and supported by data from original scientific papers. Otherwise, according to Johnson et al., the key components of sustainable diets fall into five overarching categories of analysis: (1) agriculture, (2) health, (3) culture, (4) socioeconomics, and (5) the environment [15]. For purposes of comprehensiveness, animal biodiversity and wellbeing, which are much less emphasized in international reports or scientific papers about sustainability, should be integrated together with the preservation of smallholder agriculture. Altogether, the different dimensions of sustainability are summarized in six dimensions (Figure 1) and will guide the discussion and analyses of this review. In a paper entitled “Production and processing of foods as core aspects of nutrition-sensitive agriculture and sustainable diets”, Keding et al. interestingly emphasize the relevant role that food processing could play in food system sustainability, specifically regarding a sustainable diet [1]. Notably, they write, “When moving along the value chain, agriculture will encounter its limits at some point where food processing starts. 1. Introduction The processing of foods is very important for ensuring food security and safety [1]. For a long time, the security and safety of food have been ensured by salting, drying, smoking, sugaring, pasteurizing, or fermenting. At present, numerous additives, namely, preservatives and antioxidants, are also used. Their use makes it possible to preserve foods during long periods of transport in trucks or boats from a production site to supply megalopolises worldwide and to help typical consumers cover, for example, seasonal gaps or if food storage at the household level is poorly managed [1]. Therefore, to feed humanity, food processing is essential. In addition, some foods require processing to be palatable (e.g., grains), safe (e.g., pasteurized milk), or available year-round (e.g., canned, dried, and frozen fruits and vegetables) [1,2]. Processed foods, especially those of recognized multinational brands [3], in developing countries have a modern image. Importantly, improvements have been made in addressing food toxicity, notably in developed and emerging countries. However, food nutritional security has deteriorated, as seen from the triple burden of malnutrition that affects all countries worldwide, i.e., under- and over-nutrition and nutritional deficiencies [4]. In particular, over-nutrition has led to explosions in the prevalence of chronic diseases. In 2016, the World Health Organization (WHO) estimated that approximately 650 million adults were Sustainability 2020, 12, 6280; doi:10.3390/su12156280 www.mdpi.com/journal/sustainability Sustainability 2020, 12, 6280 2 of 29 obese [5]. According to the same estimates, the rate of type 2 diabetes, currently at 9%, is projected to rise by three percentage points over the next 25 years [6]. Additionally, excess body weight affects over two billion people worldwide [7]. Chronic diseases have progressively replaced infectious diseases. p p p g y p Since 2009, the concept of ultra-processed foods (UPFs) has rapidly emerged and is now recognized and used by both public institutions (e.g., Food and Agriculture Organization of the United Nations (FAO), World Health Organization (WHO), Pan American Health Organization (PAHO), United Nations Children’s Fund (UNICEF), and The World Bank) and academic researchers worldwide [8–10]. In brief, within the proposed NOVA classification of four technological groups, UPFs belong to NOVA group 4 and are notably described in the 2014 Brazilian Dietary Guidelines [11]. They are characterized as having undergone excessive processing and containing additional ‘cosmetic’ ingredients and/or additives of primarily industrial use to mimic, exacerbate, mask or restore sensory properties (aroma, texture, taste and colour) [11–13]. 1. Introduction While a fluent transition between the different fields of responsibilities without clear boundaries exists, it is important to investigate explicitly the food processing part for its nutrition-sensitiveness similarly to that of agriculture” (pages 826–827) [1], suggesting that processing may play a relevant role in food system sustainability, which has yet to be explored. In addition, although there may be exceptions depending on country [16], UPFs appear globally less expensive than minimally processed foods [17–19], and the growth rates of UPFs worldwide are very high, especially in emerging countries—notably those in Latin America and South Asia—where sales are continuously increasing [8,20]. In addition, it has been shown that the lower the cost of food is, the lower the nutrient density [21]. For example, Maillot et al. reported that a low energy density and a high nutritional quality are each associated with higher diet costs in French adults [22]. 3 of 29 Sustainability 2020, 12, 6280 UPF Health Culinary tradition Figure 1. The potential impacts of ultra-processed foods (UPFs) on the six dimensions of food system sustainability. Figure 1. The potential impacts of ultra-processed foods (UPFs) on the six dimensions of food system sustainability. From these findings, substantial questions arise: How are the high amounts of UPF ingredients produced and supplied at low cost? How can such a low price be obtained to address such a rapid growth rate worldwide, notably when UPFs are animal based? Therefore, the question addressed in this paper concerns the links between UPFs and food system sustainability, beyond the increased risk of chronic diseases, and regarding the degradation of the other five dimensions of the food system (Figure 1), i.e., environment, biodiversity and animal welfare (Section 3), and cultural and socio-economic dimensions (Section 4). However, before addressing these five dimensions, it is important to determine the ingredients frequently used in UPFs (see Section 2). This narrative review did not use specific methodology. The main feature of it is describing and appraising published articles, and gathering very sparse and scattered data about UPF regarding food system sustainability, UPF being designated - before the arrival of the UPF concept in 2009 [9,10]—as discretionary, non-core, or junk foods. 1. Introduction For this purpose, we used the ISI Web of Science database with notably the following Boolean operators: “ultraprocessed* food* OR ultra-processed food* OR discretionary OR non-core food* OR process* OR diet*” AND “sustainab* OR greenhouse OR water OR environment* OR animal* OR biodiversity OR life cycle OR socioeconomic* OR farmer* . . . ” (among other keywords linked to ‘processing and sustainability dimensions’ as shown in Figure 1). 2. Which Are the Ingredients/Additives Characteristic of Ultra-Processing, and What Is Their Origin? UPFs are made from many recombined ingredients and/or additives, and we suggested that the link between UPF and food system sustainability is first driven by the massive production of these compounds. This question is addressed by identifying the ingredients/additives characteristic of ultra-processing within the list of UPF ingredients used in these products. Based on the UPF 4 of 29 Sustainability 2020, 12, 6280 definition by NOVA, Figure 2 schematically represents the way in which a UPF is generally constructed, i.e., through the cracking of raw foods into isolated ingredients that are then recombined in artificial matrices with the addition of industrial ‘cosmetic’ additives that are not commonly used in the kitchen [9,23]. Depending on food products, e.g., ready-to-eat dishes, UPFs may also contain more or less real foods. The processes used to create these markers of ultra-processing include refining, extraction, purification, hydrolysis, and/or chemical modification. Such ingredients include processed carbohydrates such as sugar syrups, maltodextrins, dextrose, malt extracts and polyols, mainly extracted from maize, and wheat, rice, and potato; processed lipids such as refined and/or hydrogenated and inter-esterified oils; and processed proteins such as isolates from soy, milk, pea, egg, and meat, derived hydrolysates, and gluten. In addition to these ingredients, UPFs also contain “cosmetic” additives extracted directly from natural ingredients or chemically synthesized; there are more than 316 authorized at the European level and more than 2500 at the world level, as evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). The 690,499 foods referenced in the French Open Food Facts database (https://world.openfoodfacts.org/, retrieved on 20 June 2020) make it possible to determine an initial approximation of the frequency of these main ingredients/additives in UPFs (Tables 1 and 2). This database is today the most comprehensive one about packaged foods, and that gives the list of ingredients for most registered foods. Notably, this database has been previously used for retrieving lists of additives from approximatively 126,000 foods [24]. Starches and glucose-fructose/glucose syrup are by far the most commonly used carbohydrate-based ingredients in UPFs, being found in at least 7.6% and 3.2% of all referenced products, respectively. Ranking third are dextrose (>3.1%) and lactose (>1.6%), followed by malt extract (>1.2%), dextrins/maltodextrins (>1.1%), and invert sugar (>0.6%). For lipids, refined oils are extensively used and are found in at least 9.4% of referenced products, while hydrogenated oils are less commonly used (0.01%). 2. Which Are the Ingredients/Additives Characteristic of Ultra-Processing, and What Is Their Origin? In addition, for proteins, gluten (>1.7%) and milk protein isolates (>3.7%) are the most commonly used, while egg white proteins, gelatine, as well as pea and soy protein are less commonly used, falling in the range of 0.01–0.6%, and protein hydrolysates are used in a minimum of 0.04% of referenced products. Aromas are much more commonly used, being found in at least 10.5% of all referenced products (Table 1). Concerning additives, the most commonly used are texturing agents such as lecithins (>3.4%), modified starches (>2.4%), xanthan gum (>1.7%), mono- and diglycerides of fatty acids (>1.7%), pectins (>1.5%), diphosphates/pyrophosphates (>1.5%), guar gum (>1.3%), and carraghenans (>1.2%); colouring agents such as capsanthin (>0.7%), carotenes (>0.6%), carmines (>0.5%), and plain caramel (>0.5%); and taste modifiers such as monosodium glutamate (>0.5%), sucralose (>0.4%), acesulfame potassium (>0.3%), aspartame (>0.2%), and steviol glycosides (>0.1%) (Table 2). Mass production of ultra-processed non-additive ingredients, and of numerous additives processed from the cracking of raw foods, mainly comes from intensive monocultures or livestock of only a few plant/animal varieties (see Section 3.3. related to industrial farming/agriculture). At minimum, their percentage use in foods varies from 0.03 to 12.6 of all foods (Tables 1 and 2), suggesting a high level of consumption, notably due to the rapid increase in worldwide UPF consumption, especially in Latin America [25]. In the following section, we will therefore analyse how the agricultural system at the basis of these ingredients is linked with sustainability or not, and the impacts of UPF-like product consumption on environmental indicators such as greenhouse gas emissions (GHGEs). 5 of 29 Sustainability 2020, 12, 6280 Food matrix unstructuration and ingredient isolation Ingredient recombination Addition of purified (loss of protective bioactive compounds) and cosmetic (markers of ultra-processing) ingredients/compensatory additives: - Texture agents - Taste enhancers - Dyes - Aromas - Sweeteners ... Original raw food A Original raw food B Original raw food C Recombinant artificial ultra- processed food made of food ingredients A, B, C ... + cosmetic additives (± real foods) … Figure 2. Schematic representation of UPFs through fractionation of original raw foods and ingredient recombination with ‘cosmetic’ additives. Figure was originally supplied by the Siga Society©. Original raw food A Recombinant artificial ultra- processed food made of food ingredients A, B, C ... 2. Which Are the Ingredients/Additives Characteristic of Ultra-Processing, and What Is Their Origin? Additives Number of Food Products 2 Percentage of all Products in the Open Food Facts Database 2 T 1 Collected from the French Open Food Fact database, which contains 690,499 products (on 20 June 2020, as described previously [24]); 2 Ingredient lists are not given for all products in the Open Food Facts database: therefore, given values are only minimum values. 3 Refined oils are not strictly characteristic of UPFs in NOVA classification; however, due to the high level of processing that refined oils undergo, they were considered in this analysis, as in the Siga score methodology [13]. 4 Includes artificial and natural aromas. 1 Collected from the French Open Food Fact database, which contains 690,499 products (on 20 June 2020, as described previously [24]); 2 Ingredient lists are not given for all products in the Open Food Facts database: therefore, given values are only minimum values. 3 Refined oils are not strictly characteristic of UPFs in NOVA classification; however, due to the high level of processing that refined oils undergo, they were considered in this analysis, as in the Siga score methodology [13]. 4 Includes artificial and natural aromas. ble 2. Number of food products for the different ‘cosmetic’ additives characteristic of ultra-processing 1. Table 2. Number of food products for the different ‘cosmetic’ additives characteristic of ultra-processing 1. 2. Which Are the Ingredients/Additives Characteristic of Ultra-Processing, and What Is Their Origin? + cosmetic additives (± real foods) Ingredient recombination g … Addition of purified (loss of protective bioactive compounds) and cosmetic (markers of ultra-processing) ingredients/compensatory additives: - Texture agents - Sweeteners … Figure 2. Schematic representation of UPFs through fractionation of original raw foods and ingredient recombination with ‘cosmetic’ additives. Figure was originally supplied by the Siga Society©. Figure 2. Schematic representation of UPFs through fractionation of original raw foods and ingredient recombination with ‘cosmetic’ additives. Figure was originally supplied by the Siga Society©. 6 of 29 Sustainability 2020, 12, 6280 Table 1. Numberoffoodproductsforthedifferentnon-additiveingredientscharacteristicofultra-processing 1. Ingredients Number of Food Products 2 Percentage of All Products in the Open Food Facts Database 2 Ultra-processed carbohydrates: Glucose-fructose syrup/glucose syrup/(oligo)fructose >52,154 >7.6 Starch >22,389 >3.2 Dextrose >21,340 >3.1 Lactose >11,232 >1.6 Malt (extract) >8292 >1.2 Maltodextrins/dextrins >7756 >1.1 Invert sugar >4349 >0.6 Ultra-processed lipids: Refined plant-based oils and fats 3 >64,811 >9.4 Hydrogenated oils >99 >0.01 Ultra-processed proteins: Milk/whey/casein protein >11,789 >1.7 Gluten >11,428 >1.7 Gelatine >3970 >0.6 Soy protein >1953 >0.3 Pea protein >1289 >0.2 Protein hydrolysate/hydrolysed proteins >307 >0.04 Egg white and protein >62 >0.01 Aroma 4: >72,348 >10.5 1 Collected from the French Open Food Fact database, which contains 690,499 products (on 20 June 2020, as described previously [24]); 2 Ingredient lists are not given for all products in the Open Food Facts database: therefore, given values are only minimum values. 3 Refined oils are not strictly characteristic of UPFs in NOVA classification; however, due to the high level of processing that refined oils undergo, they were considered in this analysis, as in the Siga score methodology [13]. 4 Includes artificial and natural aromas. Table 2. Number of food products for the different ‘cosmetic’ additives characteristic of ultra-processing 1. 1 Collected from the French Open Food Fact database, which contains 690,499 products (on 20 June 2020, as described previously [24]); 2 Ingredient lists are not given for all products in the Open Food Facts database; therefore, given values are only minimum values. 2. Which Are the Ingredients/Additives Characteristic of Ultra-Processing, and What Is Their Origin? Additives Number of Food Products 2 Percentage of all Products in the Open Food Facts Database 2 Texture: E322: lecithins >23,640 >3.4 E14XX: modified starches >16,405 >2.4 E415: xanthan gum >12,015 >1.7 E471: mono and diglycerides of fatty acids >11,828 >1.7 E440: pectin >10,172 >1.5 E450: diphosphates, pyrophosphates >10,644 >1.5 E412: guar gum >9177 >1.3 E407: carraghenans >8616 >1.2 E420: sorbitol >4285 >0.6 E406: agar-agar >842 >0.1 E1200: polydextrose >375 >0.1 E421: mannitol >235 >0.03 Colour: E160c: paprika extract, capsanthin, capsorubin >5101 >0.7 E160a: carotenes >4347 >0.6 E120: cochineal, carmines, carminic acid >3560 >0.5 E150a: plain caramel >3097 >0.5 E133: Brilliant blue FCF >1450 >0.2 Flavour/taste: E621: monosodium glutamate >3710 >0.5 E955: sucralose >2436 >0.4 E950: acesulfame potassium >2329 >0.3 E951: aspartame >1249 >0.2 E960: steviol glycosides >880 >0.1 E953: isomalt >443 >0.06 E967: xylitol >394 >0.06 E954: saccharine >238 >0.03 1 Collected from the French Open Food Fact database, which contains 690,499 products (on 20 June 2020, as described previously [24]); 2 Ingredient lists are not given for all products in the Open Food Facts database; therefore, given values are only minimum values. Table 1. Numberoffoodproductsforthedifferentnon-additiveingredientscharacteristicofultra-processing 1. Table 1. Numberoffoodproductsforthedifferentnon-additiveingredientscharacteristicofultra-processing 1. Ingredients Number of Food Products 2 Percentage of All Products in the Open Food Facts Database 2 Ultra-processed carbohydrates: Glucose-fructose syrup/glucose syrup/(oligo)fructose >52,154 >7.6 Starch >22,389 >3.2 Dextrose >21,340 >3.1 Lactose >11,232 >1.6 Malt (extract) >8292 >1.2 Maltodextrins/dextrins >7756 >1.1 Invert sugar >4349 >0.6 Ultra-processed lipids: Refined plant-based oils and fats 3 >64,811 >9.4 Hydrogenated oils >99 >0.01 Ultra-processed proteins: Milk/whey/casein protein >11,789 >1.7 Gluten >11,428 >1.7 Gelatine >3970 >0.6 Soy protein >1953 >0.3 Pea protein >1289 >0.2 Protein hydrolysate/hydrolysed proteins >307 >0.04 Egg white and protein >62 >0.01 Aroma 4: >72,348 >10.5 1 Collected from the French Open Food Fact database, which contains 690,499 products (on 20 June 2020, as described previously [24]); 2 Ingredient lists are not given for all products in the Open Food Facts database: therefore, given values are only minimum values. 3 Refined oils are not strictly characteristic of UPFs in NOVA classification; however, due to the high level of processing that refined oils undergo, they were considered in this analysis, as in the Siga score methodology [13]. 4 Includes artificial and natural aromas. Table 2. Number of food products for the different ‘cosmetic’ additives characteristic of ultra-processing 1. 3.1. General Considerations In 2013, ten key recommendations were formulated following an extensive review of the available guidance on agricultural programming for nutrition conducted by the FAO [27] and through consultation with a broad range of partners (civil society organizations, non-governmental organizations, government staff, donors, United Nations agencies), particularly through the “Community of Practice: Agriculture-Nutrition” (Ag2Nut) [28]. Three of the recommendations highlighted were to “maintain or improve the natural resource base (water, soil, air, climate, biodiversity)”, to “facilitate production diversification, and increase production of nutrient-dense crops and small-scale livestock” and to “improve processing, storage and preservation”. Otherwise, an increasing number of scientific observers from public institutions or private agencies (e.g., FAO [29,30], Solagro [31], INRAE-Cirad [32], The Lancet Commission [7]) underline, albeit with different wording, the unsustainability of our food systems worldwide. Concerning more specifically the impact of UPFs on the environment, the Brazilian Dietary Guidelines (2014) intuitively conclude that UPFs can impact the sustained survival of the planet [11]. Based on the first version of the NOVA classification (in three technological groups) [33], Keding et al. schematized three types of food processing within the food system [1]. From this description (Figure 3), it appears that the processing underlying UPFs involves more processing steps, more packaging, and longer transport distances. Thereafter, authors distinguished processing at the household, village and factory levels with different impacts on the environment, with the main risk of the factory level being “the community as a whole does not often share the profit, which is the main drawback of shifting towards factory food processing [34]” [1]. This is “why Clarke [34] demands that ‘factories need to be well planned and should be not too big as otherwise massive investments may be lost and local lifestyles, cultures and traditions can be seriously and often irretrievably affected’. An alternative approach for factory processing might be village processing [34]” [1]. More generally, in a presentation given at the International Sustainability Conference in 2005 entitled “Nutrition ecological assessment of processed foods”, Riegel et al. [1,35] gave a framework to rate the impact of processed foods not only on health but also on the other different dimension of sustainability, additionally including social, economic and environmental impacts. 2. Which Are the Ingredients/Additives Characteristic of Ultra-Processing, and What Is Their Origin? Additives Number of Food Products 2 Percentage of all Products in the Open Food Facts Database 2 Texture: E322: lecithins >23,640 >3.4 E14XX: modified starches >16,405 >2.4 E415: xanthan gum >12,015 >1.7 E471: mono and diglycerides of fatty acids >11,828 >1.7 E440: pectin >10,172 >1.5 E450: diphosphates, pyrophosphates >10,644 >1.5 E412: guar gum >9177 >1.3 E407: carraghenans >8616 >1.2 E420: sorbitol >4285 >0.6 E406: agar-agar >842 >0.1 E1200: polydextrose >375 >0.1 E421: mannitol >235 >0.03 Colour: E160c: paprika extract, capsanthin, capsorubin >5101 >0.7 E160a: carotenes >4347 >0.6 E120: cochineal, carmines, carminic acid >3560 >0.5 E150a: plain caramel >3097 >0.5 E133: Brilliant blue FCF >1450 >0.2 Flavour/taste: E621: monosodium glutamate >3710 >0.5 E955: sucralose >2436 >0.4 E950: acesulfame potassium >2329 >0.3 E951: aspartame >1249 >0.2 E960: steviol glycosides >880 >0.1 E953: isomalt >443 >0.06 E967: xylitol >394 >0.06 E954: saccharine >238 >0.03 1 Collected from the French Open Food Fact database, which contains 690,499 products (on 20 June 2020, as described previously [24]); 2 Ingredient lists are not given for all products in the Open Food Facts database; therefore, given values are only minimum values. 7 of 29 Sustainability 2020, 12, 6280 3. Ultra-Processing, Environment, Biodiversity and Animal Welfare In this section, we addressed the links between massive production and consumption of UPFs and the environment as a whole, including GHGEs, animal/plant biodiversity and animal wellbeing (Figure 1). The issue addressed is mainly the following: “Can diets be healthy and sustainable?” [26], but taking into account the level of food processing, especially UPFs in diets, an issue very rarely considered in previous analyses about food system sustainability. 3.1. General Considerations Concerning the environmental impact, authors proposed to consider agriculture (i.e., favouring low input and organic agriculture), transport (i.e., means of transport and miles per output unit), energy (i.e., consumption per output unit), and water (i.e., consumption per output unit and pollution) [1]. Sustainability 2020, 12, 6280 8 of 29 8 of 29 Unprocessed foods Minimally processed foods (G1) Processed culinary or food industry ingredients (G2) Ultra-processed food products (G3) Foodstuffs suitable for consumption Food processing type 1 Food processing type 2 Food processing type 3 No or minimally culinary preparation Culinary/kitchen processing Out of home sector (commercial level) Processing /Utilisation (household level) Commercial level Commercial level Retail Production Figure 3. Three types of food processing within the food system (adapted from Monteiro [33] and Keding et al. [1]). Ultra-processed food products (G3) Food processing type 2 Processed culinary or food industry ingredients (G2) No or minimally culinary preparation Minimally processed foods (G1) Food processing type 1 Foodstuffs suitable for consumption Culinary/kitchen processing Unprocessed foods Production Figure 3. Three types of food processing within the food system (adapted from Monteiro [33] and Keding et al. [1]). Sustainability 2020, 12, 6280 9 of 29 3.2. Food Processing and Carbon/Water Footprint 3.2. Food Processing and Carbon/Water Footprint First, it should be reminded that, in theory, when calculating the carbon footprint of a food product, it is necessary to take into account its entire “life cycle assessment” (LCA), from research and development to the final production of the product, including conditioning until final recycling. In France, the ADEME (French Environment & Energy Management Agency) has analysed the carbon footprint and energy footprint of French foods [36]. The results show that the agricultural production phase generates the most emissions (65%), followed by freight transport (19%), processing (6%), distribution and catering (5%), and consumption (5%). Thus, the ADEME advocates agroecology to reduce GHGEs. However, the LCA index is limited, favouring “high-input intensive agricultural systems and misrepresenting less intensive agro-ecological systems such as organic agriculture”, notably due to a narrow perspective on the holistic functions of global agricultural systems, e.g., operational indicators for environmental issues are lacking [37]. Recently, the FAO reported that UPF consumption in Australia (40% of the total dietary energy consumed, i.e., ≈20% by weight) contributes to more than one-third of all diet-related environmental effects (35% of land and water use, 39% of energy use, 33% of CO2 equivalents) [9]. Such empty caloric dietary trends will lead to nearly double per capita GHG emissions by 2050 [9]. 3.2.1. Discretionary Foods Discretionary foods are very similar to UPFs, as they are defined as energy-dense foods and drinks that are high in saturated fats, sugars, salt and/or alcohol and are not necessary to provide the nutrients that the body needs. At first glance, eating too many calories favours more GHGEs [38,39]. Therefore, UPFs, which lead to consume more calories than minimally processed foods [40], indirectly generate more GHGEs. Under this assumption, the study by Hendrie et al. [39] is particularly interesting. These authors showed that the overconsumption of energy and excessive discretionary foods contributes 29.4% to the total GHGEs of the Australian population. However, other food groups probably containing UPFs (see Appendix A of their paper) may contribute even more than 30% of the GHGEs of the overall diet. Furthermore, their study shows that reducing discretionary food intake would allow for small increases in emissions from core foods (particularly vegetables, dairy and grains), thereby providing a nutritional benefit at little environmental expense. However, the GHGE calculations in this study are derived from typical LCA and misrepresent less intensive agro-ecological systems [39]. Altogether, a first strategy for reducing GHGEs and to simply fulfil recommended energy needs would be to limit caloric intake from UPFs, i.e., at a level of 15% of recommended ≈2000 kcal/day [41]. Beyond GHGEs, water use is another indicator of the environmental impact of foods. In a recent study about core and discretionary foods consumed daily by a large (>9000) population of self-selected adults, a potential association between a healthier diet and lower environmental impacts was emphasized [42]. Indeed, this study concluded that “excessive consumption of discretionary foods–i.e., energy-dense and nutrient-poor foods high in saturated fat, added sugars and salt, and alcohol–contributes up to 36% of the water-scarcity impacts and is the primary factor differentiating healthier diets with lower water-scarcity footprint from poorer quality diets with higher water-scarcity footprint” [42]. The authors added that very large reductions in the dietary water-scarcity footprint are therefore possible, notably through technological change, product reformulation, and procurement strategies in the agricultural and food industries. 3.2.2. Ultra-Processed Food-Like Products within Dietary Patterns Because a global evaluation of processing on food system sustainability is not yet available, we first reported the GHGE impacts of dietary patterns, knowing that some diets contain more ultra-processed and/or discretionary foods than others do, e.g., the Western diet [43]. 10 of 29 Sustainability 2020, 12, 6280 First, Pradhan et al. defined sixteen global food consumption patterns: three low-calorie diets, five moderate-calorie diets, three high-calorie diets, and five very-high-calorie diets (i.e., above 2850 kcal/cap/day)–mostly found in Western countries and the Middle East [44]. Notably, the diet designated ‘#14′ is rich in animal products, sweeteners, and cereals. The results clearly show that diets richer in calories (#11–#16) produce the most GHGEs (>4 kg CO2eq./cap/day). Diets #1 (with cereals contributing to more than 50% of the total energy supply), #3 (with the highest amount of starchy roots), #6 (with the highest fraction of animal products and sugar-sweeteners), and #7 (with the highest amount of vegetable oils) also yield high levels of CO2eq./cap/day. The authors explained that non-CO2 GHGEs from enteric fermentation, rice cultivation, manure management and agricultural soils accounted for their high level of CO2eq./cap/day (>3 kg CO2eq./cap/day) [44]. More generally, countries characterized by high-calorie diets exhibit a production mode that needs high fossil energy inputs (1800–3500 kcal/cap/day) [44]. Then, an Australian study by Hadjikakou et al. [43] evaluated the environmental impact of discretionary foods (generally composed of UPFs) and found that they account for a significant 35%, 39%, 33% and 35% of overall diet-related life cycle water use, energy use, carbon dioxide equivalent and land use, respectively. The authors suggested a ‘divestment’ from discretionary food products by “food substitutions to minimize environmental and social impacts whilst maximizing nutritional quality–especially amongst poorer socioeconomic groups” (page 119) [43]. Otherwise, the contribution of UPF-like products to GHGEs is evaluated in the French survey by Barré et al., where high-fat/sugar/salt foods and mixed dishes contribute approximately 22–23% to GHGEs [45]. The same research team previously showed that soft drinks were the food group with the lowest GHGEs, whereas mixed dishes and sandwiches as well as foods high in fat/salt/sugar produced more GHGEs, air acidification and freshwater eutrophication than fats and condiments, starchy foods, and fruits and vegetables [46] but less than meat, fish, and eggs. 3.2.2. Ultra-Processed Food-Like Products within Dietary Patterns In another study, albeit one in which the degree of processing is not specifically mentioned, considering unhealthy foods close to UPFs, few differences were found for unhealthy food (alcohol or sweet/fatty food) consumption across the categories of dietary GHGEs [47]. However, the percentages of UPFs in other food categories, e.g., eggs, fruits and vegetables, red meat, fat, and dairy products, are very likely not to be 0%. In the UK study by Murakami & Livingstone [48], fats and oils, sugar and confectioneries, and soft drinks corresponded to 18.8% of GHGEs. In the study by Wickramasinghe et al. [49], fatty and sugary foods, either in school lunches or in packed lunches, represented approximately 8.5% of all GHGEs of the meal. The National Health and Nutrition Examination Survey is a more relevant study because its authors built a food impact database from an exhaustive review of food LCA studies and linked it to over 6000 as-consumed foods and dishes from one-day dietary recall data on nationally representative adults (n = 16,800, follow-up 2005–2010) [50]. Meats, dairy and beverages represented an approximately 80% contribution to total GHGEs; the proportion of UPF within these food categories remains to be determined. Another similar study about the Chinese diet, showing substantially increasing GHGEs from 1989 to 2009 through more fruit, vegetables, meat and dairy, also did not differentiate foods according to the degree of processing [51]. However, the Indian study by Green et al. distinguished GHGEs from primary production, processing, packaging, and waste for each food group [52]. Unsurprisingly, the GHGEs from primary production accounted for between 50% and 75% of GHGE emissions for all food groups, although in some foods, such as dairy and highly processed foods, processing and packaging also make substantial contributions. The authors also observed that GHGEs were highly variable across the thirty-six food groups, with mutton, butter and high-fat dairy products showing the greatest emissions per kg, followed by the “other” (mostly highly processed) food groups. Concerning dietary optimization with regard to GHGEs, other authors concluded that reducing the consumption of animal-based products, switching to meats and dairy products with lower associated emissions (e.g., pork, chicken and milk), reducing the consumption of savoury snacks, switching to fruits and vegetables with lower emissions, and increasing the consumption of cereals would reduce 11 of 29 Sustainability 2020, 12, 6280 GHGEs [53]. Similarly, in the Australian study by Hendrie et al. 3.3. Ultra-Processed Foods and Intensive Agriculture and Livestock Due to their massive supply at very low cost, which leads to massive consumption, the probability that UPFs are associated with intensive agriculture and livestock appears very high. 3.2.3. What to Do When Ultra-Processed/Discretionary Foods Are Not Available? Another final issue arises from the following question: “What would be the difference in environmental impact of foods people might consume if UPFs were not available?”. This is the case in some lower-income developing countries where plant-based diets are increasingly supplemented with animal-based calories, which are still mildly processed. Generally, such introduction of animal-based foods might threaten the environment and biodiversity, considering the sourcing of animals coming from either local hunting involving deforestation and/or growing intensive livestock, which demands high energy, land, chemicals, and water. For example, in emerging countries such as China, the increasing demands for meat and dairy drive up GHGEs [51], but increasing the intake of fruits and vegetables for a healthier diet may cancel out the environmental benefits from reducing meat intake [58]. Therefore, not increasing UPF consumption for more non-UPF foods such as animal products is not necessarily a guarantee of any reduction of the environmental footprint, particularly if nutrition transition consumption is based on animal products, independent of their level of processing. 3.3. Ultra-Processed Foods and Intensive Agriculture and Livestock 3.2.2. Ultra-Processed Food-Like Products within Dietary Patterns [39], foods wereclustered into core and non-core foods (similar to discretionary foods or UPFs). Non-core foods represented 27.1% of all GHGEs of the diet (3.9 kg CO2eq./cap/day), and by suppressing them from the average diet–with excess calories–to reach a balanced diet (called the “foundation diet”), GHGEs could be reduced by 25%. In Japan, exploring the factors differentiating the household food carbon footprint, Kanemoto et al. reported high emission intensities for some markers of ultra-processing, i.e., 7.06, 4.57, 7.61, 3.90, and 5.97 t-CO2eq./million yen for sugar, starch, dextrose/syrup/isomerized sugar, vegetable oils and meal, and animal oils and fats, respectively, compared to other typical minimally processed food groups. They noted that soft drinks are associated with a moderate carbon footprint (2.42) [54]. Finally, as pointed out by Aleksandrowicz et al. [55], these studies show that a convergence of healthy, low-GHGE and low-water footprint diets may be possible, though with a careful and realistic substitution of foods processed and supplied to populations [53]. Additionally, UPFs containing no or small amounts of animal source foods tend to have lower environmental impacts [56]. A recent global analysis, based on fifteen different food groups associated with five health outcomes and five aspects of environmental degradation, found that foods associated with improved adult health also often have low environmental impacts [57]. However, as mentioned above, reducing UPF consumption (which can reach up to 29% of the GHGEs of the diet) without substituting core food remains an interesting lever for more sustainable food systems. 3.2.3. What to Do When Ultra-Processed/Discretionary Foods Are Not Available? 3.3.1. Industrial Farming/Agriculture Six years ago, Keding et al. wrote, “Maximizing the nutrient output of farming systems for a culturally acceptable and balanced diet, however, has unfortunately never been an objective of agriculture, rather the objective has been to maximize production while minimizing costs [59]. Companies and breeders have influenced food crops, both through the introduction of varieties requiring certain inputs and by encouraging the growth of crops that may be industrially processed [60]. In some areas, replacement of traditional crops, such as legumes, by high yielding modern varieties has badly affected food resilience through the incorrect application of fertilizers and pesticides owing to lack of knowledge or financial resources, resulting in low or no yields at all [61].” [1]. In the same way, Johnston et al. reported that the “ . . . same successful global agro-food system is the dominant force behind many environmental threats, including climate change, simplification of diets, biodiversity loss, and degradation of land, soil, and freshwater [62]. If the current global food system Sustainability 2020, 12, 6280 12 of 29 continues to produce and process foods at the current amount and speed, it will continue to degrade the environment and compromise the capacity of the world to produce food in the future and will have irreversible effects on ecosystems [62–65].” [15]. The FAO also issues a warning with regard to the loss of plant biodiversity: of the 10,000 plant species that can be used as food for humans, only approximately 150 have been commercially cultivated, and only four (rice, wheat, maize, and potatoes) supply 50% of the world’s energy needs [66], with the latter being used for the massive production of starches, modified starches, and sugar syrups used in UPFs. The restricted diversity of highly cultivated crops has also led to intensive agriculture that is very demanding in terms of pesticides (herbicides, pesticides, etc.) and fertilizers. The French ADEME (Agence de l’environnement et de la maîtrise de l’énergie) specifically calculated the average GHGEs (CO2, N2O and CH4 in kg per kg of active ingredient) of these chemical substances (Table 3). Table 3. Average GHGEs for the main pesticides and fertilizers 1. 3.3.1. Industrial Farming/Agriculture kg CO2/kg of Active Ingredient kg CH4/kg of Active Ingredient kg N2O/kg of Active Ingredient Pesticides: Herbicides 8.33217 0.02548 0.00022 Fungicides 5.537 0.01855 0.00015 Insecticides 23.7 0.0543 0.00063 Growth regulators 7.86 0.0241 0.00021 Fertilizers 2: kg CO2/unit kg CO2/unit kg CO2/unit Manure in heap (ton) 2940.000 0.0647 9.120 Liquid manure (m3) 2920.000 0.0988 6.960 kg CO2eq./kg of nutrient Nitrogen fertilizer 5.34 Phosphate fertilizer 0.57 Potassium fertilizer 0.45 1 Retrieved from the ADEME website (on 20 June 2020) at http://www.bilans-ges.ademe.fr/documentation/UPLOAD_ DOC_FR/index.htm?pesticides_et_autres_produits_.htm, and http://www.bilans-ges.ademe.fr/documentation/ UPLOAD_DOC_FR/index.htm?engrais_et_composes_azotes.htm. Values are from GES’TIM, the methodological guide for estimating the impacts of agricultural activities on the greenhouse effect; 2 GHGE per kg of nitrogen in the fertilizer. Table 3. Average GHGEs for the main pesticides and fertilizers 1. le 3. Average GHGEs for the main pesticides and fertilizers 1. 1 Retrieved from the ADEME website (on 20 June 2020) at http://www.bilans-ges.ademe.fr/documentation/UPLOAD_ DOC_FR/index.htm?pesticides_et_autres_produits_.htm, and http://www.bilans-ges.ademe.fr/documentation/ UPLOAD_DOC_FR/index.htm?engrais_et_composes_azotes.htm. Values are from GES’TIM, the methodological guide for estimating the impacts of agricultural activities on the greenhouse effect; 2 GHGE per kg of nitrogen in the fertilizer. Among pesticides, insecticides clearly emit the most GHGs, approximately 2–3 times more than the others. Manure in heap emits slightly more GHGs than liquid manure. Finally, among fertilizes, nitrogen is by far the greatest emitter of GHGs. From these simple and synthetic data, it is clear that developing more organic agriculture may significantly reduce the level of GHGEs. Among pesticides, insecticides clearly emit the most GHGs, approximately 2–3 times more than the others. Manure in heap emits slightly more GHGs than liquid manure. Finally, among fertilizes, nitrogen is by far the greatest emitter of GHGs. From these simple and synthetic data, it is clear that developing more organic agriculture may significantly reduce the level of GHGEs. More generally, in 2018, the FAO published a report entitled “Soil pollution, a hidden reality” [67]. The cycle of soil pollution includes pesticides, livestock wastes, fertilizers, and/or irrigation with untreated water. Conventional intensive monocultures are therefore highly demanding in insecticides, pesticides, and fertilizers, and notably serve as the basis for the massive production of ingredients contained in UPFs, producing high amounts of GHGE. 3.3.2. Intensive Livestock Due to the generally high quantity and very low cost of animal-based UPFs, animal ingredients of these products are very likely to come from intensive livestock, very often associated with animal suffering and/or abuse [68]. According to the FAO, livestock production is widespread around the world, with up to 26% of terrestrial areas dedicated to rangelands and 33% of croplands dedicated to fodder production. Although intensive livestock systems use less land by unit of product, they are often associated with a higher use of inputs and higher concentrations of animals. Such an association can lead to nutrient 13 of 29 13 of 29 Sustainability 2020, 12, 6280 pollution if the system does not incorporate nutrient capture and recycling technologies; it can also lead to habitat destruction by heavily fertilized feed crops with an impact on biodiversity [69]. According to another FAO report [70], GHGEs along livestock supply chains were estimated at 7.1 gigatons CO2eq./year, representing 14.5% of all human-induced emissions. This sector plays an important role in climate change through feed production, and processing and enteric fermentation from ruminants are the two main sources of GHGEs, representing 45% and 39% of sector emissions, respectively. Land-use change for feed production, i.e., the expansion of pasture and feed crops into forests, accounts for approximately 9% of sector GHGEs. Manure storage and processing represent 10% of emissions, whereas the remainder are attributable to the processing and transportation of animal products, including the consumption of fossil fuel along the sector supply chain, contributing approximately 20% of GHGEs. In this sector, beef and cattle milk contribute 41% and 20% of the sector’s emissions, respectively, while pig and poultry meat and eggs contribute 9% and 8%, respectively. Finally, enteric CH4 accounts for 39.1% of global emissions from livestock supply chains. Intensive livestock systems can also concentrate manure at the site of production, which, if improperly managed, can adversely impact soil and water quality [71]. Conversely, it is also important to note that extensively managed grassland-based systems can provide crucial biodiversity habitats extended to wildlife species [71] but with higher GHGEs per unit of product compared to intensively managed systems [71]. The reason lies in the fact that “these ‘units of product’ usually focus on food or proteins and do not take into account other social and ecosystem services” (page 19) [71], i.e., lacking a holistic perspective, as also discussed by van der Werf [37]. 3.3.2. Intensive Livestock The FAO also reported that high-yielding animals producing more milk per lactation generally exhibit lower GHGE intensities [70]. Notably, the main reason is that the impact per unit of milk is reduced at both the individual cow and dairy herd levels due to the reduced standing biomass (both in lactating and in replacement herds) per unit of milk produced. However, it seems that in such calculations based on LCA [72], the GHGEs produced by deforestation for intensive monocultures to feed animals were not considered–nor was animal suffering in intensive and concentrating conditions (see below). Conversely, on a per cow basis, GHGEs increase with higher yields because higher productivity is usually associated with larger animals and a higher feed intake [72]. Concerning pigs, industrial production produces more GHGEs than backyard production (approximately 8% less) [70]. Otherwise, the role of agriculture as a driver of deforestation has gained recognition in UNFCCC (United Nations Framework Convention on Climate Change) REDD+ (Reducing Emissions from Deforestation and Forest Degradation) negotiations since 2012 [73]. In addition, soybeans and corn for feed are estimated to produce 340 and 1000 kg CO2eq./acre [74]. According to the Friends of the Earth Europe association [68], the intensive production of meat is not healthy because of the use of antibiotics and hormones and because of the overuse of chemicals in food production. In contrast, small-scale urban and rural livestock can make an important contribution to reducing poverty and to healthy food–not just in developing countries. From the 1960s to the 1970s, “people began to pay attention to animal welfare in intensive breeding after livestock and poultry husbandry changed from extensive range to intensive animal husbandry” [75]. In intensive livestock, including sow confinement and poultry breeding, animal welfare is no longer guaranteed, affecting the quality of animal products [75]. Behind this situation, there is the idea of refusing to sanction change unless supported by scientific evidence, even if ethical considerations can be considered sufficient per se [76]. Since the management of farm animals must take into account their physiological, social and behavioural needs, organic systems are probably a relevant solution for optimal welfare [77]. 3.3.3. Loss of Farming Animal Biodiversity Animal-based UPFs are linked to intensive livestock, and intensive livestock is also reported to be linked to loss of animal biodiversity, which means that UPF massive production is also related to loss of animal biodiversity. 14 of 29 Sustainability 2020, 12, 6280 One out of five breeds of livestock are threatened with extinction, and an alert was issued by the FAO in 2008 [78]. Of the 6300 domestic animal breeds, 1350 are threatened with extinction or have already disappeared. Their replacement is for the benefit of a small number of breeding breeds mostly selected for their productivity. A dozen animal species alone provides 90% of the animal protein consumed worldwide. In this respect, there is a race to control animal genetics by a handful of economic actors within the context of industrial agriculture [79]. According to the International Livestock Research Institute (https://www.ilri.org/), five breeds, all from Europe and North America, presently dominate world breeders. The carefully selected Prim’Holstein dairy cow of Dutch–German–American origin [80] is present in 128 countries and provides two-thirds of milk production in the world [79]. Similarly, Large White pigs, which are of English origin, are present in 117 countries, accounting for one-third of the global supply of pigs in the world. The top five also include Saanen goats, which are Swiss in origin (81 countries), the Spanish Merino sheep (60 countries), and white Leghorn laying hens, which are of Italian origin and raised all over the world [79]. Ultimately, as reported by the FAO, virtually one breed has disappeared per month over the last six years, and livestock production around the world is increasingly based on a limited number of breeds [78]. This approach of highly efficient breeds can be questioned in regard to sustainable food systems, particularly specific diseases that can affect these animals, which are selected for their production but not for their disease resistance and are maintained through the use of vaccines and antibiotics. 3.4.1. Energy by Food Groups and Processes This section will focus on the energy spent for food processing, mainly based on the recent and exhaustive review by Ladha-Sabur et al. [87]. First, the authors reported that the food sector consumes approximately 200 EJ (exajoule = 1018 J) globally per year [90,91], of which 45% corresponds to processing and distribution activities [92,93]. Ladha-Sabur et al. found that products that are freeze-dried–such as instant coffee (average of 50.20 MJ/kg) and milk powder (average of 16.22 MJ/kg)–or dried–such as French fries (average of 15.16 MJ/kg) and crisps (average of 17.30 MJ/kg)–consume among the highest amounts of energy [87]. However, value ranges are highly variable according to the energy origin, especially electricity versus fossil fuels (coal, petroleum, and gas), with electricity being much less energy demanding. Among processed and more processed foods, the highest maximum values are observable for chocolate, sugar, breakfast cereals, instant coffee, factory roasted and wrapped beef, deboned beef meat, beef pies, smoked and cooked pig joints, and distilled spirits. For other foods, notably some candy, cocoa butter, processed cereals, processed fats, food ingredients, light alcohols, soft drinks, pig ham, beef burger and bacon, and tomato-based products, the energy demand ranges from 0.07 to 11.11 MJ/kg. More specifically, taking food groups separately, the following striking conclusions are drawn [87]: (1) When including grinding, milling, wetting, drying, and baking, data from 1975 to 1996 report that 66 MJ/kg was used for the manufacture of breakfast cereals. The milling of flour appears to be an energy-intensive process [94]. (2) Potato-based products, notably dried products, consume the most energy among vegetables (Figure 4A) [95]. (3) Baking and freezing are the most energy-demanding steps for breads and rolls, biscuits and crackers, cakes, and frozen cakes, pies, and other pastries, i.e., 4.07 (67%), 4.17 (78%), 0.94 (38%) and 1.68 (32%) MJ/kg, respectively [87,96]. (4) Dairy processing is considered one of the most energy-intensive sectors within the food industry [97]. Cheese, including ripening, is the most energy intensive (13.85 MJ/kg), followed by powdered milk (10.30 MJ/kg) (Figure 4B), notably requiring over nine times more water, four times as much raw milk and electricity, and three times more fuel than processed milk [98]. For the latter, UHT and sterilization processes are also energy intensive since higher temperatures are required [87]. 3.4. Energy Consumption in Food Manufacturing, Packaging and Transport 3.4. Energy Consumption in Food Manufacturing, Packaging and Transport .4. Energy Consumption in Food Manufacturing, Packaging and Transport Overall, energy is intensively used both for manufacturing and for product transport to consumers [87], and the importance of the processing stage in the whole life cycle of elaborated food products has been emphasized by several authors [88,89]. 3.4.1. Energy by Food Groups and Processes 3.3.4. Plastic Pollution Overall, the consumption of UPFs is high in Western countries, especially Anglo-Saxon countries, with 307 kg/year per capita in the USA, followed by Canada (230 kg), Germany 219 kg), Mexico (214 kg), Belgium (210 kg), Australia, Norway and the UK (>200 kg/year) [20]. Conversely, it is still low in India (7 kg) and some African, South America and Asian countries (<100 kg) [20]. However, the growth rate of sales is very large in emerging countries, with a 115% increase in sales between 2000 and 2013 for Asian and Pacific regions, 71% in the Middle East and Africa, and 73% in Eastern Europe [20]. Overall, world growth was 44% during this period. Finally, the market share of UPFs is the highest in Asian and Pacific countries, with 29.2%. Therefore, our massive consumption of over-packaged UPFs worldwide is very likely to generate massive plastic pollution [67] without neglecting plastic bags to bring products from market to home. Indeed, over-packaged UPFs are designed to be consumed while travelling, in isolated situations, and rapidly [11]. Overall, the largest source of plastic production is packaging, driven by the pervasive commercial use of single-use containers destined for immediate disposal [81]. Worldwide, primary plastic waste generation has grown from nearly 0 in 1950 to 300 million metric tons (Mt) in 2015, with approximately 42% being used for food packaging [82] and approximately 79% being accumulated in landfills or the natural environment [83], with dramatic impacts on marine life [84]. In supermarkets, UPFs constitute more than two-thirds of packaged foods in France [13], more than 70% in the USA [85], and even more than 83% in New Zealand [16]. Therefore, it is very likely that returning to more fresh food should drastically alleviate plastic waste. Notably, marine animals are mostly affected through entanglement in and ingestion of plastic litter, and the absorption of polychlorinated biphenyls from ingested plastics is another threat [84]. As reported recently, there is also growing evidence that many single-use materials in contact with food, including plastics, can pose health risks to consumers through chemical migration [86]. It has been shown that harmful chemicals, such as endocrine disruptors, migrate not only in plastic packaging, but also in other materials, such as recycled paperboard. 15 of 29 Sustainability 2020, 12, 6280 15 of 29 3.4.4. Emerging Techniques Pardo and Zufía [100] proposed that emerging techniques may reduce the environmental impacts of preservation processing, such as lower energy demand and GHGEs, compared with traditional thermal processes. Environmental impacts may also be reduced with nonthermal technologies, including modified atmosphere packaging or high hydrostatic pressure, requiring less water than equivalent thermal processes [100]. Altogether, the two main targets of emerging techniques are as follows: - Their capacity to preserve foods by avoiding successive conditions of severe heating/cooling, which contribute to considerable water and heat consumption minimization; and - Their capacity to preserve foods by avoiding successive conditions of severe heating/cooling, which contribute to considerable water and heat consumption minimization; and - Electricity as the basis of the energy consumption source of such techniques, with an important contribution of renewable resources instead of the direct combustion of fossil fuels required for heat generation in conventional thermal treatments [100]. - Electricity as the basis of the energy consumption source of such techniques, with an important contribution of renewable resources instead of the direct combustion of fossil fuels required for heat generation in conventional thermal treatments [100]. 3.4.1. Energy by Food Groups and Processes (5) Poultry products are the most energy intensive (due to hair and feather removal and singeing operations), while beef, veal and sheep are the least energy intensive [87]. Overall, less processed products, such as butter, fish, eggs, pasta, poultry, beef and milk, consume less end use energy than processed products (e.g., fruit juices, yogurts, cheese, processed vegetable, sugar, bread, bacon, and ham) and ultra-processed-like products (e.g., soft drinks, biscuits, cakes, buns, pastries, crispbreads). Cheese is largely the more demanding in end use energy. Regarding processes, overall, thermal processes are energy intensive and responsible for a large proportion of the energy consumed in food processing [87]. Then, the highest maximum values are obtained for cooling (depending on temperature differences), drying, freeze-drying, packaging, microwave drying and milk pasteurization. Other preservation processes, such as dehydration or sterilization, have been estimated to account for approximately 29% of the total energy used in the food sector [99]. Globally, the LCA of processed foods is significantly impacted by preservation techniques, as stated by Pardo et al.: “This can be attributed to the large energy and water resources demanded during the preservation treatment. Since heat and electricity production steps often implies hydrocarbon combustion processes, this stage involves most of the air emissions to the atmosphere affecting categories such as climate change or acidification potential” (page 203) [100]. 16 of 29 16 of 29 Sustainability 2020, 12, 6280 A) B) Figure 4. Energy consumed in processing: (A) fruits and vegetables, and (B) dairy products (from Ladha-Sabur et al. [87], with permission of Elsevier under the terms of the Creative Commons CC-BY license©). A) B) A) A) gy consumed in processing: (A) fruits and vegetables, and (B) dairy products (from Ladha-Sabur et al. [87], with permission of Elsevier under the terms C CC BY li ©) Figure 4. Energy consumed in processing: (A) fruits and vegetables, and (B) dairy products (from Ladha-Sabur et al. [87], with permission of Elsevier under the terms of the Creative Commons CC-BY license©). 17 of 29 Sustainability 2020, 12, 6280 17 of 29 3.4.2. Packaging and Transport In the study by Pardo and Zufía [100], packaging implies a considerable share of the total evaluated impacts on the environment, particularly for preserving foods through pasteurization with either high hydrostatic processing, autoclaves, modified atmosphere packaging or microwaves, i.e., up to 80% for global warming potential. For example, the GHGE rates from food packaging through the use of fossil fuel (natural gas, coal, and petroleum) may reach 70.54 kg/MMBtu (million British thermal units) for petroleum and 94.67 kg/MMBtu for coal [74]. Transportation represents 19% of GHGEs when evaluated from farm to fork [36]. Thus, solutions to reduce transport can be sought by developing localized food supply systems [101] or developing food processing at the farm level. 3.4.3. Ultra-Processing? Strictly speaking, UPFs are recombination of processed ingredients that already consume energy, as described above, notably through intensive agriculture, transportation, and packaging. Thus, it is tempting to conclude that dispatching them worldwide might produce more GHGEs than consuming local raw and mildly processed foods. However, as described above, discretionary foods, with most of them being UPFs, do not produce the highest level of GHGEs, provided that LCA calculations are sufficiently holistic to consider all sources of GHGEs, from farm to fork. In addition, fragmentation of their production leads authors to assign trends in energy consumption to general food groups rather than specific food products [87]. Otherwise, very few data have been found on the level of energy use for UPF ready-to-eat meals that need to be cooked, preserved, and chilled or frozen [87]. 3.5. Partial Conclusions UPFs appear associated with a poor level of biodiversity, notably due to the few plant and animal varieties that supplied the ingredients used for their production and processing. Moreover, intensive monocultures are very demanding in high input energy, and animal calories found in UPF are associated with high levels of GHGE, as well as deforestation with feed animals in intensive conditions, that are otherwise far from respecting their basic needs and wellbeing. In addition, fractionating raw foods into massive amounts of ingredients for producing UPFs all around the world appears more energy demanding than locally consuming raw or minimally processed foods. Plant-based UPFs are clearly not so energy demanding than animal-based UPFs, but they are not yet associated with a better food system sustainability, especially regarding intensive monocultures. In the following section, we intended to go beyond agricultural and environmental considerations, and to analyse and discuss the impacts of massive UPF consumption on cultural and socio-economic dimensions. Sustainability 2020, 12, 6280 18 of 29 18 of 29 4. Ultra-Processed Foods, and Cultural and Socio-Economic Dimensions Beyond supplying nutrients and pleasure, diets are influenced not only by social/cultural traditions [102] (e.g., rice in Asia, cheeses in France) but also by religious traditions (e.g., vegetarianism in Hinduism) and socio-economic dimensions, including fair trade, the preservation of small farmers, and healthy food affordability [15]. Therefore, in this section, we addressed the links between massive production and consumption of UPFs, and culinary traditions, social life, and small farmers (Figure 1). 4.1. Ultra-Processed Foods and Culinary Traditions 4.1. Ultra-Processed Foods and Culinary Traditions 4.2. Ultra-Processed Foods and Socioeconomics Regarding social life, the Brazilian Dietary Guidelines [11] note that ready-to-consume UPFs, which can be consumed anytime and anywhere, “makes meals and sharing of food at table unnecessary”, leads to the isolation of the consumer even if these foods “are disguised by advertisements suggesting that such products promote social interaction, which they do not”. 4.2.1. The Socioeconomic Profiles of the High UPF Consumers 4.1. Ultra-Processed Foods and Culinary Traditions Regarding social and culinary traditions, the Brazilian Dietary Guidelines warn about the loss of culinary habits in the confrontation of the country with industrialized and standardized products disseminated by means of intensive and aggressive advertising campaigns, leading consumers, particularly younger consumers, to consider genuine food cultures to be uninteresting [11]. If food standardization obviously allows strict and efficient toxicological and hygienic control, conversely, it is also a basis for ultra-processed and unhealthy foods. Indeed, the food safety paradigm has somewhat replaced food diversity and substitutes for healthier foods, as demonstrated in Western and emerging countries where consumers no longer die from food toxins but from chronic diseases and suffer from deficiencies because the empty calories from UPFs do not supply enough protective micronutrients (i.e., hidden hunger) [8,16,17,103–106]. Food standardization is also accompanied by standardized tastes worldwide [107,108]. Consequently, vacationers and travellers may prefer to buy UPFs abroad with no risk of disliking the product rather than testing a local dish with the risk of not liking it. The same is true for children, who are accustomed at a very young age to a standardized taste and who, upon reaching adulthood, reject real foods with subtler tastes. One can also observe that in numerous emerging and developing countries where the standard of living increases, this translates into the decline of traditional foods, i.e., there is a shift towards a certain homogenization of the way of eating, i.e., towards more animal and UPF calories, which are often considered outward signs of wealth [8]. However, if UPFs are very standardized foods marketed worldwide, there is also a tendency towards diet diversification due to world exchange [108]. At present, it is clear that several countries have access to a much higher food diversity than was available several hundred years ago, but this diversification has more to do with real or gastronomic foods than with UPFs. Moreover, the hyper-palatability of the latter increases the frequency of their consumption, to the detriment of traditional foods, resulting in a real addiction, as observed in obese children in Brazil [109]. 4.2.1. The Socioeconomic Profiles of the High UPF Consumers In Westernized countries, populations with higher incomes can purchase foods of greater variety and nutritional value [15]. Thus, it is well demonstrated that there are more obese and diabetic people in low-income populations [110,111] or countries [112,113], notably because less healthy foods (often imported) are less expensive than locally produced foods [15]. Because of the numerous published studies on UPFs, it is now possible to start depicting the socioeconomic status of high-UPF consumers according to country. Sustainability 2020, 12, 6280 19 of 29 - In France, a higher consumption of UPFs was independently associated with being male, being younger, having a lower income level, smoking, being overweight, being obese, and having a lower level of education [114]. - In France, a higher consumption of UPFs was independently associated with being male, being younger, having a lower income level, smoking, being overweight, being obese, and having a lower level of education [114]. - In France, a higher consumption of UPFs was independently associated with being male, being younger, having a lower income level, smoking, being overweight, being obese, and having a lower level of education [114]. - The Spanish SUN cohort of young university graduates, who have a high level of education, revealed other associated factors, including sedentary activities (computer, television) and a high total fat intake together with a low protein and carbohydrate intake [115]. - The Spanish SUN cohort of young university graduates, who have a high level of education, revealed other associated factors, including sedentary activities (computer, television) and a high total fat intake together with a low protein and carbohydrate intake [115]. - In the USA, the highest consumers of UPFs (NHANES cohort, 1988–1994) are more likely to be younger, male, non-Hispanic White and current smokers and are less likely to have less than a high school level of education or to have a household income of more than 350% of the poverty level [116]. Similar results in the USA were obtained in the NHANES cohort (2009–2014), showing that subjects who have an income-to-poverty ratio <3.5, 12 years of education, and low physical activity and who are current smokers present the highest UPF consumption [117]. - In South Korea, energy drink intake in Korean adolescents, in isolation or in combination with junk food consumption, was shown to have detrimental effects related to stress, sleep dissatisfaction, mood, and suicidality [118]. Concerning social isolation, Bae et al. 4.2.1. The Socioeconomic Profiles of the High UPF Consumers showed that adolescent female rats’ body weight gain and daily chow intake were significantly increased by this stress, suggesting that social isolation during adolescence may increase food intake, perhaps preferentially towards palatable food [119]. This result was confirmed in mice that become obese under social isolation stress [120]. Surprisingly, however, although social isolation generally increases the risk of type 2 diabetes, socially connected obese participants pose a higher risk of type 2 diabetes than socially isolated obese participants, potentially because the stigmatization of obesity leads to negative social interactions [121]. Indeed, overweight youth are more likely to experience verbal victimization, feel less supported by their peers, and are less likely to date than youth who are not overweight from mid-adolescence into early young adulthood [122]. Data reported in France and the USA showed that the highest UPF consumers had lower income and educational levels. Since higher UPF consumption is associated with a higher prevalence of obesity [123], this may be related to the well-known fact that lower-income populations in high-income countries often have higher rates of obesity and diabetes than do high-income populations in high-income countries [124]. However, lower-income countries often have lower rates of obesity and diabetes than higher-income countries [125], although conditions will worsen due to the rapid nutrition transition that includes a significant level of UPFs, as shown in developing and emerging countries [20]. 5.1. A Global Synthesis from Published Data In this review, we intended to answer to the following issue: “are UPFs linked to food system sustainability regarding, beyond human health, the degradation of the other five dimensions of the food system as shown on Figure 1?” First, UPFs, encompassing other designations such as junk, discretionary, non-core, or sometimes street foods, is an updated concept that explains why it was difficult to obtain specific information about their potential associations with the different dimensions of food systems worldwide (Figure 1). Nevertheless, on Figure 5, in reference to Figure 1 and based on the gathered data, we built the potential links between excess UPF consumption and the alteration of the different dimensions of the food system sustainability. More generally, by combining both the low cost at purchase and increased consumption worldwide, most of these products appear potentially associated with intensive agriculture/livestock, a loss of culinary traditions, the progressive disappearance of small farmers/peasants, increased animal suffering, a loss of biodiversity, and social inequalities (Figure 5). 4.3. Partial Conclusions Overall, UPFs do not appear associated with a high level of social life, being consumed in isolated situations, e.g., in front of screens or on the move. On the contrary, real meals mostly made of real foods are associated with moments of festivity and family sharing. Due to their very low cost, some of them may also threaten small farmers and producers in many countries worldwide, especially in developing countries where local foods may be more expensive. In our developed societies, UPFs are generally more consumed by the poorest and less educated people, contrary to emerging and developing countries where they may appear as outward signs of wealth. Finally, through the high level of standardization, and their lower cost, many of them are progressively replacing some culinary traditions worldwide, especially among the youngest, such traditions appearing less attractive, with more subtle, risky, and demanding tastes. 4.2.2. Ultra-Processed Foods and Small Farmers Low-price, ready-to-eat, and highly attractive UPFs may lead to a partial or complete substitution of local and traditional foods, especially in emerging and developing countries. For example, in Africa, it has been observed that the import of chicken wings destroys local companies [68]. Indeed, the processing of slaughtering by-products into animal feed is prohibited for European poultry companies, and as a result, these countries export them cheaply to developing countries. This is only one example among others, e.g., excess milk in Europe is dried, defatted and exported to Africa, where it is cheaper than local milk. As reported by Johnston et al. [15], the reason lies in the fact that “current government subsidies to farmers in the United States and parts of Europe enable developed countries to produce large quantities of cheap staple and ultra-processed foods at 40–60% below the cost of local production of similar goods in developing countries [126]. In turn, these less healthy foods as massive imports are considerably less expensive than the locally produced foods, distorting local markets and depressing demand for the more expensive, locally produced, and often times healthier food options [63]”. Sustainability 2020, 12, 6280 20 of 29 Therefore, the adoption of imported UPFs from developed countries may directly threaten small farmers in developing countries, who are then obliged ‘to put the key under the door’ and to feed the slums. 5.2. Non-UPF Versus UPF? Although present studies suggest that UPFs do not necessarily produce the highest GHGEs, within a context of overconsumption of animal calories, their contribution to GHGEs could be importantly reduced without negative health effects. It should also be recognized that some non-UPFs may be produced at low cost [127] and/or environmental impact [128] while being highly consumed worldwide, e.g., refined sugars, oils and cereals, but to the detriment of health outcomes (e.g., obesity [129,130] or type 2 diabetes [131]). However, the contribution of some non-UPF food (e.g., palm oil, banana, avocado . . . ) to the degradation of food system sustainability is already well recognized, notably through intensive monocultures with large amounts of inputs and loss of biodiversity. 21 of 29 Sustainability 2020, 12, 6280 ↑ UPF consumption worldwide (low cost, highly standardized and overpackaged) ↑ Risk of chronic diseases ↓Healthy life years ↑ medical and pharmacological health care ↓Culinary traditions by substituting real foods and local dishes ↑ Standardisation of taste ↓Group, friends and family meals ↓Social life ↑ Chronic diseases within the poorest ↑ Intensive agriculture/livestock ↑ Deforestation ↑ Pollution: plastic, fertlizers, pesticides, herbicides… ↓Plant and animal biodiversity ↑ GHGE and temperature ↑ Animal abuse and suffering ↓ Smallholder agriculture ↑ Slums of big cities with unemployed small famers ↑ Worldwide distribution of highly processed ingredients/additives ↑ GHGE and temperature Recombination ↑ Raw food cracking: - Pea and soya - Maize, wheat and rice - Potato - Eggs and milk - Meat Small farmers Culinary traditions Human health Animal welfare Environment Biodiversity Environment Environment Social life Figure 5. A summary of the impact of increased UPF consumption on food system sustainability. ↑ GHGE and temperature Environment ↑ Raw food cracking: - Pea and soya - Maize, wheat and rice - Potato - Eggs and milk - Meat ↑ Worldwide distribution of highly processed ingredients/additives ↑ GHGE and temperature Recombination ↑ medical and pharmacological health care ↑ Risk of chronic diseases ↓Healthy life years Human health ↓Culinary traditions by substituting real foods and local dishes ↑ Standardisation of taste Figure 5. A summary of the impact of increased UPF consumption on food system sustainability. 22 of 29 Sustainability 2020, 12, 6280 6.1. Better Consideration of the Degree of Processing in Science and Food Policy 6.1. Better Consideration of the Degree of Processing in Science and Food Policy If agriculture is considered to produce too many GHGEs, future evaluations from farm to fork should further analyse the level of contribution of UPF processing, packaging, and transport. Similarly, when analysing the associations between food groups and GHGEs, it is important to discriminate the degree of processing of each of the foods included in those groups. Meanwhile, the available data appear sufficient to extend the application of the precautionary principle (applied to human health [132]) and to urgently implement policy regulations for agro-industrials to include nutritional and environmental criteria with regard to processed foods and policy incentives for consumers to shift from UPFs to real raw and mildly processed foods, preferably seasonal, organic and local products. 6.2. The 3V’s RULE Proposal to Counteract Excess UPF Consumption On that basis and extended to an ethical and sustainable diet, three golden rules for designing a protective diet food system sustainability have been elaborated in our laboratory, and taking into consideration the neglected dimension of the degree of processing (second rule). In French, this new concept is called the 3Vs Rule for Végétal (animal calories not exceeding 15% per day), Vrai (real: ultra-processed calories not exceeding 15% per day), and Varié (varied real foods), using, if possible, local, seasonal, and organic products [42,133]. In line with previous collective experience searching for a generic complex diet protecting both human health and the planet as a whole with a time horizon of 2050 [7,31,32,134–140], the 3Vs concept is based on a holistic view in that, through its application, it protects humans, animals, and the environment as a whole. Therefore, if removing the second rule concerning the degree of processing, and based on the data of this review, a diet appears no more fully sustainable. Finally, the 3Vs-based diet sustains several, if not all, dimensions of the regionally adapted food systems. Author Contributions: A.F. conceptualized the review, carried out the data extraction from the literature, and wrote the original draft of the manuscript. E.R. analysed the data and participated in the final writing and validation of the manuscript. A.F. and E.R. performed funding acquisition. All authors have read and agreed to the published version of the manuscript. Funding: This review article has been funded by the INRAE/Cirad’s GloFoodS (“Transitions for World Food Security”) metaprogramme. Conflicts of Interest: Anthony Fardet has been a member of the scientific committee of the Siga Society since 2017. Siga has developed a holistic food score based on the degree of processing and is specialized in UPF characterization. He is also a consultant for Wuji & Co. society, and co-president of the scientific committee of the Holistic Care Association. Edmond Rock declares no conflict of interest. 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https://periodicorease.pro.br/rease/article/download/4624/1735
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A ATUAÇÃO DO FARMACÊUTICO NA AUTOMEDICAÇÃO
Revista Ibero-Americana de Humanidades, Ciências e Educação
2,022
cc-by
2,916
doi.org/10.51891/rease.v8i3.4624 doi.org/10.51891/rease.v8i3.4624 Universidade Iguaçu no Estado do Rio de Janeiro. Palavras-chave: Automedicação. Atenção Farmacêutica. Riscos da automedicação. Palavras-chave: Automedicação. Atenção Farmacêutica. Riscos da automedicação. ABSTRACT: Self-medication is the act of taking a medication without the supervision of a qualified professional. In other words, the use of medicines without a prescription. This article aims to point out the dangers of self-medication. The main risks were analyzed in order to alert individuals about this practice. Self-medication has consequences. Consumption without knowledge leads to intoxication, addiction and even death. Every drug has side effects, and its long-term use can be harmful to health. 662 Keyword: Self-medication. Pharmaceutical attention. Risks of self-medication. 1 Graduação em Farmácia na Universidade Iguaçu. 2 Mestre em Ciências do Meio Ambiente na Universidade Veiga de Almeida. Faz parte do corpo ç g ç 2 Mestre em Ciências do Meio Ambiente na Universidade Veiga de Almeida. Faz parte do corpo docente da Universidade Iguaçu no Estado do Rio de Janeiro tre em Ciências do Meio Ambiente na Universidade Veiga de Almeida. Faz parte do corpo docente d ersidade Iguaçu no Estado do Rio de Janeiro. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.03. mar. 2022. ISSN - 2675 – 3375 1 Graduação em Farmácia na Universidade Iguaçu. d b d d d l d d d A ATUAÇÃO DO FARMACÊUTICO NA AUTOMEDICAÇÃO THE PHARMACIST'S PERFORMANCE IN AUTOMEDICATION Ana Caroline Moraes de Souza1 Leonardo Guimarães de Andrade2 RESUMO: A automedicação é o ato de tomar um medicamento sem a supervisão de um profissional habilitado. Em outras palavras, o uso de medicamentos sem prescrição médica. Esse artigo, tem como objetivo apontar os perigos da automedicação. Foram analisados os principais riscos, a fim de alertar aos indivíduos sobre essa prática. A automedicação traz consequências. O consumo sem conhecimento leva a intoxicação, dependência e até mesmo a morte. Todo medicamento possui efeito colateral, e seu uso a longo prazo pode ser prejudicial à saúde. Universidade Iguaçu no Estado do Rio de Janeiro. g 2 Mestre em Ciências do Meio Ambiente na Universidade V OBJETIVOS ESPECÍFICO • Orientar a automedicação; • Orientar a automedicação; • Reações adversas estimuladas pelo uso de medicamentos sem a orientação de um profissional não habilitado; • Debater prescrições de medicamentos; • Esclarecer que o objetivo do farmacêutico é orientar adequadamente o paciente durante o tratamento, a fim de obter um resultado satisfatório. OBJETIVO GERAL 663 O objetivo deste trabalho foi argumentar a responsabilidade do farmacêutico no combate à automedicação e os riscos à saúde decorrentes dessa prática. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.03. mar. 2022. ISSN - 2675 – 3375 INTRODUÇÃO A automedicação é o hábito de tomar medicamentos por conta própria ou por recomendação de amigos, familiares e conhecidos. No Brasil, a automedicação tem origem na época colonial portuguesa, quando os farmacêuticos eram responsáveis pela prescrição, processamento e produção de medicamentos. Dificuldade em marcar consultas na rede pública, venda de medicamentos sem receita médica, publicidade e buscas na web são alguns dos motivos da prática, que pode ser mais prejudicial à saúde do que se imagina. Os perigos da automedicação não só ç g ç 2 Mestre em Ciências do Meio Ambiente na Universidade Veiga de Almeida. Faz parte do corpo docente da Universidade Iguaçu no Estado do Rio de Janeiro. Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE agravam a doença, mas também levam à dependência, envenenamento, reações alérgicas, resistência aos medicamentos e até a morte. O papel do farmacêutico vai muito além da bancada, ele é responsável por todos os processos de produção de medicamentos, desde a fabricação até o consumidor final. Além de utilizar o próprio conhecimento para orientar os pacientes quanto ao tratamento, dosagem, efeitos colaterais, ou seja, o uso correto do insumo, a fim de alcançar resultados satisfatórios. 1 Graduação em Farmácia na Universidade Iguaçu. 1 Graduação em Farmácia na Universidade Iguaçu. 1 Graduação em Farmácia na Universidade Iguaçu. 2 Mestre em Ciências do Meio Ambiente na Universidade Veiga de Almeida (2016). Graduação em Enfermagem na Universidade Iguaçu. Faz parte do corpo docente da Universidade Iguaçu no Estado do Rio de Janeiro. 2 Mestre em Ciências do Meio Ambiente na Universidade Veiga de Almeida (2016). Graduação em Enfermagem na Universidade Iguaçu. Faz parte do corpo docente da Universidade Iguaçu no Estado do Rio de Janeiro. JUSTIFICATIVA O uso de medicamentos sem prescrição é um hábito muito comum, passado de geração em geração. Para evitar os riscos associados ao uso correto do medicamento, deve- se seguir as orientações de um profissional habilitado. É importante que o farmacêutico forneça informações claras esclarecendo dúvidas sobre o tratamento, eficácia, quantidade e dosagem. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.03. mar. 2022. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE A atenção farmacêutica tem como objetivo principal o paciente, promove o uso racional de medicamentos, obtendo eficácia em seu resultado. A atenção farmacêutica tem como objetivo principal o paciente, promove o uso racional de medicamentos, obtendo eficácia em seu resultado. Um costume cultural que ganhou forma com o passar do tempo. A automedicação no Brasil teve origem no período colonial, em plena colonização portuguesa. Na época, a saúde ficava nas mãos dos boticários, que prescrevem receitas sem embasamento científico para a população. Quase três séculos depois, muitos brasileiros se dirigem diretamente às farmácias para solucionar problemas de saúde, como dores de cabeça e crises de pressão arterial. Porém, longe de ser apenas uma prática cultural, a automedicação é responsável pela morte de 20 mil pessoas por ano no país, segundo dados da Associação Brasileira das Indústrias Farmacêuticas (Abifarma). (CORREIO BRAZILIENSE, 2010). Em uma sociedade com hábitos de consumo de medicamentos pode ser positiva a criação de políticas nacionais que promovem a regulamentação do suprimento e a disponibilização de medicamentos essenciais, pressupondo o acesso ao diagnóstico e prescrição por profissionais habilitados. Porém, por outro lado, o consumo pode ser influenciado negativamente pelo acesso sem barreiras e pela promoção e publicidade de medicamentos, que em sua maioria, estimulam a utilização desnecessária dos mesmos.” (NAVES et al., 2010). 664 O setor privado é o responsável por fornecer fármacos e medicamentos no Brasil, enquanto as farmácias são responsáveis pela comercialização de medicamentos e distribuição para a população em geral, o que coloca em maioria nas mãos de leigos, ou seja, de proprietários e balconistas.” (NAVES et al., 2010). Figura 1 Figura 1 Fonte : https://ictq.com.br/pesquisa-do-ictq/871-pesquisa-automedicacao-no-brasil-2018 Figura 1 Fonte : https://ictq.com.br/pesquisa-do-ictq/871-pesquisa-automedicacao-no-brasil-2018 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.03. mar. 2022. METODOLOGIA Para o desenvolvimento dessa pesquisa foi realizado um levantamento bibliográfico, em outras palavras, artigos, livros e monografias já publicados por outros autores em relação ao assunto pesquisado. 665 O presente estudo se apoia em artigos, revistas eletrônicas, monografia, PDF, dissertações, páginas de web sites, obtidos através de ferramentas eletrônicas, como SciELO-Scientific Eletronic Library, Google Acadêmico, biblioteca virtual, Anvisa e Ministério da Saúde. Sendo publicados nos períodos de 2018 até 2022. De modo, a obter informações sobre a atuação do farmacêutico na automedicação. JUSTIFICATIVA ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Os sintomas que levam as pessoas a se automedicarem, segundo os estudos são: dor de cabeça, resfriado/gripe, dor muscular e dor de garganta.” (MONTANARI et al., 2014; RODRIGUES et al., 2015; LIMA et al., 2017). Quando o médico prescreve a receita de um medicamento, ele leva em consideração sintomas e indicações necessárias para o seu tratamento, fatores genéticos, idade e condições de funcionamento dos rins e fígado, além de fatores como hábitos de alimentação e tabagismo, por exemplo, para assim indicar a medicação e a dosagem correta. No caso da automedicação, quando uma pessoa usa medicamentos sem prescrição ou supervisão de um médico, essa orientação não existe, e isto pode ser mais prejudicial do que se imagina, inclusive para doenças do coração e tratamentos contínuos (EUROFARMA, 2022). Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.03. mar. 2022. ISSN - 2675 – 3375 AS COMPLICAÇÕES CAUSADAS PELA AUTOMEDICAÇÃO AS COMPLICAÇÕES CAUSADAS PELA AUTOMEDICAÇÃO Figura 2 Fonte: http://neuroclinicasantafe.com.br/blog/treinamento/ Figura 2 DESENVOLVIMENTO Nenhuma droga é inofensiva ao organismo, muitos fatores levam ao consumo de insumos sem orientação trazendo consequências à saúde. O ideal é que o paciente siga sempre as orientações do farmacêutico. Para esse trabalho foram realizados estudos e pesquisas sobre o assunto abordado a fim de, trazer um melhor conhecimento sobre automedicação e os riscos causados por essa prática. Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE AS COMPLICAÇÕES CAUSADAS PELA AUTOMEDICAÇÃO Figura 2 AS COMPLICAÇÕES CAUSADAS PELA AUTOMEDICAÇÃO Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.03. mar. 2022. ISSN - 2675 – 3375 666 Fonte: http://neuroclinicasantafe.com.br/blog/treinamento/ O desenvolvimento de fármacos possibilitou transformações nas atividades de assistência à saúde e sendo assim, o medicamento é uma tecnologia difundida e muito utilizada. Contudo, com o advento do capitalismo e com o expressivo crescimento do consumo desses itens farmacêuticos em conjunto com o modelo de atenção à saúde focado no tratamento de doenças, fez com que o uso de medicamentos se tornasse cada vez maior tornando-se em muitos casos, abusivo e colocando a população diante de riscos relacionados ao seu uso (GUEDES, 2021). Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.03. mar. 2022. ISSN - 2675 – 3375 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.03. mar. 2022. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Segundo a publicação no site da empresa Pfizer, esses são as possíveis complicações causadas: Intoxicação - usar doses inadequadas de remédios pode causar diversos impactos na saúde, desde a ineficácia do tratamento, até overdose da substância no organismo, que leva a intoxicação (PFIZER, 2020). Interação medicamentosa - há risco de um medicamento ingerido reagir em contato com outro que a pessoa usa de forma contínua. Neste caso, um pode anular ou potencializar os efeitos do outro (PFIZER, 2020). Alívio dos sintomas que mascara o diagnóstico correto da doença - usar remédios para aliviar imediatamente dor e mal-estar pode esconder a real causa daqueles sintomas. Dessa forma, a doença não é tratada corretamente e pode se agravar (PFIZER, 2020). Reação alérgica - ingerir medicamentos que não foram prescritos por um profissional da saúde pode causar reações não esperadas no organismo (PFIZER, 2020). Dependência - algumas substâncias proporcionam mais chances de vício quando tomadas em doses incorretas e por tempo além do indicado por um médico (PFIZER, 2020). 667 Resistência ao medicamento - o uso indiscriminado de um remédio pode facilitar o aumento da resistência dos microrganismos àquela substância. No caso dos antibióticos, por exemplo, pode prejudicar a eficácia de tratamentos em infecções futuras (PFIZER, 2020). Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.03. mar. 2022. ISSN - 2675 – 3375 A ATUAÇÃO DO FARMACÊUTICO NO COMBATE A AUTOMEDICAÇÃO Segundo o Conselho Federal de Farmácia (2011) os farmacêuticos são profissionais da área da saúde, e tem uma função na sociedade, uma vez que é fundamental o trabalho do profissional na manipulação de fármacos e medicamentos e isso requer um saber específico já que trazem consequências ao organismo humano e animal. Desse modo, o trabalho de um farmacêutico vai desde indicar, aconselhar, deve ser o também de atuar na prevenção da automedicação. Podemos dizer assim, que este é um profissional multicomponente na sociedade. A inclusão do farmacêutico no processo de automedicação responsável começa com a percepção do problema de saúde pelo usuário, dessa maneira, é preciso que o , ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE profissional tenha a noção da competência e dos limites de sua intervenção no processo saúde/doença para que possa então assumir uma postura que esteja condizente com as determinações do seu conselho em relação ao que deve ou não ser feito, e ser ainda capaz de avaliar a situação do usuário, conduzindo-o quando preciso a uma consulta médica (ZUBIOLI, 2000, p.45). A prática da atenção farmacêutica é fundamental ao paciente, a fim de que haja o uso racional de medicamentos através de boa comunicação entre os profissionais farmacêuticos e usuários com relação às dosagens, posologias, informações importantes sobre os medicamentos, para que possam ser utilizados de maneira racional (PEDRO, 2020). Quando o medicamento é dispensado de maneira racional, ele está prescrito corretamente, com doseamento e tratamento perfeitos, pois conceder fármacos apropriados, de maneira segura, de acordo com o diagnóstico dado pelo médico, através de uma prescrição legível, a qual ao ser dispensada, deve sempre ser explicada ao paciente, tanto pelo farmacêutico, quanto pelo atendente de balcão, é fundamental para adesão ao tratamento (PEDRO, 2020). 668 A dispensação é ação de proporcionar medicamentos a pacientes, como resposta a uma receita, além disso, o farmacêutico deve informar e orienta o paciente sobre o uso adequado do medicamento, de forma que essas informações e orientações sobre a importância do respeito a dosagem, a influência da alimentação no resultado, além da interação com outros medicamentos, reações adversas e ainda sobre a conservação dos fármacos. Dessa maneira, podemos entender que como boas práticas, existe um conjunto de normas que estabelecem regras que garantem um bom trabalho (MINISTÉRIO DA SAÚDE, 2001). Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.03. mar. 2022. ISSN - 2675 – 3375 A ATUAÇÃO DO FARMACÊUTICO NO COMBATE A AUTOMEDICAÇÃO Importante frisar que os farmacêuticos são os únicos profissionais da saúde que possuem potencial formação para exercer a Atenção Farmacêutica no uso racional de medicamentos, já que toda sua bagagem de conhecimento acerca do medicamento está direcionada na base da sua formação acadêmica ao bem-estar físico, mental e social dos indivíduos, permitindo uma visão humanizada do paciente e usuário do medicamento e serviço (ENFAR, 2013). Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.03. mar. 2022. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE CONCLUSÃO Considerando o que foi observado no decorrer deste estudo, concluiu-se que o ato de tomar um medicamento sem receita médica, ou seja, tomar o medicamento sozinho, tem implicações para a saúde. No entanto, a coleta de informações sobre uma doença permanece válida desde que seja uma fonte puramente de conhecimento, e o diagnóstico, e a prescrição de medicamentos estejam sempre nas mãos dos profissionais certos. Os farmacêuticos atuam em todas as áreas da produção farmacêutica e, além de serem responsáveis pelas compras, garantem a qualidade dos produtos e serviços. Diante dessas informações, disponibiliza seu conhecimento para fornecer avaliação, monitoramento e distribuição de medicamentos. A atenção farmacêutica é uma estratégia que promove o uso racional de medicamentos e visa estabelecer uma relação direta com os pacientes. Cabe, portanto, ao farmacêutico atuar fora do balcão, no desempenho de suas funções, ao mesmo tempo que se volta para o atendimento farmacêutico e seu saber como aliado contra a automedicação. 669 REFERÊNCIAS Associação Paulista para o Desenvolvimento da Medicina. Riscos e consequências da automedicação. São Paulo; 2016. Associação Paulista para o Desenvolvimento da Medicina. Riscos e consequências da automedicação. São Paulo; 2016. CONSELHO FEDERAL DE FARMÁCIA. Atuação do Farmacêutico. Brasília, 2011. FERNANDES. CONSELHO FEDERAL DE FARMÁCIA. Atuação do Farmacêutico. Brasília, 2011. FERNANDES. CORREIO BRAZILIENSE. Automedicação é responsável pela morte de 20 mil pessoas por ano no Brasil. 2010. Cordeiro Júnior, E. M.., & Abreu, T. (2021). ATUAÇÃO DO PROFISSIONAL FARMACÊUTICO NA AUTOMEDICAÇÃO. Revista Ibero-Americana De Humanidades, Ciências E Educação, 7(9), 216–229. Domingues PHF, Galvão TF, Andrade KRC, Sá PTT, Silva MT, Pereira M. Prevalência da automedicação na população adulta do Brasil: revisão sistemática. Rev Saúde Pública. 2015; 49 (36): 1-8. FERREIRA, R. L.; TERRA JÚNIOR, A. T. ESTUDO SOBRE A AUTOMEDICAÇÃO, O USO IRRACIONAL DE MEDICAMENTOS E O PAPEL DO FARMACÊUTICO NA SUA PREVENÇÃO. 2018. 7 f. v. 9, n., Revista Científica FAEMA. Faculdade de Educação e Meio Ambiente, 2018. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.03. mar. 2022. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE GUEDES, A. C. S. A ATUAÇÃO DO FARMACÊUTICO NO COMBATE A AUTOMEDICAÇÃO. 2021. v. 7, Nova Iguaçu, Revista Ibero-Americana de Humanidades, Ciências e Educação, 2021. MARINHO, R. A.; CARDOSO, G. P.; FERREIRA, W. A. VANTAGENS E DESVANTAGENS DA AUTOMEDICAÇÃO: PRINCÍPIOS GERAIS. 2018. 6 f. v. 23, Rondônia, Brazilian Journal of Surgery and Clinical Research - BJSCR, 2018. FREITAS, MARIA ROSALINA SANA DE; GERON, Vera Lúcia Gomes. O PAPEL DO FARMACÊUTICO NO COMBATE A AUTOMEDICAÇÃO. 2020. MELO, D. O. D.; CASTRO, L. L. C. D. A contribuição do farmacêutico para a promoção do acesso e uso racional de medicamentos essenciais no SUS. 2015. v. 22, Diadema, SciELO Brasil Scientific Electronic Library Online, 2017. NAVES, Janeth de Oliveira Silva et al. Automedicação: uma abordagem qualitativa de suas motivações. Ciência & Saúde Coletiva [online].2010, v. 15, suppl 1, pp. 1751-1762. OLIVEIRA, Kamilla de; DUTRA, Ana Carolina Garcez; AZEVEDO, Arielly Cristina de. OS IMPACTOS DA AUTOMEDICAÇÃO NA SAÚDE. Episteme Transversalis, [S.l.], v. 12, n. 2, set. 2021. ISSN 2236-2649. SANTANA, D. P. H.; TAVEIRA, J. D. C. F.; EDUARDO, A. M. D. L. E. N. A Importância da Atenção Farmacêutica na Prevenção de Problemas de Saúde. Souza, R. C. de O. ., & Andrade, L. G. de . (2021). 670 TEIXEIRA, Daiele Ratin et al. AUTOMEDICAÇÃO. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.03. mar. 2022. ISSN - 2675 – 3375 REFERÊNCIAS Anais do Salão de Iniciação Cientifica Tecnológica ISSN-2358-8446, n. 1, 2021. ZUBIOLI, Arnaldo. O Farmacêutico e a automedicação responsável. Pharmacia Brasileira, 2000.
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Silver-catalyzed intramolecular oxidative decarboxylative C-H arylation reactions for synthesis of biaryl sultams
ARKIVOC
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Paper Archive for Organic Chemistry Archive for Organic Chemistry Arkivoc 2019, part vi, 105-115 Arkivoc 2019, part vi, 105-115 Silver-catalyzed intramolecular oxidative decarboxylative C-H arylation reactions for synthesis of biaryl sultams Silver-catalyzed intramolecular oxidative decarboxylative C-H arylation reactions for synthesis of biaryl sultams Junmin Chen,*a Qiuhong Wang,a Xiangkang Li,a,b and Yongli Zhaoa aKey Laboratory of Functional Small Organic Molecules, Ministry of Education and College of Chemistry & Chemical Engineering, Jiangxi Normal University, Nanchang, Jiangxi 330022, China; b Key Laboratory of Organo-Pharmaceutical Chemistry of Jiangxi Province, Gannan Normal University, Ganzhou 341000, China Email: jxnuchenjm@163.com Received 12-12-2018 Published on line 09-08-2019 Accepted 08-21-2019 The Free Internet Journal for Organic Chemistry The Free Internet Journal for Organic Chemistry Abstract A mild, versatile and efficient method to form biaryl sultams through silver-catalyzed intramolecular oxidative decarboxylative C-H arylation reactions has been developed. The present protocol features a broad substrate scope and very good tolerance to different substituent groups with satisfactory yields, giving access to a wide range of biaryl sultam derivatives. S O O N Ag2SO4 (10 mol%) K2S2O8 (2 equiv) CH3CN, 100 oC, 12 h S O O 1 2 COOH R2 N R2 R1 R1 13 examples, 73-89% yields 2 13 examples, 73-89% yields 13 examples, 73-89% yields Keywords: silver-catalyzed; decarboxylative; C-H arylation; biaryl sultam Keywords: silver-catalyzed; decarboxylative; C-H arylation; biaryl sultam Keywords: silver-catalyzed; decarboxylative; C-H arylation; biaryl sultam DOI: https://doi.org/10.24820/ark.5550190.p010.846 Page 105 ©ARKAT USA, Inc ©ARKAT USA, Inc Arkivoc 2019, vi, 105-115 Chen, J. et al. Introduction The sulfonamides have been extensively used as pharmaceutical and agricultural agents because of their diverse biological properties.1-5 Among them, sultams (cyclic sulfonamides) are important structural scaffolds. Thus the pharmaceutically relevant molecules A, B and C (Figure 1) have been found to exhibit broad inhibitory properties against a variety of enzymes as COX-2 ,6 HIV integrase,7 lipoxygenase,8 Calpain I,9 and MMP-2.10 Furthermore, biaryls embedded in cyclic sulfonamide (biaryl sultams) have emerged as privileged structures in drug discovery. For example, carbapenem derived biaryl sultam D provides for potent binding to the target penicillin binding proteins (PBPs).11 The related quinolinederived biaryl sultam E plays an active role in the NF-κB pathway, which has provided a favorable target for pharmacological intervention for chronic inflammation, neurodegenerative diseases, and certain types of cancer.6 As a consequence, a variety of strategies have been developed for the synthesis of sultams.12-17 However, it was less reported for the synthesis of biaryl based sultams. Recently, some methodologies have also been developed including intramolecular C-H arylation of the 2-halobenzenesulfonamides catalyzed by palladium or with the assistance of a single-electron-transfer pathway18-21 and intramolecular oxidative C-H amination of 2- phenylarylsulfonamides under metal-free conditions.22 In addtion, palladium catalyzed intramolecular oxidative coupling (IOC) of two C(sp2 )-H bonds was also developed for the synthesis of biaryl sultams.23 Very recently, an alterative approach has been reported for the preparation of biaryl sultams using visible-light- promoted denitrogenative cyclization of 1,2,3,4-benzothiatriazine-1,1-dioxides.24 Because of its significance in pharmaceutical development, the exploration of a novel and simple synthetic method for the construction of biaryl sultams would be highly desirable. N S O O OH O H N N S O O S O H2NO2S N S O O O H N O O O H NH S O O N N S O O N COONa Me O H H Me OH antibiotics D NF-κB inhibitor E Piroxicam (COX-2) A calpain 1 inhibitor B brinzolamide C N S O O OH O H N Piroxicam (COX-2) A N S O O S O H2NO2S N S O O O H N O O O H calpain 1 inhibitor B brinzolamide C H calpain 1 inhibitor B Piroxicam (COX-2) A Piroxicam (COX-2) A B brinzolamide C brinzolamide C N S O O N COONa Me O H H Me OH antibiotics D NH S O O N NF-κB inhibitor E COONa NH NF-κB inhibitor E antibiotics D Figure 1 Figure 1 Transition-metal-catalyzed decarboxylative transformations of arenecarboxylic acids through extrusion of the traceless CO2 have drawn considerable attention in the past decade.25 Since the pioneering work of Myers and Gooßen, decarboxylative coupling of benzoic acids with aryl halides or triflates using Pd/Cu or Pd/Ag bimetallic catalyst systems have been extensively studied.26-28 Crabtree developed an elegant method for the synthesis of biaryl compounds via transition-metal-catalyzed decarboxylative C-H arylation reaction.29 Subsequently, Larrosa,30 Su,31 Greaney,32 and others 33-36 reported palladium-catalyzed intramolecular and intermolecular decarboxylative arylation of activated heteroarenes for the synthsis of biaryl motifs. Notably, Page 106 Arkivoc 2019, vi, 105-115 Chen, J. et al. silver-catalyzed or visible-light-enabled decarboxylative transformations via aryl radical generation have also been successfully achieved.37-39 silver-catalyzed or visible-light-enabled decarboxylative transformations via aryl radical generation have also been successfully achieved.37-39 Inspired by recently elegant works on the intramolecular or intermolecular decarboxylative C-H arylation of (hetero)arenes with aromatic carboxylic acids.40 In the course of our ongoing investigation on transition- meta-catalyzed C-H activation reactions to access sulfonamide derivatives.41 We report herein the first example of silver-catalyzed intramolecular oxidative decarboxylative C-H arylation for synthesis of biaryl sultams. Results and Discussion Results and Discussion Initially, we selected the intramolecular oxidative decarboxylative C-H arylation of 2-phenylsulfamoyl-benzoic acid 1a as a model reaction for optimization studies (Table 1). To our delight, a 72% yield of the desired product 2a was obtained when the reaction was conducted in CH3CN at 100 oC for 12h in the presence of AgOAc (20 mol%) with K2S2O8 (2 equiv) as an oxidant (Table 1, entry 1). And then, various oxidants were investigated, we found the yield of 2a dropped to 37% (Table 1, entry 2) and 26% (Table 1, entry 3) when (NH4)2S2O8 and Cu(OAc)2 was used, respectively. Surprisingly, No desired product could be detected using PhI(OAc)2 as the oxidant (Table 1, entry 4). Subsequently, Investigation of different silver catalysts for the reaction revealed that Ag2SO4 was superior, giving 89% yield (Table 1, entries 5-8). It is interesting to find that reducing the amount of catalyst loading from 20 to 10 mol % did not effect on the activity and gave the comparable yield (87%) (Table 1, entry 9). Whereas further reducing the amount of catalyst loading to 5% mol % only affords 64% yield (Table 1, entry 10). Temperature has a great impact on this reaction; the product 2a was obtained in 76% yield at 80 °C (Table 1, entry 11). Control experiments revealed that no such coupling reaction occurred in the absence of the silver catalyst (Table 1, entry 12). Finally, the optimized reaction conditions were identified as follows: Ag2SO4 (10 mol %), and K2S2O8 (2 equiv) in CH3CN at 100 °C for 12 h under air. Page 107 ©ARKAT USA, Inc Table 1. Optimization of reaction conditions S O O N H Ag salt Oxidant (2 equiv) CH3CN 100 oC, 12 h S O O 1a 2a COOH NH Entry a Ag salt (mol%) Oxidant Yield b (%) 1 AgOAc (20) K2S2O8 72 2 AgOAc (20) (NH4)2S2O8 37 3 AgOAc (20) Cu(OAc)2 26 4 AgOAc (20) PhI(OAc)2 n.r 5 Ag2CO3 (20) K2S2O8 63 6 AgTFA (20) K2S2O8 51 7 AgNO3(20) K2S2O8 53 8 Ag2SO4 (20) K2S2O8 89 9 Ag2SO4 (10) K2S2O8 87 Table 1. Optimization of reaction conditions ©ARKAT USA, Inc Arkivoc 2019, vi, 105-115 Chen, J. et al. Chen, J. et al. Chen, J. et al. Arkivoc 2019, vi, 105-115 Chen, J. et al. Table 1. Results and Discussion Continued Entry a Ag salt (mol%) Oxidant Yield b (%) 10 Ag2SO4 (5) K2S2O8 64 11 c Ag2SO4 (10) K2S2O8 76 12 \ K2S2O8 n.r a Reaction conditions: 1a (0.2 mmol), Ag salt (0.02 mmol), oxidant (0.4 mmol), and CH3CN (1.0 mL), heated at 100 oC for 12 h under air. bIsolated yields. c at 80 oC. Table 1. Continued With the optimized reaction conditions in hand, we then evaluated the influence of the aniline moiety bearing various substituents on the reactivity and regioselectivity (Table 2). The electronic nature of the substituents seemed to have a little effect on the product yields, for example, substrate 1 with a electron- donating para-methyl or chrolo group on the N-aryl ring afforded the biaryl sultams 2b-c in 83% or 82% yield, respectively (Table 2, entries 2,3), while the slightly higher yield (87%) was obtained for 2d containing an electron-withdrawing para-CF3 group (Table 2, entry 4). As for substrates 1e-g bearing a meta-group, such as chrolo, floro or methoxyl, Cyclisation only occurs regioselectively on the less hindered position and the desired biaryl sultams 2e-g were obtained in good yields (Table 2, entries 5-7). It should be noted that ortho-chloro or ortho-methoxyl substituted substrates 1g, 1h, and 1i worked well to generate the corresponding desired products 2g-i in excellent yields without the cleavage of the C-Cl bond even though aryl halides are well- known to participate in Pd-catalyzed decarboxylative coupling reactions (Table 2, entries 8-10). Thus, these halogens can provide the opportunity for further transition metal catalyzed syntheses, thereby broadening the diversity of the products. Furtheromre, methyl protected amino group substrates 1k and 1l also provided the desired products 2k and 2l in very high yield (Table 2, entries 11,12). Notably, heteroaromatic thiophene on the acid moiety 1m was also successfully employed to provide the corresponding biaryl sultam 2m in good yield (Table 2, entry 13). Page 108 ©ARKAT USA, Inc e 2. Silver-catalyzed intramolecular oxidative decarboxylative C-H arylation S O O N Ag2SO4 (10 mol%) K2S2O8 (2 equiv) CH3CN, 100 oC, 12 h S O O 1 2 COOH R2 N R2 R1 R1 Entry a Substrate 1 Product 2 Yield b (%) 1 S O O N H 1a COOH S O O 2a NH 89 Table 2. Silver-catalyzed intramolecular oxidative decarboxylative C-H arylation Table 2. Results and Discussion Entry a Substrate 1 Product 2 Yield b (%) 11 S O O N 1k COOH S O O 2k N 82 12 S O O N 1l COOH S O O 2l N 77 13 S O O N H 1m S COOH S O O 2m NH S 73 O 12 2l 1l 2l 13 S O O N H 1m S COOH S O O 2m NH S 73 1l a Reaction conditions: 1 (0.2 mmol), Ag2SO4(0.02 mmol), ( ) g ( ) K2S2O8 (0.4 mmol), and CH3CN (1.0 mL), heated at 100 oC for 12 h under air. bIsolated yields. K2S2O8 (0.4 mmol), and CH3CN (1.0 mL), heated at 100 oC K2S2O8 (0.4 mmol), and CH3CN (1.0 mL), heated at 100 oC for 12 h under air K2S2O8 (0.4 mmol), and CH3CN (1.0 mL), heated at 100 oC for 12 h under air. b Conclusions In summary, we have developed a novel and efficient protocol for the synthesis of biaryl sultams via silver- catalyzed intramolecular oxidative decarboxylative C-H arylation. The reaction proceeds under mild conditions without the use of expensive transition metals or ligands. Many functional groups are tolerated, giving access to a wide range of biaryl sultam derivatives. The cyclization was proposed to proceed via a radical mechanism. Further investigations to the reaction mechanism and utilization of this catalyzed system in other biaryl based heterocyclic compounds are currently in progress. In summary, we have developed a novel and efficient protocol for the synthesis of biaryl sultams via silver- catalyzed intramolecular oxidative decarboxylative C-H arylation. The reaction proceeds under mild conditions without the use of expensive transition metals or ligands. Many functional groups are tolerated, giving access to a wide range of biaryl sultam derivatives. The cyclization was proposed to proceed via a radical mechanism. Further investigations to the reaction mechanism and utilization of this catalyzed system in other biaryl based heterocyclic compounds are currently in progress. Results and Discussion Silver-catalyzed intramolecular oxidative decarboxylative C-H arylatio Page 108 N K2S2O8 (2 equiv) CH3CN, 100 oC, 12 h 1 2 COOH N R2 Entry a Substrate 1 Product 2 Yield b (%) 1 S O O N H 1a COOH S O O 2a NH 89 Entry a Substrate 1 Product 2 Yield b (%) Entry a Substrate 1 a Substrate 1 NH ©ARKAT USA, Inc Arkivoc 2019, vi, 105-115 Chen, J. et al. Table 2. Continued Entry a Substrate 1 Product 2 Yield b (%) 2 S O O N H 1b COOH S O O 2b NH 83 3 S O O N H 1c COOH Cl S O O 2c NH Cl 82 4 S O O N H 1d COOH CF3 S O O 2c NH CF3 87 5 S O O NH 1e COOH Cl S O O 2e NH Cl 79 6 S O O NH 1f COOH F S O O 2f NH F 84 7 S O O NH 1g COOH O S O O 2g NH O 75 8 S O O NH 1h COOH Cl S O O 2h NH Cl 83 9 S O O NH 1i COOH Cl Cl S O O 2i NH Cl Cl 86 10 S O O N H 1j COOH O S O O 2j N O 74 Product 2 Yield (%) S O O 2b NH 83 S O O 2c NH Cl 82 2 S O O N H 1b COOH 1b 3 S O O N H 1c COOH Cl 1b 1c 1d 1e 1f 1h ©ARKAT USA, Inc Page 109 Arkivoc 2019, vi, 105-115 Chen, J. et al. Table 2. Continued Substrate 1 Product 2 Yield b (%) Product 2 Yield b (%) a Reaction conditions: 1 (0.2 mmol), Ag2SO4(0.02 mmol), K2S2O8 (0.4 mmol), and CH3CN (1.0 mL), heated at 100 oC for 12 h under air. bIsolated yields. Experimental Section General. All reactions were carried out under air atmosphere in oven-dried glassware with magnetic stirring. Unless otherwise specified, all other reagents and solvents were purchased from Energy Chemical, Alfa Aesar or J&K Chemical Company and used without any further purification. TLC information was recorded on GF-254 (Qingdao Haiyang Chemical Co., Ltd. P. R. China) plates. Purification of reaction products was carried out by flash chromatography using Silica gel (200-300 mesh, Qingdao Haiyang Chemical Co. Ltd. P. R. China). All products were recorded using Bruker Avance-400 instruments, calibrated to TMS as the internal reference (0.00 ppm for 1H NMR spectra and 100.00 ppm for 13C NMR spectra). High-resolution mass spectra (HRMS) were recorded on a Bruker Apex IV FTMS mass spectrometer using ESI (electrospray ionization). Melting points were measured uncorrected. Page 110 ©ARKAT USA, Inc ©ARKAT USA, Inc Page 110 Page 110 Arkivoc 2019, vi, 105-115 Chen, J. et al. Silver-catalyzed intramolecular oxidative decarboxylative C-H arylation 9-chloro-6H-dibenzo[c,e][1,2]thiazine 5,5-Dioxide (2c) White solid, isolated yield 85% (43 mg); mp: 156-158 oC; 1H NMR (400 MHz, d6-DMSO): δ 8.43 (d, J = 4.0 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 8.18-8.08 (m, 2H), 7.77-7.73 (m, 2H), 7.66-7.63 (m, 2H), 13C NMR (100 MHz, d6-DMSO): δ 158, 136.99, 136.58, 135.90, 131.25, 130.56, 128.01, 126.86, 126.04, 122.34. HRMS (ESI) m/z calcd for C12H9ClNO2S (M+H)+ 266.0043, found 266.0045. 9-(trifluromethyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-Dioxide (2d)42 White solid, isolated yield 87% ( 52 mg); mp: 234-235 oC; 1H NMR (400 MHz, d6-DMSO): δ 8.47 (d, J = 8.0 Hz, 8.27 (d, J = 8.0 Hz,) 8.20-7.07 (m, 4H), 7.89 (d, J = 8.0 Hz,). 13C NMR (100 MHz, d6-DMSO): δ 158.36, 136.89, 136.69, 136.01, 133.28, 130.76, 130.44, 129.81, 127.60, 127.67, 126.15, 125.57, 122.85, 122.37. 8-(chloro)-6H-dibenzo[c,e][1,2]thiazine 5,5-Dioxide (2e) White solid, isolated yield 79% (42 mg); mp: 155-157 oC; 1H NMR (400 MHz, d6-DMSO): δ 8.42 (d, J = 8.0 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.16-8.08 (m, 4H), 8.07-7.84 (m, 2H). 13C NMR (100 MHz, d6-DMSO): δ 158.36, 136.92, 136.69, 136.00, 133.29, 130.43, 129.79, 127.60, 127.56, 125.82, 125.16, 122.36. HRMS (ESI) m/z C12H9ClNO2S (M+H)+ 266.0043, found 266.0045. 8-(chloro)-6H-dibenzo[c,e][1,2]thiazine 5,5-Dioxide (2e) White solid, isolated yield 79% (42 mg); mp: 155-157 oC; 1H NMR (400 MHz, d6-DMSO): δ 8.42 (d, J = 8.0 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.16-8.08 (m, 4H), 8.07-7.84 (m, 2H). 13C NMR (100 MHz, d6-DMSO): δ 158.36, 136.92, 136.69, 136.00, 133.29, 130.43, 129.79, 127.60, 127.56, 125.82, 125.16, 122.36. HRMS (ESI) m/z C12H9ClNO2S (M+H)+ 266.0043, found 266.0045. 8-(fluoro)-6H-dibenzo[c,e][1,2]thiazine 5,5-Dioxide (2f) White solid, isolated yield 84% (42 mg); mp: 152-154 oC; 1H NMR (400 MHz, d6-DMSO): δ 8.28 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 8.02-7.92 (m, 2H), 7.60-7.54 (m, 1H), 7.41-7.32 (m, 3H), 13C NMR (100 MHz, d6-DMSO): δ 167.42 (d, J = 244 Hz), 163.11, 141.53 (d, J = 32 Hz), 140.69, 136.79, 135.37, 131.55, 130.81, 130.26, 127.09, 122.50 (d, J = 21 Hz), 121.46 (d, J = 25 Hz). HRMS (ESI) m/z calcd for C12H9O2NFS: 250.0333; found: 250.0330. Silver-catalyzed intramolecular oxidative decarboxylative C-H arylation y y y An oven-dried 10 mL Schlenk tube was charged with 1 (0.20 mmol), Ag2SO4 (6.22 mg, 0.02 mmol, 10 mol %), K2S2O8 (108 mg, 0.4 mmol, 2 equiv), and CH3CN (1 mL). It was then closed with a Teflon-lined cap and kept for stirring at 100 °C (preheated oil bath temperature) for 12 h. After the mixture cooled to room temperature, the reaction mixture was filtered through a short pad of Celite; the solvent was removed under reduced pressure and the residue was purified by silica gel chromatography using petroleum ether/EtOAc to afford the desired product 2. 6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide (2a)23 White solid, isolated yield 89% (41 mg); mp: 195-196 oC; 1H NMR (400 MHz, d6-DMSO): δ 8.42 (d, J = 8.0 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.16-8.08 (m, 4H), 8.06-7.84 (m, 2H). 13C NMR (100 MHz, d6-DMSO): δ 158.36, 136.92, 136.69, 136.00, 133.29, 130.43, 129.79, 127.60, 127.56, 126.82, 126.16, 122.36. 6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide (2a)23 White solid, isolated yield 89% (41 mg); mp: 195-196 oC; 1H NMR (400 MHz, d6-DMSO): δ 8.42 (d, J = 8.0 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.16-8.08 (m, 4H), 8.06-7.84 (m, 2H). 13C NMR (100 MHz, d6-DMSO): δ 158.36, 136.92, 136.69, 136.00, 133.29, 130.43, 129.79, 127.60, 127.56, 126.82, 126.16, 122.36. 9-Methyl-6H-dibenzo[c,e][1,2]thiazine 5,5-Dioxide (2b)42 White solid, isolated yield 82% (40 mg); mp: 217- 218 oC; 1H NMR (400 MHz, d6-DMSO): δ 8.15 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.94-7.86 (m, 2H), 7.41(d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 2.43 (s, 3H). 13C NMR (100 MHz, d6-DMSO): δ 158.52, 140.51, 137.71, 135.01, 134.40, 130.62, 128.66, 127.32, 125.83, 125.61, 121.25, 21.34. 9-chloro-6H-dibenzo[c,e][1,2]thiazine 5,5-Dioxide (2c) White solid, isolated yield 85% (43 mg); mp: 156-158 oC; 1H NMR (400 MHz, d6-DMSO): δ 8.43 (d, J = 4.0 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 8.18-8.08 (m, 2H), 7.77-7.73 (m, 2H), 7.66-7.63 (m, 2H), 13C NMR (100 MHz, d6-DMSO): δ 158, 136.99, 136.58, 135.90, 131.25, 130.56, 128.01, 126.86, 126.04, 122.34. HRMS (ESI) m/z calcd for C12H9ClNO2S (M+H)+ 266.0043, found 266.0045. Silver-catalyzed intramolecular oxidative decarboxylative C-H arylation 8-(fluoro)-6H-dibenzo[c,e][1,2]thiazine 5,5-Dioxide (2f) White solid, isolated yield 84% (42 mg); mp: 152-154 oC; 1H NMR (400 MHz, d6-DMSO): δ 8.28 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 8.02-7.92 (m, 2H), 7.60-7.54 (m, 1H), 7.41-7.32 (m, 3H), 13C NMR (100 MHz, d6-DMSO): δ 167.42 (d, J = 244 Hz), 163.11, 141.53 (d, J = 32 Hz), 140.69, 136.79, 135.37, 131.55, 130.81, 130.26, 127.09, 122.50 (d, J = 21 Hz), 121.46 (d, J = 25 Hz). HRMS (ESI) m/z calcd for C12H9O2NFS: 250.0333; found: 250.0330. 8-methoxy-6H-dibenzo[c,e][1,2]thiazine 5,5-Dioxide (2g)24 White solid, isolated yield 71% (64 mg); mp: 86-88 oC; 1H NMR (400 MHz, d6-DMSO): δ 8.44 (d, J = 8.0 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 8.19-8.09 (m, 2H), 7.52-7.48 (m, 2H), 7.24-7.21 (m, 2H), 3.90 (s, 1H). 13C NMR (100 MHz, d6-DMSO): δ 158.44, 137.08, 136.53, 135.85, 131.14, 130.10, 126.85, 125.98, 122.26, 121.36, 116.24, 115.19, 56.03. 8-methoxy-6H-dibenzo[c,e][1,2]thiazine 5,5-Dioxide (2g)24 White solid, isolated yield 71% (64 mg); mp: 86-88 oC; 1H NMR (400 MHz, d6-DMSO): δ 8.44 (d, J = 8.0 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 8.19-8.09 (m, 2H), 7.52-7.48 (m, 2H), 7.24-7.21 (m, 2H), 3.90 (s, 1H). 13C NMR (100 MHz, d6-DMSO): δ 158.44, 137.08, 136.53, 135.85, 131.14, 130.10, 126.85, 125.98, 122.26, 121.36, 116.24, 115.19, 56.03. 7-chloro-6H-dibenzo[c,e][1,2]thiazine 5,5-Dioxide (2h) White solid, isolated yield 83% (43 mg); mp: 141-143 oC; 1H NMR (400 MHz, d6-DMSO): δ 8.45 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.18-8.07 (m, 2H), 7.81 (d, J = 8.0 Hz, 1H), 7.72-7.62 (m, 3H), 13C NMR (100 MHz, d6-DMSO): δ 157.78, 137.51, 136.89, 136.11, 134.63, Page 111 ©ARKAT USA, Inc ©ARKAT USA, Inc Page 111 Arkivoc 2019, vi, 105-115 Chen, J. et al. 133.20, 132.95, 131.37, 129.26, 126.26, 122.61. HRMS (ESI) m/z calcd for C12H9ClNO2S (M+H)+ 266.0043, found 266.0044. 133.20, 132.95, 131.37, 129.26, 126.26, 122.61. HRMS (ESI) m/z calcd for C12H9ClNO2S (M+H)+ 266.0043, found 266.0044. 133.20, 132.95, 131.37, 129.26, 126.26, 122.61. HRMS (ESI) m/z calcd for C12H9ClNO2S (M+H)+ 266.0043, found 266.0044. Silver-catalyzed intramolecular oxidative decarboxylative C-H arylation 8-Methyl-5H-1,4-dithia-5-aza-cyclopenta[a]naphthalene 4,4-dioxide (2m) White solid, isolated yield 73% (37mg); mp: 158-160oC; 1H NMR (400 MHz, d6-DMSO): δ 8.47 (d, J = 4.0 Hz, 1H), 7.94-7.92 (m, 1H), 7.43-7.38 (m, 3H), 2.40 (s, 3H), 13C NMR (100 MHz, d6-DMSO): δ 140.48, 134.06, 131.74, 130.37, 129.62, 129.58, 128.18, 128.08, 127.74, 125.80, 21.34. HRMS (ESI) m/z calcd for C11H10NO2S2 (M+H)+ 252.0154, found 252.0148. 8-Methyl-5H-1,4-dithia-5-aza-cyclopenta[a]naphthalene 4,4-dioxide (2m) White solid, isolated yield 73% (37mg); mp: 158-160oC; 1H NMR (400 MHz, d6-DMSO): δ 8.47 (d, J = 4.0 Hz, 1H), 7.94-7.92 (m, 1H), 7.43-7.38 (m, 3H), 2.40 (s, 3H), 13C NMR (100 MHz, d6-DMSO): δ 140.48, 134.06, 131.74, 130.37, 129.62, 129.58, 128.18, 128.08, 127.74, 125.80, 21.34. HRMS (ESI) m/z calcd for C11H10NO2S2 (M+H)+ 252.0154, found 252.0148. 8-Methyl-5H-1,4-dithia-5-aza-cyclopenta[a]naphthalene 4,4-dioxide (2m) White solid, isolated yield 73% (37mg); mp: 158-160oC; 1H NMR (400 MHz, d6-DMSO): δ 8.47 (d, J = 4.0 Hz, 1H), 7.94-7.92 (m, 1H), 7.43-7.38 (m, 3H), 2.40 (s, 3H), 13C NMR (100 MHz, d6-DMSO): δ 140.48, 134.06, 131.74, 130.37, 129.62, 129.58, 128.18, 128.08, 127.74, 125.80, 21.34. HRMS (ESI) m/z calcd for C11H10NO2S2 (M+H)+ 252.0154, found 252.0148. Acknowledgements This work is supported by the National Natural Science Foundation of China (21762022 and 51463002) and the Foundation of Jiangxi Educational Committee (Grant No. GJJ160287). Acknowledgements This work is supported by the National Natural Science Foundation of China (21762022 and 51463002) and the Foundation of Jiangxi Educational Committee (Grant No. GJJ160287). Supplementary Material 1H and 13C NMR spectra of products 2 can be found in the online Supplementary Material. Silver-catalyzed intramolecular oxidative decarboxylative C-H arylation 7,8-(dichloro)-6H-dibenzo[c,e][1,2]thiazine 5,5-Dioxide (2i) White solid, isolated yield 86% (52 mg); mp: 219-- 221 oC; 1H NMR (400 MHz, d6-DMSO): δ 8.46 (d, J = 8.0 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.19-8.08 (m, 2H), 7.97 (d, J = 8.0 Hz, 1H), 7.73-7.63 (m, 2H), 13C NMR (100 MHz, d6-DMSO): δ 157.64, 137.48, 136.98, 136.18, 133.83, 133.71, 133.37, 131.78, 129.82, 126.36, 125.19, 122.69. HRMS (ESI) m/z calcd for C12H8Cl2NO2S (M+H)+ 299.9654, found 299.9651. 7-methoxy-6H-dibenzo[c,e][1,2]thiazine 5,5-Dioxide (2j)24 White solid, isolated yield 74% (39 mg); mp: 211- 213 oC; 1H NMR (400 MHz, d6-DMSO): δ 8.37 (d, J = 8.0 Hz, 1H), 8.20 (d, J = 4.0 Hz, 1H), 8.12-8.06 (m, 2H), 7.61 (s, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.17 (q, J = 4.0 Hz, 1H), 3.77 (s, 3H), 13C NMR (100 MHz, d6-DMSO): δ 157.15, 137.67, 136.58, 135.88, 132.93, 131.64, 126.55, 125.98, 122.40, 121.54, 116.70, 113.79, 56.64. HRMS (ESI) m/z calcd for C13H12O3NS (M+H)+ 262.0532; found: 262.0534. 5-Methylphenanthridin-6(5H)-one (2k)43 White solid, isolated yield 82% (35 mg); mp: mp 79-81oC; 1H NMR (400 MHz, CDCl3): δ 7.93 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.12-7.18 (m, 2H), 13C NMR (100 MHz, CDCl3): δ 139.52, 134.27, 132.27, 130.45, 128.24, 125.52, 125.44, 124.69, 124.01, 122.51, 119.43, 32.77 5-Methylphenanthridin-6(5H)-one (2k)43 White solid, isolated yield 82% (35 mg); mp: mp 79-81oC; 1H NMR (400 MHz, CDCl3): δ 7.93 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.12-7.18 (m, 2H), 13C NMR (100 MHz, CDCl3): δ 139.52, 134.27, 132.27, 130.45, 128.24, 125.52, 125.44, 124.69, 124.01, 122.51, 119.43, 32.77 6,9-Dimethyl-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide (2l)44 White solid, isolated yield 82% (43mg); mp: 185- 187oC; 1H NMR (400 MHz, d6-DMSO): δ 7.93 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.12-7.18 (m, 2H), 13C NMR (100 MHz, d6-DMSO): δ 139.52, 134.27, 132.27, 130.45, 128.24, 125.52, 125.44, 124.69, 124.01, 122.51, 119.43, 32.77. https://doi.org/doi/10.1021/jm9906015 6. 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Phytochemical and Safety Evaluations of Zingiber ottensii Valeton Essential Oil in Zebrafish Embryos and Rats
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  Academic Editor: Aleksandra Zielinska Received: 18 March 2021 Accepted: 27 April 2021 Published: 3 May 2021 Keywords: Zingiber ottensii Valeton; zebrafish; embryotoxicity; teratogenicity; rats; acute oral toxicity Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Citation: Thitinarongwate, W.; Mektrirat, R.; Nimlamool, W.; Khonsung, P.; Pikulkaew, S.; Okonogi, S.; Kunanusorn, P. Phytochemical and Safety Evaluations of Zingiber ottensii Valeton Essential Oil in Zebrafish Embryos and Rats. Toxics 2021, 9, 102. https://doi.org/10.3390/toxics9050102 Wisit Thitinarongwate 1,2 , Raktham Mektrirat 3,4 , Wutigri Nimlamool 1,4 , Parirat Khonsung 1, Surachai Pikulkaew 4,5, Siriporn Okonogi 5,6 and Puongtip Kunanusorn 1,* tinarongwate 1,2 , Raktham Mektrirat 3,4 , Wutigri Nimlamool 1,4 , Parirat Khonsung 1, Pikulkaew 4,5, Siriporn Okonogi 5,6 and Puongtip Kunanusorn 1,* Wisit Thitinarongwate 1,2 , Raktham Mektrirat 3,4 , Wutigri Nimlamool 1,4 , Parirat Khonsung 1, Surachai Pikulkaew 4,5, Siriporn Okonogi 5,6 and Puongtip Kunanusorn 1,* g y g 4 Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Chiang Mai Univer Chiang Mai 50200, Thailand; surapikulkaew@gmail.com g p g 5 Department of Food Animal Clinic, Faculty of Veterinary Medicine, Chiang Mai University Chiang Mai 50100, Thailand; okng2000@gmail.com g g g 6 Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Ch h l d g , * Correspondence: puongtip.k@cmu.ac.th; Tel.: +66-53-935-353 Abstract: Zingiber ottensii Valeton (ZO) exhibits pharmacological activity and has long been used in traditional medicine. However, reports about its safety profiles are limited. The present study aimed to evaluate the phytochemical profile and the toxic effects of ZO essential oil on the development of zebrafish and acute oral toxicity in rats. The essential oil was isolated from ZO rhizomes, and phytochemicals were analyzed using a gas chromatography–mass spectrometer (GC–MS). The embryotoxic and teratogenic effects of ZO essential oil were evaluated in zebrafish embryos and larvae and the acute oral toxicity was determined in rats. GC–MS results showed the essential oil contained zerumbone as a major phytoconstituent (24.73%). The zebrafish embryotoxicity of ZO essential oil appeared to be concentration- and time-dependent manner, with a moderate LC50 (1.003 µg/mL). Teratogenicity in zebrafish embryos also included morphological defects, decreased hatchability, and reduced heart rate. In rats, ZO essential oil (2000 mg/kg, p.o.) resulted in no mortality or significant toxicities. These findings suggest that ZO has embryotoxic and teratogenic effects in zebrafish embryos but does not result in death or acute oral toxicity in rats. Further long- term toxicity studies are needed to confirm the safety of products developed from ZO essential oil. toxics toxics 1. Introduction Various plants have been shown to possess significant pharmacological activity and to provide many health benefits, both in preclinical studies (in vitro and in vivo models) and in clinical studies in humans [1–3]. Additionally, medicinal plants have been used as alternative treatments to cure illness in Ayurvedic and Thai traditional medicine. Presently, the use of medicinal plants is increasing as their natural compounds are believed to be safer than modern medicines. However, the toxicological profiles of most medicinal plants have not been completely determined. Many plant-derived treatments may result in harmful effects in humans, including carcinogenic, mutagenic, and teratogenic effects [4,5]. Toxicity testing in a diverse range of in vitro studies using animal models is crucial and includes experimental screening methods for determining the safety profile of medicinal plants. For example, acute oral toxicity test in rodents is widely used to evaluate acute toxicity of a drug or an herb. These tests can include evaluation of the LD50 of the tested substance. Although rodents, rabbits, and sheep have traditionally been used for studying toxicity Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/toxics Toxics 2021, 9, 102. https://doi.org/10.3390/toxics9050102 Toxics 2021, 9, 102 2 of 17 in embryonic development. However, using these animals to study consumes time and expense. Thus, there is an effort to find an alternative animal model for the toxicity study of medicinal plants in embryonic development. Danio rerio, commonly called zebrafish, is a small tropical freshwater fish in the Cyprinidae family. In recent times, animal models using zebrafish are becoming popular and reliable models widely used in biological researches such as in the fields of genetic development, transgenesis, and toxicology [6]. Zebrafish embryos are useful for evalu- ating vertebrate development because the developmental steps in the zebrafish embryo correspond to other higher vertebrates’ embryogenesis, including humans [7]. The egg of zebrafish was transparent, which allows the direct observation of developmental stages (from fertilization, embryogenesis, and organogenesis to larva hatching) and assessment of endpoint morphological changes in toxicity studies [8]. 1. Introduction Moreover, animal models using zebrafish have many advantages including low husbandry cost, requiring small housing spaces, having a small number of chemical compounds and test drugs, short breeding cycle (5–7 days) and higher fecundity (with 200–300 eggs per one pair of adult fish), and are suitable for high throughput screening. These benefits of the zebrafish model are the reasons for the popularity of using these models as alternative models, in comparison to some vertebrate toxicity assessment models [9–11]. y Zingiber ottensii (ZO) Valeton, locally famous as Plai Dam or Plai Muang (Bangkok) and Pu Loei Dum (northern Thailand), can be found in Southeast Asia, including Indone- sia, Malaysia, and Thailand [12,13]. In Malay traditional medicine, midwives in Perak commonly make the poultice from leaves and rhizomes of ZO before applying it to the body of the confinement’s women for postpartum care. ZO leaves are also used as the poultice for lumbago [14]. In Thai traditional medicine, ZO rhizomes are used to treat gastrointestinal diseases (peptic ulcers and stomachache), constipation, myalgia, sprain, bruising/contusion, and wounds. Moreover, essential oil from ZO rhizomes has been used as a topical agent for Thai traditional massage. Although various pharmacological activities including antidiabetic, anticancer, and antimicrobial activities of ZO have been reported [15,16]. In addition, the major phytochemical components of essential oil of ZO rhizomes of Malaysia are reported to be terpene compounds [17]. However, the reports about phytochemical characteristics of essential oil of ZO rhizomes found in Thailand and its toxicological profiles including embryotoxicity, teratogenicity, and acute oral toxicity in animal models are still lacking. Thus, the present study aimed to identify the phytochemi- cal profile of ZO essential oil from ZO grown in Thailand and evaluate its toxicity in in vivo models using both zebrafish embryos and rats. These findings of this study are intended to provide part of the data necessary to confirm the safety of new products developed using ZO essential oil in the future. 2. Materials and Methods 2.1. Plant Material Rhizomes of Zingiber ottensii (ZO) Valeton were harvested at Saluang Subdistrict, Mae Rim District, Chiang Mai, Thailand (Lat 19◦01′57.719” N and Long 98◦88′46.0119” E) in March 2020, two years after planting. Plant authentication was carried out by the Faculty of Pharmacy, Chiang Mai University where the voucher specimen (000109) was deposited. The fresh rhizomes were washed, chopped into very small pieces, then kept in a storage box at room temperature before the extraction. 2.2. Distillation and Chemical Composition Analysis of ZO Essential Oil 2.3. Laboratory Animal Care and Maintenance Wild-type zebrafish (Danio rerio) were purchased from a local ornamental fish shop, that normally supplies zebrafish used for studying in the aquatic laboratory of the Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, Thailand. Maintenance of zebrafish was in accordance with the internationally accepted guideline (OECD 236) [18], with minor modifications. Adult zebrafish were maintained in an 80 L glass tank with a maximum density of 1 g fish/L filtered tap water (pH 6.9–7.4, 26 ± 1 ◦C) with a 14:10 h of the light–dark cycle in the Aquatic Medicine Room located of the Faculty of Veterinary Medicine, Chiang Mai University. The zebrafish were fed twice a day: frozen brine shrimps (Artemia) in the morning and commercial dry flake food in the afternoon. Water was changed and feces removed from each tank daily. Water parameters, including temperature, pH, nitrite, nitrate, and ammonia content, were also monitored daily. All zebrafish were quarantined for at least 3 months before using them in the experiments. They were selected and graded into many groups depending on health status and fertility. Only zebrafish with the best quality were selected to be used as the breeders. These zebrafish were acclimatized for 4 weeks before initial breeding. All experiments were approved by the Animal Ethics Committee of the Faculty of Veterinary Medicine, Chiang Mai University, Thailand (Permit No. R8/2563, 17 July 2020). Female Sprague Dawley rats (Rattus norvegicus) (age 8–12 weeks/180–200 g) were purchased from Nomura Siam International Co. Ltd., Bangkok, Thailand. All the animals were kept in an animal room maintained under environmentally controlled conditions of 24 ± 1 ◦C, 50 ± 10% relative humidity, and a 12:12 h light–dark cycle. They had free access to drinking water and a standard pelleted diet and were acclimatized for at least one week before the start of the experiments. All experiments were approved by the Animal Ethics Committee of the Faculty of Medicine, Chiang Mai University, Thailand (Permit No. 22/2563, 9 July 2020). 2.2. Distillation and Chemical Composition Analysis of ZO Essential Oil One kg of ZO fresh rhizomes and 1 L of distilled water were used to obtain ZO essential oil by hydrodistillation extraction using a Clevenger apparatus over a period of 3 h. The ZO essential oil was then collected and stored in an airtight dark bottle at 4 ◦C until use. Gas chromatography–mass spectrometry (GC–MS) was used to identify the chemical components found in ZO essential oil. The system was comprised of a 7890A gas chromatograph (Agilent, Santa Clara, CA, USA) equipped with a 5975C mass-selective Toxics 2021, 9, 102 3 of 17 3 of 17 detector (Agilent, Santa Clara, CA, USA) using a DB5-MS column (30 m × 0.25 mm i.d. × 0.25 µm film thickness). The ionization energy was 70 eV and the ion source temperature was 230 ◦C. The oven temperature was initially set at 50 ◦C, then slowly raised to 220 ◦C over a period of 45 min (4 ◦C/min) with helium as the carrier gas. The flow rate of the carrier gas was set at 1 mL/min using a split mode (split ratio 500:1). The injection temperature was 250 ◦C, and the detector temperature was 280 ◦C. Identification of the ZO essential oil components was based on the comparison of their retention times and their mass spectra by matching with standard reference database and library, which included NIST Mass Spectral Database (2008) and W8N08 library (John Wiley & Sons, Inc., Hoboken, NJ, USA). Each component’s percentage was calculated based on the total area of all peaks obtained from the ZO essential oil. 2.4. Zebrafish Breeding and Embryo Care The evening of the day before breeding the healthy and active adult male and female wild-type zebrafish (ratio 1:2) with high ability to produce fertilized eggs were selected for spawning and were moved to a spawning tank equipped with 5 L of filtered tap water and fitted with spawning enhancers consisting of marbles and a spawn trap. Mating occurred the next morning within 30–60 min after the lights were turned on. The zebrafish were then removed from the spawning tank and placed back into their resting glass tank. Spawning enhancers were removed from the spawning tank. Water was poured out slowly from one corner of the spawning tank, and the fertilized eggs were collected with a plastic pipette and transferred to clean Petri dishes containing embryo water prepared with minor modifications of the previously reported protocol [19]. The fertilized embryos were carefully washed with sea salt egg water (60 mg/L sea salt and 2 mg/L methylene blue) to remove debris [20], while unhealthy and dead embryos were removed by aspiration using a plastic pipette. Fertilized embryos were kept at 26.5 ◦C and allowed to develop for 6 h Toxics 2021, 9, 102 4 of 17 postfertilization (hpf). Microscopic observation was accomplished using a stereomicroscope (Nikon, Tokyo, Japan) to observe embryo development prior to treatment [21]. 2.5. Zebrafish Embryonic Toxicity Test 2.5. Zebrafish Embryonic Toxicity Test 2.5.1. Dose–Response Embryotoxicity and Median Lethal Concentration (LC50) Embryotoxicity in the zebrafish was assessed by measuring the mortality rate of the zebrafish embryos. Exposure of the zebrafish embryos to the extract was performed accord- ing to the method described in OECD 236 [18]. At 6 hpf, healthy zebrafish embryos were selected for subsequent experiments. Overall, 20 fertilized eggs for each concentration treatment were transferred to individual wells of 24-well plates. The embryos were exposed to various concentrations of ZO essential oil containing 0.1% dimethyl sulfoxide (DMSO) diluted in 2 mL of embryo water. It was serially diluted via twofold serial dilution to pro- duce five different concentrations of ZO essential oil (3.91, 1.95, 0.98, 0.49, and 0.24 µg/mL). The control (untreated) group was exposed to 2 mL of embryo water containing only 0.1% DMSO. The experiment was duplicated, each performed with three independent replicates. The embryos were observed under a stereomicroscope every 24 h. Death of an embryo was indicated by coagulation and/or absence of a heartbeat. 2.6. Zebrafish Teratogenicity Test 2.6.1. Evaluation of Morphological Characteristics 2.4. Zebrafish Breeding and Embryo Care The number of dead embryos in each concentration at 96 hpf was recorded, and the mortality rate was calculated. The GraphPad Prism8 [log (inhibitor) vs. normalized response-variable slope nonlinear model] was used to calculate the 50% lethal concentration (LC50) values for ZO essential oil [22]. 2.6.1. Evaluation of Morphological Characteristics The teratogenicity of the zebrafish was evaluated both in embryos and in larvae after five days (120 hpf) exposure to five different concentrations of ZO essential oil by observing morphological changes and developmental abnormalities. After treatment, the embryos and larvae were examined every 24 h using a stereomicroscope. Embryonic and larval morphology was determined according to OECD Test Guideline 236 [18], and the normal development of the embryos and larvae was compared using the method of Kimmel et al. (1995) [7], as previously described. Examples of teratogenicity identified included deformities in somite otolith and eyes, failure of tail detachment, absence of heartbeat or blood circulation, yolk sac or pericardial edema, yolk sac malabsorption, and skeletal malformation and delayed hatching [23]. The malformed embryos and larvae were captured with an Olympus digital camera (OM-D E-M10 Mark III), and the teratogenicity was evaluated by the percentage of embryos or larvae with abnormalities to the number remaining of normal embryos, as previously described [20]. 2.6.2. Evaluation of Zebrafish Embryos Hatchability 2.6.2. Evaluation of Zebrafish Embryos Hatchability The hatchability of the zebrafish embryos treated with different concentrations of ZO essential oil (0–3.91 µg/mL) was determined between 48 and 120 hpf using a stereomi- croscope. The hatching success of embryos was determined by chorion rupture releasing the larvae into the embryo water. Hatchability rates were determined by comparing the number of hatched embryos with the total number of embryos tested. 2.6.3. Evaluation of Zebrafish Larvae Heart Rates 2.5.2. Time–Kill Analysis The time–kill kinetics of ZO essential oil were determined by analyzing the time–kill rates of the zebrafish embryos. Dead embryos were examined under a stereomicroscope, and the time–kill analysis was conducted at 24, 48, 72, 96, and 120 hpf. Survival rates and the median survival time were also evaluated [20]. 2.6.3. Evaluation of Zebrafish Larvae Heart Rates The number of heartbeats of larvae between 48 and 120 hpf treatments with ZO essential oil (0–3.91 µg/mL) was determined in this experiment. At 72 hpf, the heartbeat count was conducted using a stereomicroscope connected to a computer and digital camera Toxics 2021, 9, 102 5 of 17 device for video recording. Counting was performed using a mechanical counter and stopwatch. The heart rate was expressed as beats per minute (bpm) [23,24]. device for video recording. Counting was performed using a mechanical counter and stopwatch. The heart rate was expressed as beats per minute (bpm) [23,24]. Relative organ weight = organ weight (g)/body weight of the rat (g) × 100 Relative organ weight = organ weight (g)/body weight of the rat (g) × 100 2.8. Statistical Analysis In zebrafish embryotoxicity and teratogenicity tests, the experiments were conducted with three independent replications. The mortality of zebrafish embryos and teratogenicity test results were compared by means of one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test. Student’s t-test was used for comparisons between two experimental groups in the acute oral toxicity study using SPSS Statistical Package version 22 (IBM, Armonk, NY, USA). Data are presented as mean ± standard deviation (SD). Statistical significance of differences was set at p < 0.05. Differences between the five groups in rates of time to outcome were compared using the Kaplan–Meier curve, and the log-rank test for significance and statistical analysis of LC50 was calculated using GraphPad Prism8 Software (San Diego, CA, USA). 2.7. Acute Oral Toxicity Study in Rats The acute oral toxicity of ZO essential oil was evaluated in rats following interna- tionally accepted guidelines (OECD Test Guideline 420) [25]. The female rats used in the experiment were randomly selected and marked on the tail for individual identification. A single high dose of 2000 mg/kg of ZO essential oil was administered by oral gavage to one rat following 12 h of fasting. 48 h later, the same dose was administered to another four rats, for a total of five treated rats. The negative control group of five rats was treated in parallel with the vehicle (0.9% saline). Food was provided to all rats approximately 1 h after administration. All rats were observed in detail periodically and daily for 14 days for any toxic effects [26]. Intake of water and food, along with body weight, were measured daily [25]. Mortality, behavioral pattern, physical appearance changes, injuries, pain, and signs of illness were monitored daily during the period [27]. After 14 days, all the rats were sacrificed, and their vital organs, including heart, kidneys, liver, lung, and spleen were removed, weighed, and microscopically examined. All vital organs isolated from each rat were fixed in 10% buffered formalin before being subjected to further histopathological evaluation. The relative organ weight of each animal was calculated as follows: 3.1. Chemical Compositions of the Zingiber ottensii (ZO) Valeton Essential Oil 3.2. Dose–Response Embryotoxicity in Zebrafish and LC50 Embryotoxicity was evaluated at five different concentrations of ZO essential oil (3.91, 1 95 0 98 0 49 d 0 24 / L) C l ti d th b f h tb t i b fih Table 1. Chemical composition of essential oil from Zingiber ottensii (ZO) Valeton identified by GC–MS analysis. The ZO essential oil was extracted from the rhizomes of ZO by simultaneous steam distillation and analyzed by GC–MS. RT: Retention time; MW: molecular weight. MS analysis. The ZO essential oil was extracted from the rhizomes of ZO by simultaneous steam distillation and analyzed by GC–MS. RT: Retention time; MW: molecular weight. Peaks RT (Min) Component Formula MW (g/moL) Amount (%) Peaks RT (Min) Component Formula MW (g/moL) Amount (%) 1 6.39 α-pinene C10H16 136.23 2.94 2 7.55 sabinene C10H16 136.23 15.19 3 7.69 β-pinene C10H16 136.23 7.95 4 9.34 1,8-cineole C10H18O 154.25 3.16 5 10.18 γ-terpinene C10H16 136.23 3.73 6 14.49 terpinen-4-ol C10H18O 154.25 18.75 7 14.87 α-terpineol C10H18O 154.25 1.67 8 23.37 β-selinene C15H24 204.35 4.48 9 29.24 α-eudesmol C15H22O 222.37 0.95 10 31.50 zerumbone C15H22O 218.33 24.73 1 6.39 α-pinene C10H16 136.23 2.94 2 7.55 sabinene C10H16 136.23 15.19 3 7.69 β-pinene C10H16 136.23 7.95 4 9.34 1,8-cineole C10H18O 154.25 3.16 5 10.18 γ-terpinene C10H16 136.23 3.73 6 14.49 terpinen-4-ol C10H18O 154.25 18.75 7 14.87 α-terpineol C10H18O 154.25 1.67 8 23.37 β-selinene C15H24 204.35 4.48 9 29.24 α-eudesmol C15H22O 222.37 0.95 10 31.50 zerumbone C15H22O 218.33 24.73 Figure 1. GC–MS chromatogram of ZO essential oil. Phytochemicals were identified by the GC–MS method. Chemical structures of the five major compounds from ZO essential oil included (A) sabinene, (B) β-pinene, (C) terpinen-4-ol, (D) Figure 1. GC–MS chromatogram of ZO essential oil. Phytochemicals were identified by the GC–MS method. Chemical structures of the five major compounds from ZO essential oil included (A) sabinene, (B) β-pinene, (C) terpinen-4-ol, (D) β-selinene, and (E) zerumbone. Figure 1. GC–MS chromatogram of ZO essential oil. Phytochemicals were identified by the GC–MS method. Chemical structures of the five major compounds from ZO essential oil included (A) sabinene, (B) β-pinene, (C) terpinen-4-ol, (D) Figure 1. GC–MS chromatogram of ZO essential oil. Phytochemicals were identified by the GC–MS method. Chemical structures of the five major compounds from ZO essential oil included (A) sabinene, (B) β-pinene, (C) terpinen-4-ol, (D) β-selinene, and (E) zerumbone. 3.1. Chemical Compositions of the Zingiber ottensii (ZO) Valeton Essential Oil The rhizomes of ZO were subjected to hydrodistillation and yielded 0.24% (w/w) of essential oil with a pale yellowish color and camphoraceous odor. Phytochemicals were characterized using the GC–MS method. The peak numbers were recorded according to the retention time and percentage of each compound (Table 1). Most constituents were terpenoids, which consisted mainly of 21 monocyclic monoterpenoids and seven sesquiterpenes. Chromatograms showed major components’ identifiable spectra (Figure 1). The compound present in the highest quantity was zerumbone (24.73%), followed by terpinen-4-ol (18.75%), sabinene (15.19%), and β-pinene (7.95%). Toxics 2021, 9, 102 6 of 17 y Table 1. Chemical composition of essential oil from Zingiber ottensii (ZO) Valeton identified by GC–MS analysis. The ZO essential oil was extracted from the rhizomes of ZO by simultaneous steam distillation and analyzed by GC–MS. RT: Retention time; MW: molecular weight. Peaks RT (Min) Component Formula MW (g/moL) Amount (%) 1 6.39 α-pinene C10H16 136.23 2.94 2 7.55 sabinene C10H16 136.23 15.19 3 7.69 β-pinene C10H16 136.23 7.95 4 9.34 1,8-cineole C10H18O 154.25 3.16 5 10.18 γ-terpinene C10H16 136.23 3.73 6 14.49 terpinen-4-ol C10H18O 154.25 18.75 7 14.87 α-terpineol C10H18O 154.25 1.67 8 23.37 β-selinene C15H24 204.35 4.48 9 29.24 α-eudesmol C15H22O 222.37 0.95 10 31.50 zerumbone C15H22O 218.33 24.73 Table 1. Chemical composition of essential oil from Zingiber ottensii (ZO) Valeton identified by GC– MS analysis. The ZO essential oil was extracted from the rhizomes of ZO by simultaneous steam distillation and analyzed by GC–MS. RT: Retention time; MW: molecular weight. Peaks RT (Min) Component Formula MW (g/moL) Amount (%) 1 6.39 α-pinene C10H16 136.23 2.94 2 7.55 sabinene C10H16 136.23 15.19 3 7.69 β-pinene C10H16 136.23 7.95 4 9.34 1,8-cineole C10H18O 154.25 3.16 5 10.18 γ-terpinene C10H16 136.23 3.73 6 14.49 terpinen-4-ol C10H18O 154.25 18.75 7 14.87 α-terpineol C10H18O 154.25 1.67 8 23.37 β-selinene C15H24 204.35 4.48 9 29.24 α-eudesmol C15H22O 222.37 0.95 10 31.50 zerumbone C15H22O 218.33 24.73 Figure 1. GC–MS chromatogram of ZO essential oil. Phytochemicals were identified by the GC–MS method. Chemical structures of the five major compounds from ZO essential oil included (A) sabinene, (B) β-pinene, (C) terpinen-4-ol, (D) β-selinene, and (E) zerumbone. 3.2. Dose–Response Embryotoxicity in Zebrafish and LC50 Figure 1. GC–MS chromatogram of ZO essential oil. Phytochemicals were identified by the GC–MS method. Chemical structures of the five major compounds from ZO essential oil included (A) sabinene, (B) β-pinene, (C) terpinen-4-ol, (D) β-selinene, and (E) zerumbone. p ( ) , ( ) one. 3.2. Dose–Response Embryotoxicity in Zebrafish and LC50 p one. 3.2. Dose–Response Embryotoxicity in Zebrafish and LC50 3.2. Dose–Response Embryotoxicity in Zebrafish and LC50 Embryotoxicity was evaluated at five different concentrations of ZO essential oil (3.91, 1.95, 0.98, 0.49, and 0.24 µg/mL). Coagulation and the absence of heartbeat in zebrafish embryos were indicative of mortality [4]. The results showed that the toxic effect of ZO essential oil appeared to occur in a concentration-dependent manner. The mean mortality of zebrafish at 96 hpf is shown in Figure 2. The lowest dose of ZO essential oil (0.24 µg/mL) and 0.1% DMSO (control) caused no mortality in the zebrafish embryos. By contrast, a significantly increased mortality rate (p < 0.05) was observed in zebrafish em- bryos exposed to 0.49, 0.98, 1.95, and 3.91 µg/mL of ZO essential oil, when compared with Embryotoxicity was evaluated at five different concentrations of ZO essential oil (3.91, 1.95, 0.98, 0.49, and 0.24 µg/mL). Coagulation and the absence of heartbeat in zebrafish embryos were indicative of mortality [4]. The results showed that the toxic effect of ZO essential oil appeared to occur in a concentration-dependent manner. The mean mortality of zebrafish at 96 hpf is shown in Figure 2. The lowest dose of ZO essential oil (0.24 µg/mL) and 0.1% DMSO (control) caused no mortality in the zebrafish embryos. By contrast, a significantly increased mortality rate (p < 0.05) was observed in zebrafish embryos exposed to 0.49, 0.98, 1.95, and 3.91 µg/mL of ZO essential oil, when compared with the lowest concentration of ZO essential oil (0.24 µg/mL). No viable zebrafish embryos were observed in the groups treated with 1.95 and 3.91 µg/mL ZO essential oil. The LC50 value of ZO essential oil was 1.003 µg/mL. Toxics 2021, 9, 102 7 of 17 y LC50 Figure 2. The embryotoxicity of different concentrations of ZO essential oil in embryonic zebrafish. Zebrafish embryos were treated with a series of twofold dilution concentrations (0.24–3.91 µg/mL). Percentages of embryonic mortality were calculated from embryo deaths after exposure to ZO es- sential oil at 0.24, 0.49, 0.98,1.95, 3.91 µg/mL, and 0.1% DMSO (control). Data represent the mean ± SD of three independent experiments (n = 60 embryos/group). Experiments were analyzed using one-way ANOVA and Tukey’s multiple comparison test. Different lowercase letters indicate signif- cant differences between groups (p < 0.05). Figure 2. The embryotoxicity of different concentrations of ZO essential oil in embryonic zebrafish. p ( ) , ( ) one. 3.2. Dose–Response Embryotoxicity in Zebrafish and LC50 Zebrafish embryos were treated with a series of twofold dilution concentrations (0.24–3.91 µg/mL). Percentages of embryonic mortality were calculated from embryo deaths after exposure to ZO essential oil at 0.24, 0.49, 0.98,1.95, 3.91 µg/mL, and 0.1% DMSO (control). Data represent the mean ± SD of three independent experiments (n = 60 embryos/group). Experiments were analyzed using one-way ANOVA and Tukey’s multiple comparison test. Different lowercase letters indicate significant differences between groups (p < 0.05). 3.4. Morphological Defects of Zebrafish Embryos 3.4. Morphological Defects of Zebrafish Embryos Morphological defects included pericardial sac edema, coagulation, dented tail, poor reabsorption of the yolk sac, malformation of the yolk sac and spinal curvature (Figure 4A–D) Determination of teratogenicity was performed using five different concentrations of ZO essential oil (3.91, 1.95, 0.98, 0.49, and 0.24 µg/mL) and 0.1% DMSO (control) at various time points over the period 24–120 hpf (Figure 4). The rate of teratogenic malformation resulting from ZO essential oil in zebrafish embryos is presented in Table 2. The results showed no teratogenic abnormalities in the control group of zebrafish embryos or in the group treated with 0.24 µg/mL of ZO essential oil (Figure 4E,F). However, all embryos and larvae were found to express morphological abnormalities when 3.91, 1.95, and 0.98 µg/mL of ZO essential oil were present at 48, 72, and 96 hpf, respectively (Table 2). Notably, zebrafish embryo’s accumulative abnormalities were found in the groups treated with 0.49–3.91 µg/mL of ZO essential oil. Morphological defects included pericardial sac edema, coagulation, dented tail, poor reabsorption of the yolk sac, malformation of the yolk sac, and spinal curvature (Figure 4A–D). Table 2. Teratogenic effect of ZO essential oil on zebrafish embryos at 24, 48,72, 96, and 120 hpf, which had received ZO essential oil at dose of 0.24, 0.49, 0.98, 1.95, and 3.91 µg/mL, and 0.1% DMSO (control) at 24, 48, 72, 96, and 120 hpf. Descriptive data show the percentages (mean ± SD) of terato- genic zebrafish embryos from three independent experiments (n = 60 embryos/group). ED = embry- onic death. Table 2. Teratogenic effect of ZO essential oil on zebrafish embryos at 24, 48,72, 96, and 120 hpf, which had received ZO essential oil at dose of 0.24, 0.49, 0.98, 1.95, and 3.91 µg/mL, and 0.1% DMSO (control) at 24, 48, 72, 96, and 120 hpf. Descriptive data show the percentages (mean ± SD) of teratogenic zebrafish embryos from three independent experiments (n = 60 embryos/group). ED = embryonic death. 3.4. Morphological Defects of Zebrafish Embryos 3.4. Morphological Defects of Zebrafish Embryos However, a embryos and larvae were found to express morphological abnormalities when 3.91, 1.9 and 0.98 µg/mL of ZO essential oil were present at 48, 72, and 96 hpf, respectively (Table 2 Notably, zebrafish embryo’s accumulative abnormalities were found in the groups treate with 0.49–3.91 µg/mL of ZO essential oil. Morphological defects included pericardial sa edema, coagulation, dented tail, poor reabsorption of the yolk sac, malformation of th yolk sac, and spinal curvature (Figure 4A–D). Table 2. Teratogenic effect of ZO essential oil on zebrafish embryos at 24, 48,72, 96, and 120 hp which had received ZO essential oil at dose of 0.24, 0.49, 0.98, 1.95, and 3.91 µg/mL, and 0.1 DMSO (control) at 24, 48, 72, 96, and 120 hpf. Descriptive data show the percentages (mean ± SD of teratogenic zebrafish embryos from three independent experiments (n = 60 embryos/group ED = embryonic death. Concentrations (µg/mL) Hours Post Fertilization (hpf) 24 48 72 96 120 0 0 0 0 0 0 0.24 0 0 0 0 0 0.49 0 0 40.17 ± 2.25 75.36 ± 4.84 79.28 ± 8.24 0.98 0 18.99 ± 4.15 91.50 ± 8.10 100 100 1.95 0 47.30 ± 21.75 100 ED ED 3.91 0 100 ED ED ED p g f f f y Determination of teratogenicity was performed using five different concentrations of ZO essential oil (3.91, 1.95, 0.98, 0.49, and 0.24 µg/mL) and 0.1% DMSO (control) at various time points over the period 24–120 hpf (Figure 4). The rate of teratogenic malformation resulting from ZO essential oil in zebrafish embryos is presented in Table 2. The results showed no teratogenic abnormalities in the control group of zebrafish embryos or in the group treated with 0.24 µg/mL of ZO essential oil (Figure 4E,F). However, all embryos and larvae were found to express morphological abnormalities when 3.91, 1.95, and 0.98 µg/mL of ZO essential oil were present at 48, 72, and 96 hpf, respectively (Table 2). Nota- bly, zebrafish embryo’s accumulative abnormalities were found in the groups treated with 0.49–3.91 µg/mL of ZO essential oil. 3.3. Time–Kill Analysis in Zebrafish Embryos 3.3. Time–Kill Analysis in Zebrafish Embryos Time–kill analysis of five different concentrations of ZO essential oil in a series of wofold dilution concentrations over the range of 0.24–3.91 µg/mL was conducted to eval- uate the kinetic killing in zebrafish embryos over the course of 24–120 hpf. The Kaplan– Meier curve was used to display the relationship between time (hpf) to zebrafish embryo death (Figure 3). The results showed that the killing potency of ZO essential oil appeared o occur in a time-dependent manner. The survival rate of zebrafish embryos treated with he lowest dose of ZO essential oil or with 0.1% DMSO (control) was 1.00. On the other hand, the survival rates of zebrafish embryos treated with 0.49 and 0.98 µg/mL of ZO essential oil were reduced to 0.72 and 0.33 at 120 hpf, respectively. Interestingly, the sur- vival rates of zebrafish embryos treated with 3.91 and 1.95 µg/mL were decreased to zero at 72 and 96 hpf, respectively (log-rank test, p < 0.0001). The mean survival times of zebrafish embryos treated with 3.91 and 1.95 µg/mL of ZO essential oil were equal (72 hpf), while the mean survival time in the group treated with 0.98 µg/mL of ZO essential il 120 h f Time–kill analysis of five different concentrations of ZO essential oil in a series of twofold dilution concentrations over the range of 0.24–3.91 µg/mL was conducted to evaluate the kinetic killing in zebrafish embryos over the course of 24–120 hpf. The Kaplan– Meier curve was used to display the relationship between time (hpf) to zebrafish embryo death (Figure 3). The results showed that the killing potency of ZO essential oil appeared to occur in a time-dependent manner. The survival rate of zebrafish embryos treated with the lowest dose of ZO essential oil or with 0.1% DMSO (control) was 1.00. On the other hand, the survival rates of zebrafish embryos treated with 0.49 and 0.98 µg/mL of ZO essential oil were reduced to 0.72 and 0.33 at 120 hpf, respectively. Interestingly, the survival rates of zebrafish embryos treated with 3.91 and 1.95 µg/mL were decreased to zero at 72 and 96 hpf, respectively (log-rank test, p < 0.0001). The mean survival times of zebrafish embryos treated with 3.91 and 1.95 µg/mL of ZO essential oil were equal (72 hpf), while the mean survival time in the group treated with 0.98 µg/mL of ZO essential oil was 120 hpf. 3.3. Time–Kill Analysis in Zebrafish Embryos 3.3. Time–Kill Analysis in Zebrafish Embryos 8 of 17 8 of 17 Toxics 2021, 9, 102 Toxics 2021, 9, x FOR Figure 3. The embryotoxicity of different concentrations of ZO essential oil in the time–kill analysis of embryonic zebrafish. Zebrafish embryos were treated with 0.1% DMSO (control), and 0.24, 0.49, 0.98, 1.95 and 3.91 µg/mL of ZO essential oil. The Kaplan–Meier curve shows the average survival rate in six different groups in the three independent experiments (n = 60 embryos/group). The log-rank test was used for statistical analysis (p < 0.0001). Figure 3. The embryotoxicity of different concentrations of ZO essential oil in the time–kill analysis of embryonic zebrafish. Zebrafish embryos were treated with 0.1% DMSO (control), and 0.24, 0.49, 0.98, 1.95 and 3.91 µg/mL of ZO essential oil. The Kaplan–Meier curve shows the average survival rate in six different groups in the three independent experiments (n = 60 embryos/group). The log-rank test was used for statistical analysis (p < 0.0001). Figure 3. The embryotoxicity of different concentrations of ZO essential oil in the time–kill analysis of embryonic zebrafish. Zebrafish embryos were treated with 0.1% DMSO (control), and 0.24, 0.49, 0.98, 1.95 and 3.91 µg/mL of ZO essential oil. The Kaplan–Meier curve shows the average survival rate in six different groups in the three independent experiments (n = 60 embryos/group). The log-rank test was used for statistical analysis (p < 0.0001). Figure 3. The embryotoxicity of different concentrations of ZO essential oil in the time–kill analysis of embryonic zebrafish. Zebrafish embryos were treated with 0.1% DMSO (control), and 0.24, 0.49, 0.98, 1.95 and 3.91 µg/mL of ZO essential oil. The Kaplan–Meier curve shows the average survival rate in six different groups in the three independent experiments (n = 60 embryos/group). The log-rank test was used for statistical analysis (p < 0.0001). 3.4. Morphological Defects of Zebrafish Embryos 3.4. Morphological Defects of Zebrafish Embryos 3.4. Morphological Defects of Zebrafish Embryos Determination of teratogenicity was performed using five different concentrations o ZO essential oil (3.91, 1.95, 0.98, 0.49, and 0.24 µg/mL) and 0.1% DMSO (control) at variou time points over the period 24–120 hpf (Figure 4). The rate of teratogenic malformation resulting from ZO essential oil in zebrafish embryos is presented in Table 2. The result showed no teratogenic abnormalities in the control group of zebrafish embryos or in the group treated with 0.24 µg/mL of ZO essential oil (Figure 4E,F). However, all embryo and larvae were found to express morphological abnormalities when 3.91, 1.95, and 0.98 µg/mL of ZO essential oil were present at 48, 72, and 96 hpf, respectively (Table 2). Nota bly, zebrafish embryo’s accumulative abnormalities were found in the groups treated with 0.49–3.91 µg/mL of ZO essential oil. Morphological defects included pericardial sa edema, coagulation, dented tail, poor reabsorption of the yolk sac, malformation of the yolk sac, and spinal curvature (Figure 4A–D). Table 2. Teratogenic effect of ZO essential oil on zebrafish embryos at 24, 48,72, 96, and 120 hpf which had received ZO essential oil at dose of 0.24, 0.49, 0.98, 1.95, and 3.91 µg/mL, and 0.1% DMSO (control) at 24, 48, 72, 96, and 120 hpf. Descriptive data show the percentages (mean ± SD) of terato genic zebrafish embryos from three independent experiments (n = 60 embryos/group). ED = embry onic death. Concentrations (µg/mL) Hours Post Fertilization (hpf) 24 48 72 96 120 0 0 0 0 0 0 0.24 0 0 0 0 0 0.49 0 0 40.17 ± 2.25 75.36 ± 4.84 79.28 ± 8.24 0.98 0 18.99 ± 4.15 91.50 ± 8.10 100 100 1.95 0 47.30 ± 21.75 100 ED ED 3.91 0 100 ED ED ED 3.4. Morphological Defects of Zebrafish Embryos Determination of teratogenicity was performed using five different concentration of ZO essential oil (3.91, 1.95, 0.98, 0.49, and 0.24 µg/mL) and 0.1% DMSO (contro at various time points over the period 24–120 hpf (Figure 4). The rate of teratogen malformation resulting from ZO essential oil in zebrafish embryos is presented in Table The results showed no teratogenic abnormalities in the control group of zebrafish embryo or in the group treated with 0.24 µg/mL of ZO essential oil (Figure 4E,F). 3.4. Morphological Defects of Zebrafish Embryos 3.4. Morphological Defects of Zebrafish Embryos Concentrations (µg/mL) Hours Post Fertilization (hpf) 24 48 72 96 120 0 0 0 0 0 0 0.24 0 0 0 0 0 0.49 0 0 40.17 ± 2.25 75.36 ± 4.84 79.28 ± 8.24 0.98 0 18.99 ± 4.15 91.50 ± 8.10 100 100 1.95 0 47.30 ± 21.75 100 ED ED 3.91 0 100 ED ED ED Concentrations (µg/mL) Hours Post Fertilization (hpf) 24 48 72 96 120 0 0 0 0 0 0 0.24 0 0 0 0 0 0.49 0 0 40.17 ± 2.25 75.36 ± 4.84 79.28 ± 8.24 0.98 0 18.99 ± 4.15 91.50 ± 8.10 100 100 1.95 0 47.30 ± 21.75 100 ED ED 3.91 0 100 ED ED ED Toxics 2021, 9, 102 Toxics 2021, 9, x FOR 9 of 17 9 of 17 Figure 4. Morphological characteristics of zebrafish embryos and larvae treated with different concentrations of ZO es- sential oil or vehicle at different time points (24–120 hpf). Normal morphology was found in the group treated with the lowest concentrations and with vehicle (E,F). Typical malformations of zebrafish development were observed in embryos that had received ZO essential oil at a dose of 0.49–3.91 µg/mL (A–D). Abbreviations: PE, pericardial sac edema; C, coag- ulation; DT, dented tail; PY, poor reabsorption of yolk sac; MY, malformation of yolk sac; SC, spinal curvature. Figure 4. Morphological characteristics of zebrafish embryos and larvae treated with different concentrations of ZO essential oil or vehicle at different time points (24–120 hpf). Normal morphology was found in the group treated with the lowest concentrations and with vehicle (E,F). Typical malformations of zebrafish development were observed in embryos that had received ZO essential oil at a dose of 0.49–3.91 µg/mL (A–D). Abbreviations: PE, pericardial sac edema; C, coagulation; DT, dented tail; PY, poor reabsorption of yolk sac; MY, malformation of yolk sac; SC, spinal curvature. Figure 4. Morphological characteristics of zebrafish embryos and larvae treated with different concentrations of ZO es- sential oil or vehicle at different time points (24–120 hpf). Normal morphology was found in the group treated with the lowest concentrations and with vehicle (E,F). Typical malformations of zebrafish development were observed in embryos that had received ZO essential oil at a dose of 0.49–3.91 µg/mL (A–D). Abbreviations: PE, pericardial sac edema; C, coag- ulation; DT, dented tail; PY, poor reabsorption of yolk sac; MY, malformation of yolk sac; SC, spinal curvature. Figure 4. 3.5. Hatchability of Zebrafish Embryos 3.5. Hatchability of Zebrafish Embryos Th h t hi f b fi h b 3.5. Hatchability of Zebrafish Embryos 3.5. Hatchability of Zebrafish Embryos Th h t hi f b fi h b The hatching of zebrafish embryos indicates successful embryonic development of the embryos after 48 hpf [28]. The hatching rates of zebrafish embryos treated with five different concentrations of ZO essential oil (3.91, 1.95, 0.98, 0.49, and 0.24 µg/mL) and 0.1% DMSO (control) are presented in Figure 5A. As the concentration increased, the hatching rate of treated embryos became lower. No hatching of any zebrafish embryos was observed in the 3.91 and 1.95 µg/mL treated groups. All of the zebrafish embryos treated with 0.24 µg/mL of the ZO essential oil or vehicle were successfully hatched. The hatching of zebrafish embryos indicates successful embryonic development of the embryos after 48 hpf [28]. The hatching rates of zebrafish embryos treated with five different concentrations of ZO essential oil (3.91, 1.95, 0.98, 0.49, and 0.24 µg/mL) and 0.1% DMSO (control) are presented in Figure 5A. As the concentration increased, the hatching rate of treated embryos became lower. No hatching of any zebrafish embryos was ob- served in the 3.91 and 1.95 µg/mL treated groups. All of the zebrafish embryos treated with 0.24 µg/mL of the ZO essential oil or vehicle were successfully hatched. (B) b (A) P t f b h t hi f b fi h (A) (B) Figure 5. Assessment of hatchability and heart rate of zebrafish embryos. (A) Percentage of embryos hatching of zebrafish treated with ZO essential oil. (B) Correlation between the dose of ZO essential oil and zebrafish larvae heart rate (bpm) at 72 hpf. Data are presented as mean ± SD of three independent experiments (n = 60 embryos/group). Data were analyzed using one-way ANOVA and Tukey’s multiple comparison test. Different lowercase letters indicate significant differences between groups (p < 0.05). 3 6 H R f Z b fi h E b Figure 5. Assessment of hatchability and heart rate of zebrafish embryos. (A) Percentage of embryos hatching of zebrafish treated with ZO essential oil. (B) Correlation between the dose of ZO essential oil and zebrafish larvae heart rate (bpm) at 72 hpf. Data are presented as mean ± SD of three independent experiments (n = 60 embryos/group). Data were analyzed using one-way ANOVA and Tukey’s multiple comparison test. Different lowercase letters indicate significant differences between groups (p < 0.05). (A) (B) Figure 5. 3.5. Hatchability of Zebrafish Embryos 3.5. Hatchability of Zebrafish Embryos Th h t hi f b fi h b Assessment of hatchability and heart rate of zebrafish embryos. (A) Percentage of embryos hatching of zebrafish treated with ZO essential oil. (B) Correlation between the dose of ZO essential oil and zebrafish larvae heart rate (bpm) at 72 hpf. Data are presented as mean ± SD of three independent experiments (n = 60 embryos/group). Data were analyzed using one-way ANOVA and Tukey’s multiple comparison test. Different lowercase letters indicate significant differences between groups (p < 0.05). Figure 5. Assessment of hatchability and heart rate of zebrafish embryos. (A) Percentage of embryos hatching of zebrafish treated with ZO essential oil. (B) Correlation between the dose of ZO essential oil and zebrafish larvae heart rate (bpm) at 72 hpf. Data are presented as mean ± SD of three independent experiments (n = 60 embryos/group). Data were analyzed using one-way ANOVA and Tukey’s multiple comparison test. Different lowercase letters indicate significant differences between groups (p < 0.05). 3.6. Heart Rates of Zebrafish Embryos The normal heart rate of zebrafis 3.6. Heart Rates of Zebrafish Embryos The normal heart rate of zebrafish embryos ranges from 120 to 180 bpm [29,30]. The zebrafish heart rates at 72 hpf with different concentrations are shown in Figure 5B. There was no significant difference between the means of the heart rates of the ZO essential oil (0.24 µg/mL)-treated group and the 0.1% DMSO-treated group (control). The average heart rate declined at higher concentrations. No heartbeat was detected at 72 hpf in any zebrafish embryos treated with 3.91 µg/mL of ZO essential oil due to embryo death. The normal heart rate of zebrafish embryos ranges from 120 to 180 bpm [29,30]. The zebrafish heart rates at 72 hpf with different concentrations are shown in Figure 5B. There was no significant difference between the means of the heart rates of the ZO essential oil (0.24 µg/mL)-treated group and the 0.1% DMSO-treated group (control). The average heart rate declined at higher concentrations. No heartbeat was detected at 72 hpf in any zebrafish embryos treated with 3.91 µg/mL of ZO essential oil due to embryo death. 3.4. Morphological Defects of Zebrafish Embryos 3.4. Morphological Defects of Zebrafish Embryos Morphological characteristics of zebrafish embryos and larvae treated with different concentrations of ZO essential oil or vehicle at different time points (24–120 hpf). Normal morphology was found in the group treated with the lowest concentrations and with vehicle (E,F). Typical malformations of zebrafish development were observed in embryos that had received ZO essential oil at a dose of 0.49–3.91 µg/mL (A–D). Abbreviations: PE, pericardial sac edema; C, coagulation; DT, dented tail; PY, poor reabsorption of yolk sac; MY, malformation of yolk sac; SC, spinal curvature. Toxics 2021, 9, 102 Toxics 2021, 9, x FOR 10 of 17 10 of 17 10 of 17 10 of 17 3.5. Hatchability of Zebrafish Embryos 3.5. Hatchability of Zebrafish Embryos Th h t hi f b fi h b 3.7. Lethality and Behavioral Analysis of Rats 3.7. Lethality and Behavioral Analysis of Rats Lethality assessment of rats treated with 2000 mg/kg of ZO essential oil revealed that no death of any animal occurred over 14 days. Some changes of general appearance and behavior seemed to be found at 6 h and 12 h after ZO essential oil administration, includ- ing sedation, lethargy, and ataxia. However, all the observed behavioral changes were later recovered to normal (Table 3). Lethality assessment of rats treated with 2000 mg/kg of ZO essential oil revealed that no death of any animal occurred over 14 days. Some changes of general appearance and behavior seemed to be found at 6 h and 12 h after ZO essential oil administration, including sedation, lethargy, and ataxia. However, all the observed behavioral changes were later recovered to normal (Table 3). 11 of 17 Toxics 2021, 9, 102 Table 3. General appearance and behavioral observations of the control group (0.9% saline) and the treatment group (2000 mg/kg of ZO essential oil) over the period from 6 h to 14 days after administration. N = normal, + = detected and - = not detected. Observation Control Group (0.9% Saline) Treatment Group (2000 mg/kg of ZO Essential Oil) 6 h 12 h 24 h 7 d 14 d 6 h 12 h 24 h 7 d 14 d Behavioral patterns N N N N N N N N N N Skin and Fur N N N N N N N N N N Eye and Ears N N N N N N N N N N Mucous membrane N N N N N N N N N N Heartbeat N N N N N N N N N N Breathing N N N N N N N N N N Sedation - - - - - +/- +/- - - - Lethargy - - - - - +/- +/- - - - Ataxia - - - - - +/- +/- - - - Salivation - - - - - - - - - - Diarrhea - - - - - - - - - - Convulsion - - - - - - - - - - 3.8. Body Weight, Food and Water Consumption, and Relative Organ Weight Analysis The rats’ final weight, the percentage of body weight gain, and food and water intake of the control and treatment groups were monitored and calculated (Table 4). 3.7. Lethality and Behavioral Analysis of Rats 3.7. Lethality and Behavioral Analysis of Rats There were no significant differences observed between the control and treatment groups. Additionally, the relative organ weights of all organs examined were similar among the groups (Table 5). Table 4. Body weight and food and water intake of the control group (0.9% saline) and the treatment group (2000 mg/kg of ZO essential oil) during the 14 day observation period. Data shown are the mean ± SD (n = 5 rats/group). All data were analyzed using Student’s t-test (p < 0.05). Parameters Control Group (0.9% Saline) Treatment Group (2000 mg/kg of ZO Essential Oil) Initial weight (g) 185.00 ± 5.00 183.00 ± 4.47 Final weight (g) 213.60 ± 7.40 214.40 ± 3.43 Body weight gain (%) 12.62 ± 3.51 12.14 ± 1.67 Food intake (g/day) 13.89 ± 0.04 13.57 ± 0.73 Water intake (mL/day) 29.68 ± 2.27 31.85 ± 2.66 Table 5. Relative organ weights of the control group (0.9% saline) and the treatment group (2000 mg/kg of ZO essential oil). Data shown are the mean ± SD (n = 5 rats/group). All data were analyzed using Student’s t-test (p < 0.05). Table 5. Relative organ weights of the control group (0.9% saline) and the treatment group (2000 mg/kg of ZO essential oil). Data shown are the mean ± SD (n = 5 rats/group). All data were analyzed using Student’s t-test (p < 0.05). Organ (g/100 g Body Weight) Control Group (0.9% Saline) Treatment Group (2000 mg/kg of ZO Essential Oil) Brain 0.93 ± 0.09 0.89 ± 0.02 Heart 0.32 ± 0.01 0.30 ± 0.02 Liver 4.63 ± 0.22 4.51 ± 0.10 Kidney 0.50 ± 0.02 0.49 ± 0.01 Spleen 0.25 ± 0.02 0.25 ± 0.03 Thymus gland 0.21 ± 0.03 0.20 ± 0.04 Ovary 0.03 ± 0.01 0.03 ± 0.01 Uterus 0.25 ± 0.02 0.28 ± 0.06 Adrenal gland 0.02 ± 0.00 0.02 ± 0.00 Lung 0.52 ± 0.04 0.75 ± 0.23 Toxics 2021, 9, 102 Toxics 2021, 9, x FOR 12 of 17 12 of 17 12 of 17 12 of 17 4. Discussion GC–MS analysis revealed that the main components of Zingiber ottensii (ZO) Valeton essential oil were sesquiterpenes of which zerumbone was the major compound (24.73%), followed by monoterpenes including terpinene-4-ol (18.75%), sabinene (15.19%), and β- Pinene (7.95%) (Table 1). These findings are in line with the phytochemical compositions of ZO cultivated in Johor, Malaysia, and Phetchaburi Province, Thailand, where major constituents have been reported to be zerumbone (25.63% and 40.14%), terpinene-4-ol (16.81% and 11.17%), sabinene (7.20% and 6.48%), and β-Pinene (5.08% and 4.32%) [17,31]. The most abundant compound of ZO cultivated in Bandung, Indonesia, however, is terpinene-4-ol (16.55%), followed by zerumbone (14.23%) with some other phytochemical components including longifolenaldehyde (1.33%) and 2,5,9-trimethyl-cycloundeca-4,8- dienone (1.00%) [32]. The differences in the amounts and phytoconstituents of the essential oil may be due to differences in cultivation areas, growing seasons, harvest times, and environmental conditions [33]. Interestingly, zerumbone and terpinene-4-ol have been reported to possess a broad spectrum of beneficial biological effects, e.g., anti-inflammatory and antioxidant effects [34,35]. Those effects make it attractive to develop pharmaceutical products from ZO essential oil, which can could potentially increase the value of the herbs as well as local farmers’ income. However, an important initial step in the development of any drug or herbal product is to confirm its safety, generally by in vivo toxicity studies. In particular, there is still a lack of scientific evidence demonstrating the safety profile of ZO essential oil in terms of embryonic and teratogenic toxicity and acute oral toxicity. The zebrafish model can also be used in high throughput and valid screening models to study the embryotoxic and teratogenic effects. Such studies require only small amounts of test substances, short duration of study, are low cost and are not so complicated to perform, compared with toxicity models in rats. On the other hand, models to study the development toxicity in rats require higher amounts of test substances, longer duration of study, are higher cost and are very complicated to perform. Due to these reasons, zebrafish models were used, instead of models to study the development toxicity in rats, as the screening models to study the embryotoxic and teratogenic effects of ZO essential oil. The acute oral toxicity in rats is also another well-known model. 3.9. Macroscopic and Histopathological Analysis 3.9. Macroscopic and Histopathological Analysis 3.9. Macroscopic and Histopathological Analysis 3.9. Macroscopic and Histopathological Analysis Macroscopic evaluation of vital organs of rats treated with 2000 mg/kg of ZO essen- tial oil found no characteristic changes that were different from the control group. The histopathological analysis of vital organs, including the brain, heart, lung, liver, spleen, and kidney, revealed no significant structural differences between the treatment and control groups (Figure 6). Macroscopic evaluation of vital organs of rats treated with 2000 mg/kg of ZO essen- tial oil found no characteristic changes that were different from the control group. The histopathological analysis of vital organs, including the brain, heart, lung, liver, spleen, and kidney, revealed no significant structural differences between the treatment and con- trol groups (Figure 6). g p ( g ) Figure 6. Histopathological examination of vital organs of rat treated with 2000 mg/kg of ZO es- sential oil, compared to the control group in acute oral toxicity study (H&E; ×100). Figure 6. Histopathological examination of vital organs of rat treated with 2000 mg/kg of ZO essential oil, compared to the control group in acute oral toxicity study (H&E; ×100). Figure 6. Histopathological examination of vital organs of rat treated with 2000 mg/kg of ZO es- sential oil, compared to the control group in acute oral toxicity study (H&E; ×100). Figure 6. Histopathological examination of vital organs of rat treated with 2000 mg/kg of ZO essential oil, compared to the control group in acute oral toxicity study (H&E; ×100). Toxics 2021, 9, 102 13 of 17 13 of 17 4. Discussion It is used to provide information on health hazards likely to arise from short-term exposure by the oral route of any test substance and is a step in establishing a dosage regimen in subchronic and other toxicity studies. y Prior to performing safety studies in a rodent model, a toxicological assay of verte- brate zebrafish has been proposed as a tractable model for screening potentially suitable phytochemical concentrations in whole more complex organisms. The present study pro- vides a toxicity assessment of ZO essential oil in zebrafish embryos. This study observed no embryotoxic mortality or malformation when using the lowest concentration of ZO essential oil (0.24 µg/mL), indicating that this concentration is not toxic to fish embryos. In contrast, increases in ZO essential oil concentrations were found to be related to higher rates of embryonic mortality and death of all embryos at the concentrations of 3.91 and 1.95 µg/mL, at 96 and 72 hpf, respectively (Figure 2). Notably, higher doses of ZO essential oil were observed to have an increase in embryotoxicity over the course of ZO treatment. These results indicate that in zebrafish embryos the toxicity of ZO essential oil appears to be concentration- and time-dependent manner. These results are in line with studies of other plants in the Zingiberaceae family, including the essential oil of Zingiber cassumunar Roxb. and the methanolic extract of Curcuma longa Linn., in which embryotoxicity of zebrafish embryo was also observed to be concentration- and time-dependent manners [20,23]. Ad- ditionally, since zerumbone was found to be a major compound of ZO essential oil in this study, it could be responsible for the embryotoxic and teratogenic effects on zebrafish. Moreover, sabinene and terpinen4-ol found in ZO essential oil could also be responsible for these effects since they were also found to be the major compounds of cassumunar ginger oil that also possessed embryotoxic and teratogenic effects [20]. Toxics 2021, 9, 102 14 of 17 14 of 17 Development deformities in zebrafish embryos were observed in the groups treated with 0.49–3.91 µg/mL of ZO essential oil. Deformities of body parts or organs included a dented tail, failure of tail detachment, spinal curvature, pericardial sac edema, and poor reabsorption of yolk (Figure 4). The abnormalities increased noticeably with prolonged exposure to ZO essential oil for 24–120 hpf (Table 2). 4. Discussion These findings suggest that the accessibility of the ZO essential oil introduced into the animal’s body increases with the duration of exposure and eventually leads to embryotoxicity. This phenomenon may be caused by the alternation of the embryo protective layer (chorion) protein, which may result in opening and widening of the chorion pore channel during normal development of the zebrafish leading to weakened and damaged chorion, which, in turn, allows more ZO essential oil to pass into the embryos [23,36]. Furthermore, terpenoid found in ZO essential oil is a compound with nonpolar properties and a low molecular weight, making it easier for it to pass through the cytoplasmic membrane of zebrafish embryos [20]. Edema formation may be caused by overhydration of the zebrafish embryo due to osmoregulatory system problems related to toxicant accumulation [37]. Cardiac edema was observed in this study (Figure 4(A2,B2,B3,C2,C3)), and most embryos exhibited cardio- vascular abnormalities, including a reduction of heart rate (Figure 5B) similar to previous reports [38]. p A decrease in the hatching rate of zebrafish embryos and a delayed hatching process were observed, which are events correlated with increasing concentrations of ZO essential oil (Figure 5A). A low hatchability rate and delayed hatching process normally indicate growth retardation, which may be due to abnormal embryo development. In particular, it may be related to a decreased chorion breaking ability resulting from the inhibition of the tetraspanin gene (cd63), which causes a lack of secreted proteolytic enzymes necessary for chorion softening [39,40]. Obvious morphological abnormalities could be observed in these larvae [41]. Additionally, as an initial step in drug development, we performed an acute oral toxicity study in rats to evaluate the safe dose of this medicinal plant in an in vivo model, which provides for more toxic expression, including general appearance and behavioral changes, than in vitro assay. Specifically, in our current study, each rat received a single dose of ZO essential oil (2000 mg/kg), and the results showed no mortality of any rats, no significant changes in the percentage of body weight gain, daily food, and water consump- tion, and no differences in relative organ weight along with normal organ appearance and structures in gross pathology and histopathology analysis, when compared to the control group (Tables 3–5). 4. Discussion However, alterations in some signs of general appearance (sedation, lethargy, and ataxia) seemed to be found (Table 3), but all these signs disappeared, and the animals returned to normal 12–24 h post administration. These temporary effects could imply that ZO essential oil may possess CNS-depressant or sedative-hypnotic effects. This result is in agreement with a previous in vivo study of terpenes, e.g., terpinen-4-ol, which is a compound that has sedative and anesthetic effects in fish [36]. Furthermore, the inhalation of sabinene and 1,8 cineole has been shown to exhibit strong sedative activity and to reduce locomotor activity in mice [42]. Oral administration of α- and β-pinene have also been demonstrated to induce mild sedation [43]. The present study revealed the monoterpene and sesquiterpene compounds in ZO essential oil and determined the toxicological profiles of the essential oil in zebrafish embryos and larvae. However, in the acute oral toxicity study in rats, ZO essential oil exhibited only temporary alteration of some general appearance characteristics without other significant toxicity or inducing death. Since routes of administration of ZO essential oil in the zebrafish model (topical route) and the rat model (oral route) were different, the toxic concentrations of ZO essential oil in the zebrafish model cannot be directly compared with the doses of ZO essential oil in the rat model. Moreover, the teratogenic effect of any substance depends on the ability of that substance to cross the placental barrier. In addition, some compounds found to be toxic in a rat embryotoxicity study were also found to cause deformities in zebrafish [44]. For these reasons, the use of ZO essential oil should Toxics 2021, 9, 102 15 of 17 be concerned in pregnant women. However, further confirmation of safety profiles of ZO essential oil in long-term toxicity studies, especially with other higher-order vertebrates, is needed. Such toxicological studies using different animal species are necessary to confirm the safety of any novel commercial pharmaceutical products containing ZO essential oil. 5. Conclusions Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 3. Arpornchayanon, W.; Gomonchareonsiri, S.; Chansakaow, S.; Wongpakaran, T.; Varnado, P.; Wongpakaran, N. Acute effects of essential oil blend containing phlai oil on mood among healthy male volunteers: Randomized controlled trial. J. Complement. Integr. Med. 2019, 17, 1–7. [CrossRef] [PubMed] 1. Ruttanapattanakul, J.; Wikan, N.; Okonogi, S.; Na Takuathung, M.; Buacheen, P.; Pitchakarn, P.; Potikanond, S.; Nimlamool, W. Boesenbergia rotunda extract accelerates human keratinocyte proliferation through activating ERK1/2 and PI3K/Akt kinases. Biomed. Pharmacother. 2021, 133, 111002. [CrossRef] [PubMed] 5. Conclusions The essential oil of Zingiber ottensii (ZO) Valeton contains terpene compounds, of which zerumbone is a major constituent. The embryotoxicity of ZO essential oil in zebrafish appears to be in a concentration- and time-dependent manner and to have a moderate LC50 (1.003 µg/kg). Teratogenic effects of ZO essential oil on zebrafish embryos include morphological defects, reduced hatchability, and decreased heart rate. In the acute oral toxicity study in rats, temporary changes in general conditions including sedation, lethargy, and ataxia seem to be found, but percent body weight gain, water and food consumption, and organ characteristics were unchanged, and there were no deaths of any rats. These preclinical study results provide crucial support for the safety profile of ZO essential oil, an initial step in drug and natural pharmaceutical development. However, further long- term toxicity studies are needed to confirm the safety of any newly developed products containing ZO essential oil. Author Contributions: Conceptualization, W.T., R.M., W.N., P.K. (Parirat Khonsung) and P.K. (Puongtip Kunanusorn); methodology, W.T., R.M., S.P., S.O. and P.K. (Puongtip Kunanusorn); soft- ware, W.T.; validation, R.M., W.N., S.P. and P.K. (Puongtip Kunanusorn); formal analysis, W.T., R.M. and P.K. (Puongtip Kunanusorn); investigation, R.M., P.K. (Parirat Khonsung) and P.K. (Puongtip Kunanusorn); resources, S.P., S.O. and P.K. (Puongtip Kunanusorn); data curation, W.T., R.M., S.P. and P.K. (Puongtip Kunanusorn); writing—original draft preparation, W.T.; writing—review and editing, W.T., R.M., W.N. and P.K. (Puongtip Kunanusorn); supervision, R.M. and P.K. (Puongtip Kunanusorn); project administration, W.T. and P.K. (Puongtip Kunanusorn); funding acquisition, P.K. (Puongtip Kunanusorn). All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Faculty of Medicine, Chiang Mai University (032-2564 to P.K. (Puongtip Kunanusorn)). Institutional Review Board Statement: The study was conducted according to the internationally accepted guidelines OECD 236 and OECD 420, and approved by the Animal Ethics Committee of the Faculty of Veterinary Medicine (Permit No. R8/2563, 17 July 2020) and the Faculty of Medicine, Chiang Mai University, Thailand (Permit No. 22/2563, 9 July 2020), respectively. Acknowledgments: The authors would like to thank Kanyarat Chairaksa for assistance in zebrafish breeding and figure editing; laboratory team of Aquatic Clinic, Department of Food Animal Clinic, Faculty of Veterinary Medicine, Chiang Mai University for the support in fish maintenance; and laboratory team of Research Center for Pharmaceutical Nanotechnology, Chiang Mai University for the support in the extraction of Zingiber ottensii Valeton essential oil. 4. Romagosa, C.M.R.; David, E.S.; Dulay, R.M.R. Embryo-toxic and teratogenic effects of Tinospora cordifolia leaves and bark extracts in Zebrafish (Danio rerio) embryos. 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Trends of ozone total columns and vertical distribution from FTIR observations at 8 NDACC stations around the globe
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Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Pa Discussion Paper ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Atmos. Chem. Phys. Discuss., 14, 24623–24666, 2014 www.atmos-chem-phys-discuss.net/14/24623/2014/ doi:10.5194/acpd-14-24623-2014 © Author(s) 2014. CC Attribution 3.0 License. Atmos. Chem. Phys. Discuss., 14, 24623–24666, 2014 www.atmos-chem-phys-discuss.net/14/24623/2014/ doi:10.5194/acpd-14-24623-2014 © Author(s) 2014. CC Attribution 3.0 License. ACPD This discussion paper is/has been under review for the journal Atmospheric Chemistry and Physics (ACP). Please refer to the corresponding final paper in ACP if available. This discussion paper is/has been under review for the journal Atmospheric Chemistry and Physics (ACP). Please refer to the corresponding final paper in ACP if available. Trends of ozone total columns and vertical distribution from FTIR observations at 8 NDACC stations around the globe the globe C. Vigouroux1, T. Blumenstock2, M. Coffey3, Q. Errera1, O. García4, N. B. Jones5, J. W. Hannigan3, F. Hase2, B. Liley6, E. Mahieu7, J. Mellqvist8, J. Notholt9, M. Palm9, G. Persson8, M. Schneider4, C. Servais7, D. Smale6, L. Thölix10, and M. De Mazière1 Department of Atmospheric Composition, Belgian Institute for Space Aeronomy BIRA-IASB), Brussels, Belgium Karlsruhe Institute of Technology (KIT), Institute for Meteorology and Climate Research IMK-ASF), Karlsruhe, Germany Atmospheric Chemistry Division, National Center for Atmospheric Research (NCAR), Boulder, Colorado, USA Izaña Atmospheric Research Centre (IARC), Agencia Estatal de Meteorología (AEMET), Santa Cruz de Tenerife, Spain Centre for Atmospheric Chemistry, University of Wollongong, Wollongong, Australia 24623 C. Vigouroux1, T. Blumenstock2, M. Coffey3, Q. Errera1, O. García4, N. B. Jones5, J. W. Hannigan3, F. Hase2, B. Liley6, E. Mahieu7, J. Mellqvist8, J. Notholt9, M. Palm9, G. Persson8, M. Schneider4, C. Servais7, D. Smale6, L. Thölix10, and M. De Mazière1 1Department of Atmospheric Composition, Belgian Institute for Space Aeronomy (BIRA-IASB), Brussels, Belgium 2Karlsruhe Institute of Technology (KIT), Institute for Meteorology and Climate Research (IMK-ASF), Karlsruhe, Germany 3 3Atmospheric Chemistry Division, National Center for Atmospheric Research (NCAR), Boulder, Colorado, USA 4Izaña Atmospheric Research Centre (IARC), Agencia Estatal de Meteorología (AEMET), Santa Cruz de Tenerife, Spain 5 p 5Centre for Atmospheric Chemistry, University of Wollongong, Wollongong, Australia 24623 Discussion Paper | Discussion Paper ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 6Department of Atmosphere, National Institute of Water and Atmospheric Research Ltd (NIWA), Lauder, New Zealand 7 6Department of Atmosphere, National Institute of Water and Atmospheric Research Ltd (NIWA), Lauder, New Zealand 7 6Department of Atmosphere, National Institute of Water and Atmospheric Research Ltd (NIWA), Lauder, New Zealand 7Institute of Astrophysics and Geophysics, University of Liège (ULg), Liège, Belgium 8Department of Earth and Space Science, Chalmers University of Technology, Göteborg, Sweden 9Institute of Environmental Physics, University of Bremen, Bremen, Germany 10Climate Research, Finnish Meteorological Institute (FMI), Helsinki, Finland Received: 11 July 2014 – Accepted: 29 August 2014 – Published: 25 September 2014 Correspondence to: C. Vigouroux (corinne.vigouroux@aeronomie.be) Published by Copernicus Publications on behalf of the European Geosciences Union. Trends of ozone total columns and vertical distribution from FTIR observations at 8 NDACC stations around the globe ACPD Received: 11 July 2014 – Accepted: 29 August 2014 – Published: 25 September 2014 Correspondence to: C. Vigouroux (corinne.vigouroux@aeronomie.be) Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 24624 Discussion Paper | Discussion Paper Discussion Paper | Discussion Paper | ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Abstract Abstract ACPD Ground-based Fourier transform infrared (FTIR) measurements of solar absorption spectra can provide ozone total columns with a precision of 2 %, but also independent partial column amounts in about four vertical layers, one in the troposphere and three in the stratosphere up to about 45 km, with a precision of 5–6 %. We use eight of the 5 Network for the Detection of Atmospheric Compososition Change (NDACC) stations having a long-term time series of FTIR ozone measurements to study the total and vertical ozone trends and variability, namely: Ny-Alesund (79◦N), Thule (77◦N), Kiruna (68◦N), Harestua (60◦N), Jungfraujoch (47◦N), Izaña (28◦N), Wollongong (34◦S) and in the stratosphere up to about 45 km, with a precision of 5–6 %. We use eight of the 5 Network for the Detection of Atmospheric Compososition Change (NDACC) stations having a long-term time series of FTIR ozone measurements to study the total and vertical ozone trends and variability, namely: Ny-Alesund (79◦N), Thule (77◦N), Kiruna (68◦N), Harestua (60◦N), Jungfraujoch (47◦N), Izaña (28◦N), Wollongong (34◦S) and Lauder (45◦S). The length of the FTIR time-series varies by station, but is typically from 10 about 1995 to present. We applied to the monthly means of the ozone total and four partial columns a stepwise multiple regression model including the following proxies: solar cycle, Quasi-Biennial Oscillation (QBO), El Niño-Southern Oscillation (ENSO), Arctic and Antarctic Oscillation (AO/AAO), tropopause pressure (TP), equivalent lati- ( ), p p p ( ), q tude (EL), Eliassen-Palm flux (EPF), and volume of polar stratospheric clouds (VPSC). 15 At the Arctic stations, the trends are found mostly negative in the troposphere and lower stratosphere, very mixed in the middle stratosphere, positive in the upper stratosphere due to a large increase in the 1995–2003 period, and non-significant when considering the total columns. The trends for mid-latitude and subtropical sta- tions are all non-significant, except at Lauder in the troposphere and upper strato- 20 tude (EL), Eliassen-Palm flux (EPF), and volume of polar stratospheric clouds (VPSC). 15 At the Arctic stations, the trends are found mostly negative in the troposphere and lower stratosphere, very mixed in the middle stratosphere, positive in the upper stratosphere due to a large increase in the 1995–2003 period, and non-significant when considering the total columns. Introduction 1 ACPD While the past negative trend in the ozone layer has been successfully attributed to the increase of ozone depleting substances, and reproduced by chemistry-climate mod- els, understanding and predicting the current and future ozone layer, and especially attributing an ozone recovery to the positive effect of the Montreal Protocol and its 5 Amendments and Adjustments, is still a challenge. This results from natural variabil- ity, observation uncertainties, and changes in dynamics and temperature induced by the increase of greenhouse gases (WMO, 2010). Long-term measurements of total and vertical ozone are required to understand the ozone response to different natural and anthropogenic forcings. Since the long-term satellite experiments ceased to op- 10 erate (i.e. SAGE, HALOE), the satellite community is working on merging the past records to the new measurements performed by a number of satellite instruments launched since 2000 (e.g. Bodeker et al., 2013; Kyrölä et al., 2013; Sioris et al., 2014; Chehade et al., 2014). Ground-based stable data are needed to validate these satellite extended datasets, and to offer an alternative determination of ozone total and vertical 15 changes. Dobson, Umkehr, and ozonesondes are the traditional ground-based data for these studies, already reporting trends in the 1985 ozone report (WMO, 1985), fol- lowed in 1998 by Lidar and microwave measurements (WMO, 1998). Ground-based FTIR (Fourier Transform Infra-Red) measurements derived from high-resolution solar absorption spectra provide an additional ozone data set, and they have been used 20 for trend studies for the first time in Vigouroux et al. (2008) with 10 years of data (1995–2004) at several European stations, then updated in the WMO (2010) report. Additional similar studies have been performed at individual stations (Mikuteit, 2008; García et al., 2012). These measurements have their own advantages: for atmospheric gases such as ozone which have very narrow absorption lines, an absolute calibration 25 is not needed; the ozone absorption signatures are self-calibrated with the reference being the surrounding continuum. Abstract The trends for mid-latitude and subtropical sta- tions are all non-significant, except at Lauder in the troposphere and upper strato- 20 sphere, and at Wollongong for the total columns and the lower and middle strato- spheric columns; at Jungfraujoch, the upper stratospheric trend is close to significance (+0.9 ± 1.0 % decade−1). Therefore, some signs of the onset of ozone mid-latitude re- covery are observed only in the Southern Hemisphere, while a few more years seems tions are all non-significant, except at Lauder in the troposphere and upper strato- 20 sphere, and at Wollongong for the total columns and the lower and middle strato- spheric columns; at Jungfraujoch, the upper stratospheric trend is close to significance (+0.9 ± 1.0 % decade−1). Therefore, some signs of the onset of ozone mid-latitude re- covery are observed only in the Southern Hemisphere, while a few more years seems | Discussion Paper | to be needed to observe it at the northern mid-latitude station. 25 24625 24625 Discussion Paper | Discussion Paper Discussion Paper ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Introduction Introduction Furthermore, they can provide not only ozone total columns with a precision of 2 %, but also low vertical resolution profiles, obtained from While the past negative trend in the ozone layer has been successfully attributed to the increase of ozone depleting substances, and reproduced by chemistry-climate mod- els, understanding and predicting the current and future ozone layer, and especially attributing an ozone recovery to the positive effect of the Montreal Protocol and its 5 Amendments and Adjustments, is still a challenge. This results from natural variabil- ity, observation uncertainties, and changes in dynamics and temperature induced by the increase of greenhouse gases (WMO, 2010). Long-term measurements of total and vertical ozone are required to understand the ozone response to different natural q p and anthropogenic forcings. Since the long-term satellite experiments ceased to op- 10 erate (i.e. SAGE, HALOE), the satellite community is working on merging the past records to the new measurements performed by a number of satellite instruments launched since 2000 (e.g. Bodeker et al., 2013; Kyrölä et al., 2013; Sioris et al., 2014; Chehade et al., 2014). Ground-based stable data are needed to validate these satellite and anthropogenic forcings. Since the long-term satellite experiments ceased to op- 10 erate (i.e. SAGE, HALOE), the satellite community is working on merging the past records to the new measurements performed by a number of satellite instruments launched since 2000 (e.g. Bodeker et al., 2013; Kyrölä et al., 2013; Sioris et al., 2014; Chehade et al., 2014). Ground-based stable data are needed to validate these satellite ) extended datasets, and to offer an alternative determination of ozone total and vertical 15 changes. Dobson, Umkehr, and ozonesondes are the traditional ground-based data for these studies, already reporting trends in the 1985 ozone report (WMO, 1985), fol- lowed in 1998 by Lidar and microwave measurements (WMO, 1998). Ground-based FTIR (Fourier Transform Infra-Red) measurements derived from high-resolution solar absorption spectra provide an additional ozone data set, and they have been used 20 for trend studies for the first time in Vigouroux et al. (2008) with 10 years of data (1995–2004) at several European stations, then updated in the WMO (2010) report. Additional similar studies have been performed at individual stations (Mikuteit, 2008; García et al., 2012). Introduction (2008): it is based on longer time series, it includes FTIR data from stations outside 5 Europe, and it uses a stepwise multiple linear regression model including several ex- planatory variables for the trend evaluation. It is presented as follows: Sect. 2 provides information about the FTIR ozone observations (retrieval strategies, characterization of the vertical information, time series and seasonality). Section 3 describes the stepwise multiple linear regression model applied to the ozone time series. Section 4 presents 10 and discusses the trend results, as well as the explained part of ozone variability. Sec- tion 5 summarizes the conclusions. multiple linear regression model applied to the ozone time series. Section 4 presents 10 and discusses the trend results, as well as the explained part of ozone variability. Sec- tion 5 summarizes the conclusions. Introduction These measurements have their own advantages: for atmospheric absorption spectra provide an additional ozone data set, and they have been used 20 for trend studies for the first time in Vigouroux et al. (2008) with 10 years of data (1995–2004) at several European stations, then updated in the WMO (2010) report. Additional similar studies have been performed at individual stations (Mikuteit, 2008; García et al., 2012). These measurements have their own advantages: for atmospheric | Discussion Paper | gases such as ozone which have very narrow absorption lines, an absolute calibration 25 is not needed; the ozone absorption signatures are self-calibrated with the reference being the surrounding continuum. Furthermore, they can provide not only ozone total columns with a precision of 2 %, but also low vertical resolution profiles, obtained from 24626 Discussion Paper | Discussion Pape ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | the temperature and pressure dependence of the absorption line shapes. This leads to about four independent partial columns, one in the troposphere and three in the stratosphere up to about 45 km, with a precision of about 5–6 %. the temperature and pressure dependence of the absorption line shapes. This leads to about four independent partial columns, one in the troposphere and three in the stratosphere up to about 45 km, with a precision of about 5–6 %. ACPD The work discussed in this paper expands the previous study of Vigouroux et al. (2008): it is based on longer time series, it includes FTIR data from stations outside 5 Europe, and it uses a stepwise multiple linear regression model including several ex- planatory variables for the trend evaluation. It is presented as follows: Sect. 2 provides information about the FTIR ozone observations (retrieval strategies, characterization of the vertical information, time series and seasonality). Section 3 describes the stepwise multiple linear regression model applied to the ozone time series. Section 4 presents 10 and discusses the trend results, as well as the explained part of ozone variability. Sec- tion 5 summarizes the conclusions. The work discussed in this paper expands the previous study of Vigouroux et al. 2.1 FTIR monitoring Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD 2.1 FTIR monitoring Table 1 identifies the ground-based FTIR stations, all part of NDACC (Network for the 15 Detection of Atmospheric Composition Change), that are contributing to the present work. The latitudinal coverage is good: from 79◦N to 45◦S. These stations perform regular solar absorption measurements, under clear-sky conditions, over a wide spec- tral range (around 600–4500 cm−1) and the derived time series of total column abun- Table 1 identifies the ground-based FTIR stations, all part of NDACC (Network for the 15 Detection of Atmospheric Composition Change), that are contributing to the present work. The latitudinal coverage is good: from 79◦N to 45◦S. These stations perform regular solar absorption measurements, under clear-sky conditions, over a wide spec- tral range (around 600–4500 cm−1) and the derived time series of total column abun- g ( ) dances of many atmospheric species are available in the NDACC database (http: 20 //www.ndacc.org). While the stations are all currently active, they started their regu- lar monitoring activities at different times. The period of measurement used for ozone trend analysis at each station is summarized in Table 1, together with the instrument manufacturer and type. Some of the stations performed measurements even earlier but these older spectra, taken with different spectrometers have to be carefully re- 25 analysed first before being included in a trend study The instruments currently used g ( ) dances of many atmospheric species are available in the NDACC database (http: 20 //www.ndacc.org). While the stations are all currently active, they started their regu- lar monitoring activities at different times. The period of measurement used for ozone trend analysis at each station is summarized in Table 1, together with the instrument manufacturer and type. Some of the stations performed measurements even earlier but these older spectra, taken with different spectrometers have to be carefully re- 25 analysed first before being included in a trend study. The instruments currently used 24627 Discussion Paper | Discussion Pap Discussion Paper | Discussion Paper | D Discussion Paper are the high-resolution spectrometers Bruker 120 M, 125 M, 120 HR, and 125 HR which can achieve a spectral resolution of 0.0035 cm−1 or better. The Bomem DA8 used in the first years of Wollongong measurements has a spectral resolution of 0.004 cm−1. ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. 2.2 FTIR retrieval strategy We refer to Vigouroux et al. (2008) for more details on the ozone FTIR inversion princi- 5 ples, which are based on the optimal estimation method (Rodgers, 2000). Two profile retrieval algorithms are widely used, PROFITT9 (Hase, 2000) at Kiruna and Izaña, and SFIT2 (Pougatchev et al., 1995) at the six other stations. It has been demon- strated in Hase et al. (2004) that the profiles and total column amounts retrieved from We refer to Vigouroux et al. (2008) for more details on the ozone FTIR inversion princi- 5 ples, which are based on the optimal estimation method (Rodgers, 2000). Two profile retrieval algorithms are widely used, PROFITT9 (Hase, 2000) at Kiruna and Izaña, and SFIT2 (Pougatchev et al., 1995) at the six other stations. It has been demon- strated in Hase et al. (2004) that the profiles and total column amounts retrieved from these two different algorithms under identical conditions are in excellent agreement. In 10 Vigouroux et al. (2008), a common retrieval strategy was optimized, within a European FTIR network, to derive ozone trends in four vertical layers. In the present work, the homogenization of the retrieval strategy went one step further. First, a common source for the ozone a priori profiles is used: the model WACCM4 (Garcia et al., 2007) calcu- p p ( ) lated at each NDACC FTIR station, except at Harestua where a climatology based on 15 ozonesondes and HALOE measurements is used. Second, all stations are employing the daily pressure and temperature profiles from NCEP (National Centers for Environ- mental Predicion). Third, the interfering species fitted in the ozone retrievals, usually with a single scaling of their apriori profile, are the same for all stations, namely, H2O, per | Discussion Paper | CO2, C2H4, and the ozone isotopologues 16O16O18O and 16O18O16O. Only Harestua 20 used fixed profiles for C2H4, and the ozone isotopologues. The spectroscopic database has been updated to HITRAN 2008 (Rothman et al., 2009). We report in Table 2 the main remaining parameters that can still differ from sta- tion to station. All choices of microwindows lead to the required 4 to 5◦of freedom tion to station. All choices of microwindows lead to the required 4 to 5 of freedom for signal (DOFS), thanks to the numerous ozone lines with different intensities which 25 give information both in the stratosphere and the troposphere. 2.2 FTIR retrieval strategy In optimal estimation, the choice of the a priori covariance matrix Sa is also an impor- tant parameter of the inversion process, and together with the measurement noise error 10 covariance matrix Sϵ, it will lead to the following averaging kernel matrix A (Rodgers, 2000): tant parameter of the inversion process, and together with the measurement noise error 10 covariance matrix Sϵ, it will lead to the following averaging kernel matrix A (Rodgers, 2000): A = (KT S−1 ϵ K + S−1 a )−1KT S−1 ϵ K, (1) where K is the weighting function matrix that links the measurement vector y to the 15 state vector x: y=Kx+ϵ, with ϵ representing the measurement error. In our retrievals, we assume Sϵ to be diagonal, in which case the diagonal elements are the inverse square of the signal to noise ratio (SNR). The diagonal elements of Sa represent the assumed variability of the target gas volume mixing ratio (VMR) at a given altitude, where K is the weighting function matrix that links the measurement vector y to the 15 state vector x: y=Kx+ϵ, with ϵ representing the measurement error. In our retrievals, we assume Sϵ to be diagonal, in which case the diagonal elements are the inverse square of the signal to noise ratio (SNR). The diagonal elements of Sa represent the assumed variability of the target gas volume mixing ratio (VMR) at a given altitude, y g g g ( ) g , and the non-diagonal elements represent the correlation between the VMR at different 20 altitudes. This Sa matrix can differ from station to station as one can see in Table 2. Except at Harestua, Kiruna and Izaña, the stations are using an a priori covariance matrix with diagonal elements constant with altitude corresponding to 10, 20 or 30 % variability, the largest variabilities taking place at the high latitude stations Ny-Alesund and Thule At Harestua the diagonal elements of S correspond to 11 % in the strato- 25 y g g g g and the non-diagonal elements represent the correlation between the VMR at different 20 altitudes. This Sa matrix can differ from station to station as one can see in Table 2. 2.2 FTIR retrieval strategy The main interfering species in this spectral region is water vapor, and it has been dealt with differently de- pending on the station: at Wollongong and Lauder station, the H2O profile is retrieved 24628 q for signal (DOFS), thanks to the numerous ozone lines with different intensities which 25 give information both in the stratosphere and the troposphere. The main interfering species in this spectral region is water vapor, and it has been dealt with differently de- pending on the station: at Wollongong and Lauder station, the H2O profile is retrieved 24628 Discussion Paper | Discussion Pape ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | simultaneously with the ozone profile, adding the microwindow 896.4–896.6 cm−1 for a better H2O determination. At Kiruna, Izaña and Jungfraujoch, the H2O a priori profiles are only scaled in the ozone retrieval, but these a priori profiles have been preliminarily retrieved in dedicated H2O microwindows for each spectrum (Schneider et al. (2006) for simultaneously with the ozone profile, adding the microwindow 896.4–896.6 cm−1 for a better H2O determination. At Kiruna, Izaña and Jungfraujoch, the H2O a priori profiles are only scaled in the ozone retrieval, but these a priori profiles have been preliminarily retrieved in dedicated H2O microwindows for each spectrum (Schneider et al. (2006) for ACPD 2 p ( ( ) Kiruna and Izaña; Sussmann et al. (2009) for Jungfraujoch). For the very dry Jungfrau- 5 joch site, it has been found that preliminary H2O retrievals do not improve the quality of the ozone retrievals. At Ny-Alesund and Thule, water vapor is treated as the other interfering species: only a scaling of a single a priori profile from WACCM4 is made. Kiruna and Izaña; Sussmann et al. (2009) for Jungfraujoch). For the very dry Jungfrau- 5 joch site, it has been found that preliminary H2O retrievals do not improve the quality of the ozone retrievals. At Ny-Alesund and Thule, water vapor is treated as the other interfering species: only a scaling of a single a priori profile from WACCM4 is made. 2.2 FTIR retrieval strategy In this approach, 10 the loss of modulation efficiency and the phase error can be described (1) by 40 pa- rameters (20 for each) at equidistant optical path differences (OPDs); (2) or simply by two parameters assuming a linear decline of the modulation efficiency with OPD, and a constant phase error. For the Ny-Alesund, Harestua and Lauder stations, the LIN- gas cell with the LINEFIT code, as described in Hase et al. (1999). In this approach, 10 the loss of modulation efficiency and the phase error can be described (1) by 40 pa- rameters (20 for each) at equidistant optical path differences (OPDs); (2) or simply by two parameters assuming a linear decline of the modulation efficiency with OPD, and a constant phase error. For the Ny-Alesund, Harestua and Lauder stations, the LIN- EFIT results were close to, and thus have been approximated by, the ideal ILS: there 15 is no loss of modulation efficiency vs. OPD and no phase error. At Izaña and Thule, the ILS was not ideal and the 40 parameters obtained from LINEFIT have been used to describe the ILS. At Harestua, the second option of parameters from LINEFIT was used and the phase error, which can lead to asymmetrical ILS but which was close p y to zero, was neglected. At Jungfraujoch, where cell measurements were not available 20 for early years, and at Wollongong for the Bomem spectra, it also has been taken into account that the ILS may not be ideal: the ILS distortions have been approximated by an empirical apodization function that represents only symmetrical distortions (Barret et al., 2002). In the case of an ideal instrument, the apodization function would be constant and equal to 1. In case of a non-ideal ILS, the parameters of the empirical 25 function are retrieved together with the VMR profiles, using the ideal ILS as the a priori value. A polynomial fit of order 2 and of order 4 has been used at Jungfraujoch and to zero, was neglected. At Jungfraujoch, where cell measurements were not available 20 for early years, and at Wollongong for the Bomem spectra, it also has been taken into account that the ILS may not be ideal: the ILS distortions have been approximated by an empirical apodization function that represents only symmetrical distortions (Barret et al., 2002). 2.2 FTIR retrieval strategy Except at Harestua, Kiruna and Izaña, the stations are using an a priori covariance matrix with diagonal elements constant with altitude corresponding to 10, 20 or 30 % variability, the largest variabilities taking place at the high latitude stations Ny-Alesund and Thule. At Harestua, the diagonal elements of Sa correspond to 11 % in the strato- 25 sphere, decreasing down to 6 % in the troposphere and to 5 % above 35 km. Except at Ny-Alesund, the SNR value is not the real one coming from each individual spectrum, but an effective SNR, that is used as a regularization parameter. It is chosen differ- ently from one station to another, together with the Sa matrix, in order to obtain stable 24629 ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | retrievals with reasonable DOFS. This effective SNR is smaller than the value derived from the inherent noise in the spectra, since the residuals in a spectral fit are not only coming from pure measurement noise but also from uncertainties in the model param- eters. At Kiruna and Izaña, the regularization is made using the Tikhonov L1 constraint (Tikhonov, 1963). 5 retrievals with reasonable DOFS. This effective SNR is smaller than the value derived from the inherent noise in the spectra, since the residuals in a spectral fit are not only coming from pure measurement noise but also from uncertainties in the model param- eters. At Kiruna and Izaña, the regularization is made using the Tikhonov L1 constraint ACPD g g (Tikhonov, 1963). 5 The observed absorption line shapes also depend on the instrument line shape (ILS) which is therefore needed in the forward models of the retrieval codes. At all stations, except Jungfraujoch and Wollongong for the Bomem spectra, the ILS has been re- trieved independently from HBr or N2O absorption measurements in a low-pressure y 2 gas cell with the LINEFIT code, as described in Hase et al. (1999). 2.3 Vertical information in FTIR retrievals 1), instead of 42 km in Vigouroux et al. (2008), the altitude above which the sensitivity is below 0.5. We still gain from 0.06 (Jungfraujoch) up to 0.23 (Lauder) DOFS in this 7 km wide range with poorer sensitivity. For Harestua, the chosen layer limits give a DOFS of only 0.9 and 0.75, in the ground 10 km and in the 29–49 km layers, respectively. 25 depending on the station. Therefore, in addition to total column trends, we provide 5 ozone trends in four independent partial column layers, corresponding to the vertical information. The layer limits have been chosen such that the DOFS is at least 1.0 in each associated partial column. The adopted layers are independent according to the resolution of the averaging kernels, as can be seen in Fig. 1, where the partial depending on the station. Therefore, in addition to total column trends, we provide 5 ozone trends in four independent partial column layers, corresponding to the vertical information. The layer limits have been chosen such that the DOFS is at least 1.0 in each associated partial column. The adopted layers are independent according to the resolution of the averaging kernels, as can be seen in Fig. 1, where the partial g g g p column averaging kernels of the four layers in the case of Jungfraujoch are shown. 10 Also shown is the sensitivity which is, at each altitude k, the sum of the elements of the corresponding averaging kernel P i Aki, and represents the fraction of the retrieval that comes from the measurement rather than from the a priori information. In the present work, small changes have been made in the partial column limits in comparison to column averaging kernels of the four layers in the case of Jungfraujoch are shown. 10 Also shown is the sensitivity which is, at each altitude k, the sum of the elements of the corresponding averaging kernel P i Aki, and represents the fraction of the retrieval that comes from the measurement rather than from the a priori information. In the present work, small changes have been made in the partial column limits in comparison to , g p p Vigouroux et al. (2008): we avoid the tropopause region at each station, in order to 15 have a better separation between the layer that we call the "tropospheric" layer, and the lower stratospheric layer. 2.2 FTIR retrieval strategy In the case of an ideal instrument, the apodization function would be to zero, was neglected. At Jungfraujoch, where cell measurements were not available 20 for early years, and at Wollongong for the Bomem spectra, it also has been taken into account that the ILS may not be ideal: the ILS distortions have been approximated by an empirical apodization function that represents only symmetrical distortions (Barret et al., 2002). In the case of an ideal instrument, the apodization function would be Discussion Paper | constant and equal to 1. In case of a non-ideal ILS, the parameters of the empirical 25 function are retrieved together with the VMR profiles, using the ideal ILS as the a priori value. A polynomial fit of order 2 and of order 4 has been used at Jungfraujoch and Wollongong, respectively. The phase error, close to zero, has been neglected. constant and equal to 1. In case of a non-ideal ILS, the parameters of the empirical 25 function are retrieved together with the VMR profiles, using the ideal ILS as the a priori value. A polynomial fit of order 2 and of order 4 has been used at Jungfraujoch and Wollongong, respectively. The phase error, close to zero, has been neglected. 24630 2.3 Vertical information in FTIR retrievals 2.3 Vertical information in FTIR retrievals Discussion Paper | Discussion Pape Discussion Paper | Discussion Paper | Discussion Paper ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD The vertical information contained in the FTIR retrievals can be characterized by the averaging kernel matrix A (Eq. 1), as described in detail in Vigouroux et al. (2008). It has been shown in this previous paper that the ozone retrievals provide 4–5 DOFS, The vertical information contained in the FTIR retrievals can be characterized by the averaging kernel matrix A (Eq. 1), as described in detail in Vigouroux et al. (2008). It has been shown in this previous paper that the ozone retrievals provide 4–5 DOFS, depending on the station. Therefore, in addition to total column trends, we provide 5 ozone trends in four independent partial column layers, corresponding to the vertical information. The layer limits have been chosen such that the DOFS is at least 1.0 in each associated partial column. The adopted layers are independent according to the resolution of the averaging kernels, as can be seen in Fig. 1, where the partial column averaging kernels of the four layers in the case of Jungfraujoch are shown. 10 Also shown is the sensitivity which is, at each altitude k, the sum of the elements of the corresponding averaging kernel P i Aki, and represents the fraction of the retrieval that comes from the measurement rather than from the a priori information. In the present work, small changes have been made in the partial column limits in comparison to Vigouroux et al. (2008): we avoid the tropopause region at each station, in order to 15 have a better separation between the layer that we call the "tropospheric" layer, and the lower stratospheric layer. Due to the high tropopause heights at Izaña (14.9 km) and Wollongong (13.8 km), compared to mid- and high-latitude stations (from 10.1 km at Ny-Alesund to 11.8 km at Jungfraujoch), we use different partial column limits for these two stations. The upper limit of the upper layer is here 49 km, the altitude above which 20 the sensitivity goes to zero (see Fig. 2.4 FTIR ozone time series Figure 2 displays the time series of ozone total columns at e Figure 2 displays the time series of ozone total columns at each ground-based FTIR Figure 2 displays the time series of ozone total columns at each ground-based FTIR station. Because we consider only solar absorption measurements, the time series at 10 Ny-Alesund, Thule, and Kiruna cover only the Mid-March–September, Late-February– Mid-October and Mid-January–Mid-November periods, respectively. The seasonal vari- ation is isolated in Fig. 3 which shows the monthly mean total columns over the periods of measurements. We clearly see the well-known seasonal cycle of ozone total column having a maximum in spring at all stations, and the higher amplitude of the seasonal 15 variation at higher latitudes (Brasseur and Solomon, 1984). station. Because we consider only solar absorption measurements, the time series at 10 Ny-Alesund, Thule, and Kiruna cover only the Mid-March–September, Late-February– Mid-October and Mid-January–Mid-November periods, respectively. The seasonal vari- ation is isolated in Fig. 3 which shows the monthly mean total columns over the periods of measurements. We clearly see the well-known seasonal cycle of ozone total column station. Because we consider only solar absorption measurements, the time series at 10 Ny-Alesund, Thule, and Kiruna cover only the Mid-March–September, Late-February– Mid-October and Mid-January–Mid-November periods, respectively. The seasonal vari- ation is isolated in Fig. 3 which shows the monthly mean total columns over the periods of measurements. We clearly see the well-known seasonal cycle of ozone total column y y having a maximum in spring at all stations, and the higher amplitude of the seasonal 15 variation at higher latitudes (Brasseur and Solomon, 1984). Figure 3 shows also the monthly means of the four partial columns defined in the previous section (Table 3). In the upper stratospheric layer, the ozone maximum occurs in summer (early summer at high latitudes shifting to late summer with decreasing lati- having a maximum in spring at all stations, and the higher amplitude of the seasonal 15 variation at higher latitudes (Brasseur and Solomon, 1984). Figure 3 shows also the monthly means of the four partial columns defined in the tude), in agreement with higher photo-chemical production of ozone during this season. 20 In the lower stratospheric layer, the ozone maximum is in late winter/early spring at all latitudes. 2.3 Vertical information in FTIR retrievals As obtained in the two previous papers, and not shown here, the smoothing error is the dominant random error source for the tropospheric and lower stratospheric layer, while the temperature dominates the random error budget for the middle and upper stratospheric layers, and ACPD for total columns. Also found in these two papers, and not repeated here, is the val- idation of the FTIR total and partial columns with correlative data (Dobson, Brewer, UV-VIs, ozonesondes, Lidar). 2.3 Vertical information in FTIR retrievals Due to the high tropopause heights at Izaña (14.9 km) and Wollongong (13.8 km), compared to mid- and high-latitude stations (from 10.1 km at Ny-Alesund to 11.8 km at Jungfraujoch), we use different partial column limits for these two stations. The upper limit of the upper layer is here 49 km, the altitude above which 20 the sensitivity goes to zero (see Fig. 1), instead of 42 km in Vigouroux et al. (2008), the altitude above which the sensitivity is below 0.5. We still gain from 0.06 (Jungfraujoch) up to 0.23 (Lauder) DOFS in this 7 km wide range with poorer sensitivity. For Harestua, the chosen layer limits give a DOFS of only 0.9 and 0.75, in the ground 10 km and in th 29 49 k l ti l | Discussion Paper | the 29–49 km layers, respectively. 25 We provide in Table 3, the partial column limits for each station. The detailed error budget for ozone FTIR retrievals has been described in Vigouroux et al. (2008) and more recently in García et al. (2012) for Izaña, and we just summarize in Table 3 the y , p y We provide in Table 3, the partial column limits for each station. The detailed error budget for ozone FTIR retrievals has been described in Vigouroux et al. (2008) and more recently in García et al. (2012) for Izaña, and we just summarize in Table 3 the 24631 Discussion Paper | Discussion Paper Discussion Paper | Discussion Paper | ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | total random uncertainties obtained for the present choice of layers. As obtained in the two previous papers, and not shown here, the smoothing error is the dominant random error source for the tropospheric and lower stratospheric layer, while the temperature dominates the random error budget for the middle and upper stratospheric layers, and for total columns. Also found in these two papers, and not repeated here, is the val- idation of the FTIR total and partial columns with correlative data (Dobson, Brewer, UV-VIs, ozonesondes, Lidar). total random uncertainties obtained for the present choice of layers. Multiple regression model ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 3 ACPD The ozone FTIR total and partial column trends in Vigouroux et al. (2008); WMO (2010); García et al. (2012) were calculated with a bootstrap re-sampling method, ap- plied to the daily means time series. In these studies, only the seasonal cycle and a linear trend were taken into account, the remaining natural ozone variability was then 5 an additional noise in the ozone trend determination. To reduce the uncertainties on the trends and to better understand what drives ozone variability and trends, we use in the present study a multiple linear regression (MLR) model. To reduce the auto-correlation The ozone FTIR total and partial column trends in Vigouroux et al. (2008); WMO (2010); García et al. (2012) were calculated with a bootstrap re-sampling method, ap- plied to the daily means time series. In these studies, only the seasonal cycle and a linear trend were taken into account, the remaining natural ozone variability was then 5 an additional noise in the ozone trend determination. To reduce the uncertainties on the trends and to better understand what drives ozone variability and trends, we use in the present study a multiple linear regression (MLR) model. To reduce the auto-correlation in the residuals, we use here the monthly means time series. 2.4 FTIR ozone time series The situation is more contrasted for the middle stratospheric layer: still late winter/early spring for Harestua, Jungfraujoch, Lauder and Wollongong, but the latter shows a second maximum in late summer, and a small amplitude of the seasonal cy- | Discussion Paper | p y cle. For the three higher latitude stations Ny-Alesund, Thule and Kiruna, the maximum 25 is still in spring, extending to May for the two latter stations. At Izaña, the maximum is in summer in the middle stratosphere. For the tropospheric column, we observe a max- imum in spring at all stations, but at Jungfraujoch it extends also in summer. 24632 24632 Discussion Paper | Discussion Pape Discussion Paper | Multiple regression model Multiple regression model The two most common explanatory variables found in the literature are the solar radio flux (F10.7 index) which represents the 11 year solar cycle (following e.g. 10 Newchurch et al., 2003; Randel and Wu, 2007), and the zonal winds measured at Sin- gapore at 30 and 10 hPa (following e.g. Brunner et al., 2006) which represent the quasi- biennial oscillation (QBO). The proxy used for the El Niño-Southern Oscillation is the Multivariate ENSO Index (MEI), following Randel et al. (2009). Different time-lags (from 0 to 4 months) between ENSO and ozone time series have been tested. The other dy- 15 namical proxies that have been explored are the tropopause pressure at each station (following e.g. Appenzeller et al., 2000), the equivalent latitude at three altitude levels around each station (ELL, ELM, ELU), the Arctic Oscillation (AO) or the Antarctic Os- cillation (AAO) indices depending on the station location (e.g. Appenzeller et al., 2000; Frossard et al., 2013), and the vertical component of the Eliassen-Palm flux (EPF) at 20 100 hPa averaged over 45 to 75◦north and south, as a proxy for the Brewer-Dobson circulation (e.g. Brunner et al., 2006). Those proxies are connected (Appenzeller et al., 2000; Weber et al., 2011), but we let the stepwise regression model choose the most adapted proxy for each station and partial column. Concerning the equivalent latitude, we did not construct an integrated equivalent proxy valuable for the whole ozone “inte- 25 grated” total column as in Wohltmann el al. (2005). Here, we simply use the equivalent latitude calculated from ERA Interim reanalysis (Dee et al., 2011) at three altitude lev- els corresponding approximately to the middle of our three stratospheric layers (ELL for the solar radio flux (F10.7 index) which represents the 11 year solar cycle (following e.g. 10 Newchurch et al., 2003; Randel and Wu, 2007), and the zonal winds measured at Sin- gapore at 30 and 10 hPa (following e.g. Brunner et al., 2006) which represent the quasi- biennial oscillation (QBO). The proxy used for the El Niño-Southern Oscillation is the Multivariate ENSO Index (MEI), following Randel et al. (2009). Different time-lags (from 0 to 4 months) between ENSO and ozone time series have been tested. The other dy- 15 namical proxies that have been explored are the tropopause pressure at each station (following e.g. Multiple regression model The following regression model is applied to the monthly means of ozone total and 10 partial column time series Y (t): Y (t) =A0 + A1 · cos(2πt/12) + A2 · sin(2πt/12)+ A3 · cos(4πt/12) + A4 · sin(4πt/12)+ A5 · t + n X k=6 Ak · Xk(t) + ϵ, (2) Y (t) =A0 + A1 · cos(2πt/12) + A2 · sin(2πt/12)+ A3 · cos(4πt/12) + A4 · sin(4πt/12)+ A5 · t + n X k=6 Ak · Xk(t) + ϵ, (2) Y (t) =A0 + A1 · cos(2πt/12) + A2 · sin(2πt/12)+ A3 · cos(4πt/12) + A4 · sin(4πt/12)+ A5 · t + n X k=6 Ak · Xk(t) + ϵ, (2) (2) 15 where the A1 to A4 parameters describes the ozone seasonal cycle, A5 is the annual trend, Xk are the explanatory variables (proxies time series) and Ak their respective coefficient, and ϵ represents the residuals. where the A1 to A4 parameters describes the ozone seasonal cycle, A5 is the annual trend, Xk are the explanatory variables (proxies time series) and Ak their respective coefficient, and ϵ represents the residuals. To select the final regression model, we have included several proxies, which rep- resent processes that are known to impact ozone, in a stepwise regression procedure 20 that keeps or rejects each proxy: the initial model (seasonal cycle and trend) is fitted first. Second, iteratively, if any proxies, not already in the model, have p values less than an entrance tolerance (0.05) i.e. if it is unlikely that they would have zero coeffi- cient if added to the model, then we add the one with the smallest p value. Otherwise, if any proxies in the model have p values greater than an exit tolerance (0.10), then 25 we remove the one with the largest p value and we repeat the whole process until no if any proxies in the model have p values greater than an exit tolerance (0.10), then 25 we remove the one with the largest p value and we repeat the whole process until no if any proxies in the model have p values greater than an exit tolerance (0.10), then 25 we remove the one with the largest p value and we repeat the whole process until no 24633 Discussion Paper | Discussion Paper ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Multiple regression model Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | single step improves the model. Hence, the final set of parameters can vary with the station and with the partial columns concerned. In this paper, a proxy is called “non- significant” when it has not been retained by the stepwise procedure. This choice of not using a fixed model for all stations and partial columns avoids to over-fit the data, and is justified by the large latitudinal range of the stations (e.g., the VPSC or ENSO proxies will not impact the stations in the same way), and by the different processes driving ozone variability at different altitudes. single step improves the model. Hence, the final set of parameters can vary with the station and with the partial columns concerned. In this paper, a proxy is called “non- significant” when it has not been retained by the stepwise procedure. This choice of not using a fixed model for all stations and partial columns avoids to over-fit the data, and is justified by the large latitudinal range of the stations (e.g., the VPSC or ENSO proxies will not impact the stations in the same way), and by the different processes driving ozone variability at different altitudes. ACPD 5 The proxies that have been tested in the stepwise regression procedure are summa- rized in Table 4. The two most common explanatory variables found in the literature are The proxies that have been tested in the stepwise regression procedure are summa- rized in Table 4. The two most common explanatory variables found in the literature are the solar radio flux (F10.7 index) which represents the 11 year solar cycle (following e.g. 10 Newchurch et al., 2003; Randel and Wu, 2007), and the zonal winds measured at Sin- gapore at 30 and 10 hPa (following e.g. Brunner et al., 2006) which represent the quasi- biennial oscillation (QBO). The proxy used for the El Niño-Southern Oscillation is the Multivariate ENSO Index (MEI), following Randel et al. (2009). Different time-lags (from The proxies that have been tested in the stepwise regression procedure are summa- rized in Table 4. Multiple regression model To account for the cumulative effect over months of the EPF and the VPSC*EESC proxies on ozone, we have followed the approach of Brunner et al. (2006) (see their Eq. 4). 0 Lastly, the volume of polar stratospheric clouds (VPSC) is used as a proxy for polar ozone loss (e.g. Brunner et al., 2006). The VPSC proxy has been multiplied Lastly, the volume of polar stratospheric clouds (VPSC) is used as a proxy for polar ozone loss (e.g. Brunner et al., 2006). The VPSC proxy has been multiplied by the effective equivalent stratospheric chlorine (EESC) time series calculated with 5 a mean age of air of 5.5 years, in order to take into account the time for the ozone depleting substances to reach the poles (http://acdb-ext.gsfc.nasa.gov/Data_services/ automailer/index.html). To account for the cumulative effect over months of the EPF and the VPSC*EESC proxies on ozone, we have followed the approach of Brunner et al. (2006) (see their Eq. 4). 10 et al. (2006) (see their Eq. 4). 10 For the two QBO proxies (30 and 10 hPa), if retained in the stepwise procedure, four seasonal parameters can be added to the model. The Ak · Xk(t) term of Eq. (2) is then replaced by: ( ) ( q ) For the two QBO proxies (30 and 10 hPa), if retained in the stepwise procedure, four seasonal parameters can be added to the model. The Ak · Xk(t) term of Eq. (2) is then replaced by: (Ak + Ak+1 · cos(2πt/12) + Ak+2 · sin(2πt/12) (3) + Ak+3 · cos(4πt/12) + Ak+4 · sin(4πt/12)) · Xk(t). k + Ak+1 · cos(2πt/12) + Ak+2 · sin(2πt/12) (3) Ak+3 · cos(4πt/12) + Ak+4 · sin(4πt/12)) · Xk(t). (Ak + Ak+1 · cos(2πt/12) + Ak+2 · sin(2πt/12) + Ak+3 · cos(4πt/12) + Ak+4 · sin(4πt/12)) · Xk(t). (3) 15 Depending on the station and on the layer, none, one or both of the two proxies QBO30 and QBO10 will be retained in the model, with or without their additional sea- sonal parameters. We will call from here “QBO contribution”, the sum of all possible contributions of QBO30 and QBO10. 20 contributions of QBO30 and QBO10. Multiple regression model Appenzeller et al., 2000), the equivalent latitude at three altitude levels around each station (ELL, ELM, ELU), the Arctic Oscillation (AO) or the Antarctic Os- cillation (AAO) indices depending on the station location (e.g. Appenzeller et al., 2000; Frossard et al., 2013), and the vertical component of the Eliassen-Palm flux (EPF) at 20 100 hPa averaged over 45 to 75◦north and south, as a proxy for the Brewer-Dobson circulation (e.g. Brunner et al., 2006). Those proxies are connected (Appenzeller et al., 2000; Weber et al., 2011), but we let the stepwise regression model choose the most adapted proxy for each station and partial column. Concerning the equivalent latitude, Discussion Paper | we did not construct an integrated equivalent proxy valuable for the whole ozone “inte- 25 grated” total column as in Wohltmann el al. (2005). Here, we simply use the equivalent latitude calculated from ERA Interim reanalysis (Dee et al., 2011) at three altitude lev- els corresponding approximately to the middle of our three stratospheric layers (ELL for 24634 Discussion Paper | Discussion Pa LowS, ELM for MidS, and ELU for UppS), namely at 370, 550, and 950 K, respectively, for all stations except Izaña and Wollongong (460, 700, and 1040 K, respectively). ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | LowS, ELM for MidS, and ELU for UppS), namely at 370, 550, and 950 K, respectively, for all stations except Izaña and Wollongong (460, 700, and 1040 K, respectively). LowS, ELM for MidS, and ELU for UppS), namely at 370, 550, and 950 K, respectively, for all stations except Izaña and Wollongong (460, 700, and 1040 K, respectively). ACPD g g Lastly, the volume of polar stratospheric clouds (VPSC) is used as a proxy for polar ozone loss (e.g. Brunner et al., 2006). The VPSC proxy has been multiplied by the effective equivalent stratospheric chlorine (EESC) time series calculated with 5 a mean age of air of 5.5 years, in order to take into account the time for the ozone depleting substances to reach the poles (http://acdb-ext.gsfc.nasa.gov/Data_services/ automailer/index.html). Multiple regression model alternative to the simple linear trend for these stations. However, we preferred to adopt the same approach for all the stations. Probably, when the FTIR record will be longer, one would be able to distinguish between the EESC impact on ozone and a possible additional trend due to process(es) that are not represented in the model. ACPD To account for the auto-correlation in the residuals, a Cochrane-Orcutt transformation is applied (Cochrane and Orcutt, 1949). This gives more reliable confidence intervals for the regression parameters. Multiple regression model 0 Since the time series involved in the present study start at earliest in 1995, we do not include two commonly used explanatory variables: the aerosol optical thickness needed to represent the effect on ozone of the large volcanic eruptions of El Chichón (1982) and Mount Pinatubo (1991), and the EESC proxy which can be used as di- rect proxy for the halogen loading of the stratosphere instead of the piecewise linear 25 Since the time series involved in the present study start at earliest in 1995, we do not include two commonly used explanatory variables: the aerosol optical thickness needed to represent the effect on ozone of the large volcanic eruptions of El Chichón (1982) and Mount Pinatubo (1991), and the EESC proxy which can be used as di- rect proxy for the halogen loading of the stratosphere instead of the piecewise linear 25 trend (PWLT) with a turnaround in 1996/1997 often used in time series starting well before this turnaround point (WMO, 2010). Our linear trend estimates are therefore better comparable to the studies which use the PWLT method. At polar stations, the turnaround is occurring a few years later, so that the use of the EESC proxy could be an 24635 rect proxy for the halogen loading of the stratosphere instead of the piecewise linear 25 trend (PWLT) with a turnaround in 1996/1997 often used in time series starting well before this turnaround point (WMO, 2010). Our linear trend estimates are therefore better comparable to the studies which use the PWLT method. At polar stations, the turnaround is occurring a few years later, so that the use of the EESC proxy could be an 24635 24635 Discussion Paper | Discussion Paper Discussion Paper | Discussion Paper | ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | alternative to the simple linear trend for these stations. However, we preferred to adopt the same approach for all the stations. Probably, when the FTIR record will be longer, one would be able to distinguish between the EESC impact on ozone and a possible additional trend due to process(es) that are not represented in the model. 4.1.1 Tropospheric (Trop) columns 5 At the three other stations, 20 this VPSC impact was not found to be significant, and the main driver of tropospheric variability is found to be the tropopause pressure TP (Fig. 4). stratospheric ozone variability on the tropospheric columns. At the three other stations, 20 this VPSC impact was not found to be significant, and the main driver of tropospheric variability is found to be the tropopause pressure TP (Fig. 4). 4 Results and discussion In Fig. 4, we show the individual contribution Cfrac of each pr In Fig. 4, we show the individual contribution Cfrac of each proxy retained by the step- wise procedure to the coefficient of determination R2 = PCfrac, for each station and 10 partial column. The individual contribution Cfrac of a proxy is the product of the stan- dardized regression coefficient of this proxy with the correlation coefficient between the proxy and the observations (Scherrer, 1984). In Fig. 4, the seasonal parameters con- tribution (A1 to A4 in Eq. 2), which gives in most cases the very dominant part of the wise procedure to the coefficient of determination R2 = PCfrac, for each station and 10 partial column. The individual contribution Cfrac of a proxy is the product of the stan- dardized regression coefficient of this proxy with the correlation coefficient between the proxy and the observations (Scherrer, 1984). In Fig. 4, the seasonal parameters con- tribution (A1 to A4 in Eq. 2), which gives in most cases the very dominant part of the 1 4 explained variability, is not shown for better clarity of the other proxies contribution. But 15 we give it for completeness in Table 5, together with R2. In the following discussion, we will highlight some selected features which are visible in the ozone time series and which can be attributed to a specific proxy. The final MLR model is the sum of all the significant proxies, and therefore the effect of a specific proxy can be visible in the plots in some years, but masked in other years. 20 In Table 6, we give the annual ozone trend at each station for each layer obtained with the stepwise multiple linear regression model. The uncertainties on the trends correspond to the 95 % percent confidence interval. A trend is considered significant if it is larger than the uncertainty. | Discussion Paper | y , y In Table 6, we give the annual ozone trend at each station for each layer obtained with the stepwise multiple linear regression model. The uncertainties on the trends correspond to the 95 % percent confidence interval. A trend is considered significant if it is larger than the uncertainty. 24636 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper High latitude stations Discussion Paper ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. 4 Results and discussion Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 4.1 ACPD In addition to the three Arctic stations Ny-Alesund, Thule and Kiruna, we will consider Harestua (60◦N) as a high latitude station since, in terms of trends, Harestua appears to behave similarly to the Arctic stations. 4.1.1 Tropospheric (Trop) columns 5 In the troposphere, the high latitude stations, except Kiruna, show negative significant ozone trends (Table 6). The spatial and temporal variability in the Arctic and the differ- ent sampling at the stations Thule/Ny-Alesund due to polar night (see Fig. 2) makes it difficult to compare the trend results. At Harestua, the negative trend is occuring in the 1995–2007 period. On the contrary, we see in Fig. 5 that at Ny-Alesund the nega- 10 tive trend occurs in the second part of the period (2004–2012). We see some similar features with the work of Hess and Zbinden (2013) who provide trends at 500 hPa for Northern Europe from ozonesondes: we also observe more tropospheric ozone in 1998 and 1999 and less in 2005 (their Fig. 1 compared to our middle panel of Fig. 5 the 1995–2007 period. On the contrary, we see in Fig. 5 that at Ny-Alesund the nega- 10 tive trend occurs in the second part of the period (2004–2012). We see some similar features with the work of Hess and Zbinden (2013) who provide trends at 500 hPa for Northern Europe from ozonesondes: we also observe more tropospheric ozone in 1998 and 1999 and less in 2005 (their Fig. 1 compared to our middle panel of Fig. 5 Discussion Paper | Discussion Paper | ( g p p g where the seasonal signal is removed for emphasizing the interannual variability). We 15 have added in Fig. 5 the VPSC signal, i.e. the VPSC proxy time series multiplied by the corresponding parameter obtained in the MLR process (Ak · Xk(t) in Eq. 2). We see that the discussed features (1998, 1999, 2005), but also e.g. the lower ozone val- ues in 2011, can be explained by the VPSC proxy, therefore by the influence of lower where the seasonal signal is removed for emphasizing the interannual variability). We 15 have added in Fig. 5 the VPSC signal, i.e. the VPSC proxy time series multiplied by the corresponding parameter obtained in the MLR process (Ak · Xk(t) in Eq. 2). We see that the discussed features (1998, 1999, 2005), but also e.g. the lower ozone val- ues in 2011, can be explained by the VPSC proxy, therefore by the influence of lower per | Discussion Paper | stratospheric ozone variability on the tropospheric columns. 4.1.3 Middle stratospheric (MidS) columns The results are mixed for the middle stratospheric layers, a The results are mixed for the middle stratospheric layers, as noticed previously for the seasonal cycles. The trend is significantly negative at Ny-Alesund and non-significant 10 at Thule. The trend is non-significant at Kiruna, and significantly positive at Harestua. The EPF proxy explains about 25 % of the variability at Ny-Alesund and Thule, and about 5 % at Kiruna (Fig. 4). This is illustrated in Fig. 6 for Ny-Alesund and Thule, where we see nicely the same features at both stations in the middle stratospheric columns (e.g. higher columns in 2009, 2010; lower columns in 2011), associated with 15 the EPF time series. 4.1.2 Lower stratospheric (LowS) columns The VPSC proxy is found significant at the four high latitude stations for the lower stratospheric columns, being the main driver of ozone variability after TP (Fig. 4). We 25 24637 Discussion Paper | Discussion Paper Discussion Paper | Discussion Paper | give the example of Kiruna in Fig. 5, where the effect of large amount of VPSC in 1996, 2000, 2003, 2005, 2011 is clearly visible in both monthly means and deseasonalized time series, and we give in addition the TP signal in the bottom panel. It can be seen that the TP signal in 2005 also contributed to even lower ozone that particular year. In the lower stratosphere, at all high latitude stations, except Thule, we observe significant negative trends (Table 6). At Thule, the shorter time period associated with the high variability of this layer at high latitude gives a large uncertainty on the trend. ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD 4.1.5 Total columns Finally, we observe that the total column ozone trends are small and non-significant at all high latitude stations, except at Ny-Alesund (−3.0 ± 1.5 % decade−1). The neg- ative trend at Ny-Alesund occurs in the 2003–2012 period, as for the lowest altitude layers. At all stations, the dominant contributions to the total column variability are the Finally, we observe that the total column ozone trends are small and non-significant at all high latitude stations, except at Ny-Alesund (−3.0 ± 1.5 % decade−1). The neg- ative trend at Ny-Alesund occurs in the 2003–2012 period, as for the lowest altitude layers. At all stations, the dominant contributions to the total column variability are the ussion Paper | Discussion Paper | TP, the VPSC, the EL at 950 K, and, except at Harestua, the EPF proxies. We see 20 nicely in Table 5, how well the proxies explained the additional variability at the Arctic stations, e.g. at Ny-Alesund R2 = 0.95, compared to the contribution of the seasonal cycle Cseas = 0.68. TP, the VPSC, the EL at 950 K, and, except at Harestua, the EPF proxies. We see 20 nicely in Table 5, how well the proxies explained the additional variability at the Arctic stations, e.g. at Ny-Alesund R2 = 0.95, compared to the contribution of the seasonal cycle Cseas = 0.68. TP, the VPSC, the EL at 950 K, and, except at Harestua, the EPF proxies. We see 20 nicely in Table 5, how well the proxies explained the additional variability at the Arctic stations, e.g. at Ny-Alesund R2 = 0.95, compared to the contribution of the seasonal cycle Cseas = 0.68. | Discussion Paper | 4.1.4 Upper stratospheric (UppS) columns 7 as an illustration turns out to be non-significant after the Cochrane- ACPD solar cycle being non-significant at Kiruna, Harestua and Ny-Alesund, a trend, that 5 would be due to it, would not taken into account in the MLR and would appear in the residual “Trend” parameter. The solar cycle might be found non-significant because the expected decrease of ozone during the declining phase of the solar cycle (2002–2009) is not observed. This could be a sign that this decrease is compensated by a positive solar cycle being non-significant at Kiruna, Harestua and Ny-Alesund, a trend, that 5 would be due to it, would not taken into account in the MLR and would appear in the residual “Trend” parameter. The solar cycle might be found non-significant because the expected decrease of ozone during the declining phase of the solar cycle (2002–2009) is not observed. This could be a sign that this decrease is compensated by a positive g y linear trend, which could be due to the declining EESCs, but also to the increase of 10 greenhouse gases (WMO, 2010). More years are needed to understand unequivocally the increase in 1995–2003, followed by a leveling off, and distinguish between the ozone responses due to solar cycle, EESCs and possible proxies not included in the present study. linear trend, which could be due to the declining EESCs, but also to the increase of 10 greenhouse gases (WMO, 2010). More years are needed to understand unequivocally the increase in 1995–2003, followed by a leveling off, and distinguish between the ozone responses due to solar cycle, EESCs and possible proxies not included in the present study. 4.1.4 Upper stratospheric (UppS) columns In the upper stratosphere, the three stations with similar time periods show a significant positive trend. In the three cases, the increase in ozone partial columns occurs in the p p 1995–2003 period, after which a leveling offis observed (Fig. 7). If we run the MLR 20 model on the same time period as Thule (October 1999–2012), all the stations show non-significant trends. Since the EESCs were still increasing until about 2000 at polar regions (WMO, 2010), the significant positive trends obtained at high latitude stations in the upper stratosphere cannot be explained by the effect of Montréal Protocol on 1995–2003 period, after which a leveling offis observed (Fig. 7). If we run the MLR 20 model on the same time period as Thule (October 1999–2012), all the stations show non-significant trends. Since the EESCs were still increasing until about 2000 at polar regions (WMO, 2010), the significant positive trends obtained at high latitude stations in the upper stratosphere cannot be explained by the effect of Montréal Protocol on 20 ozone depleting substances. At present we do not have an explanation for this increase 25 in ozone during the 1995–2003 period. The 11 year solar cycle might contribute to it, ozone depleting substances. At present we do not have an explanation for this increase 25 in ozone during the 1995–2003 period. The 11 year solar cycle might contribute to it, 24638 Discussion Paper | Discussion Paper Discussion Paper | Discussion Paper | ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | since the increase in solar activity from 1996 to its maximum in 2001–2002 is in phase with the ozone increase during the same period. The solar cycle signal at Ny-Alesund shown in Fig. 7 as an illustration turns out to be non-significant after the Cochrane- since the increase in solar activity from 1996 to its maximum in 2001–2002 is in phase with the ozone increase during the same period. The solar cycle signal at Ny-Alesund shown in Fig. 4.2.1 Tropospheric (Trop) columns At Lauder at present only a short time period (2001–2012) is available Paper | Discussion Paper | for trend studies, and we hope to have more clarification on this subject with more 20 years of data. However, if we remove the solar cycle proxy from the MLR model, we still obtain a significant trend of +5.0 ± 4.4 % decade−1. 4.2.1 Tropospheric (Trop) columns Discussion Paper | Discussion Paper | D Discussion Paper ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD The tropospheric trends are non-significant at Jungfraujoch, Izaña and Wollongong, and significantly positive at Lauder. The trend at Jungfraujoch is −2.5±2.7 % decade−1, but we see in Fig. 8 that the tropospheric columns are increasing up to 1999 and then show a linear decrease, in agreement with aircraft and surface alpine sites in the 5 study of Logan et al. (2012). If we limit our time period to the 1998–2008 period as in Logan et al. (2012), we also find a significant negative trend (−6.3 ± 4.9 % decade−1). But this is largely due to the high ozone values 1998–1999, and for the period 2000– 2012 we obtain still a non-significant trend of −2.9 ± 3.4 % decade−1. At Izaña, the then show a linear decrease, in agreement with aircraft and surface alpine sites in the 5 study of Logan et al. (2012). If we limit our time period to the 1998–2008 period as in Logan et al. (2012), we also find a significant negative trend (−6.3 ± 4.9 % decade−1). But this is largely due to the high ozone values 1998–1999, and for the period 2000– 2012 we obtain still a non-significant trend of −2.9 ± 3.4 % decade−1. At Izaña, the tropospheric trends derived from ozonesondes were found non-significant in García 10 et al. (2012), in agreement with our study, but the uncertainties were large. The situation is more mixed in the Southern Hemisphere: the tropospheric trend at Wollongong is not significant while it is significantly positive at Lauder (+7.7±3.5 % decade−1). The trend at Lauder is in agreement with the study of Oltmans et al. (2013) who obtain about 1 +5 % decade−1 in the lower and middle troposphere with ozonesondes measurements 15 at Lauder. We find a significant positive impact of the solar cycle at Lauder and it is clearly seen in Fig. 8. This is not in agreement with Chandra et al. (1999), in which the solar cycle shows a strong but negative impact on tropospheric columns for non- polluted region. 4.2 Mid-latitude and subtropical stations 4.2 Mid-latitude and subtropical stations We have two mid-latitude stations in this study (Jungfraujoch, 47◦N and Lauder, 45◦S), 25 and two subtropical stations (Izaña, 28◦N and Wollongong, 34◦S). We have two mid-latitude stations in this study (Jungfraujoch, 47◦N and Lauder, 45◦S), 25 and two subtropical stations (Izaña, 28◦N and Wollongong, 34◦S). We have two mid-latitude stations in this study (Jungfraujoch, 47◦N and Lauder, 45◦S), 25 and two subtropical stations (Izaña, 28◦N and Wollongong, 34◦S). 24639 Discussion Paper | Discussion Paper 4.2.2 Lower stratospheric (LowS) columns he trends in the lower stratosphere are non-significant at Jung The trends in the lower stratosphere are non-significant at Jungfraujoch, Izaña and Lauder, and significantly positive at Wollongong. The dominant proxy is TP for all sta- 25 tions. At the Jungfraujoch station, the VPSC proxy, which in the case of Jungfraujoch corresponds to the transport of polar ozone loss to mid-latitudes, explains about 8 % of 24640 24640 Discussion Paper | Discussion Paper ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | the variability (Fig. 4). The VPSC proxy is non-significant at the southern hemispheric station Lauder, in agreement with more stable and isolated vortex in the Antarctic com- pared to the Arctic. The Arctic Oscillation (AO) proxy is found significant at Jungfraujoch while the corresponding AAO proxy is non-significant at Lauder. the variability (Fig. 4). The VPSC proxy is non-significant at the southern hemispheric station Lauder, in agreement with more stable and isolated vortex in the Antarctic com- pared to the Arctic. The Arctic Oscillation (AO) proxy is found significant at Jungfraujoch while the corresponding AAO proxy is non-significant at Lauder. ACPD g y g We show the time series of the lower stratospheric columns at Jungfraujoch in Fig. 9 5 together with the AO and QBO signals. We see that in 2010 ozone shows larger val- ues, and that this is explained by the combination of a very negative AO index (the corresponding parameter in the MLR is negative and gives the positive signal in 2010 shown in Fig. 9) and easterly phase of the QBO. This is in agreement with Nair et al. We show the time series of the lower stratospheric columns at Jungfraujoch in Fig. 9 5 together with the AO and QBO signals. We see that in 2010 ozone shows larger val- ues, and that this is explained by the combination of a very negative AO index (the corresponding parameter in the MLR is negative and gives the positive signal in 2010 shown in Fig. 9) and easterly phase of the QBO. This is in agreement with Nair et al. 4.2.2 Lower stratospheric (LowS) columns g ) y p g (2013), who applied a MLR model to the mean of ozone anomalies at Observatoire de 10 Haute-Provence (OHP) from different instruments (Lidar, ozonesondes and satellites). However, we did not find a significant contribution from the EPF proxy, which according to Nair et al. (2013) also contributed to the high ozone values in 2010. We can state that our vertical and total column ozone trends are in agreement with the Nair et al. (2013) g results when taking the error bars into account, but the latter study found significant 15 positive trends at OHP while our trends at Jungfraujoch are all non-significant. As expected, the QBO contribution to ozone variability is more important at the sub- results when taking the error bars into account, but the latter study found significant 15 positive trends at OHP while our trends at Jungfraujoch are all non-significant. As expected, the QBO contribution to ozone variability is more important at the sub- tropical station Izaña, which is also the only station where the ENSO proxy was found to make a significant, but small, contribution to the variability (Fig. 4). We illustrate the Discussion Paper | Discussion Paper | QBO effect at Izaña in Fig. 9, for total columns. The cause of the significant positive 20 trend at Wollongong is not fully explained at present. A part of it is due to a small neg- ative trend in the ELL proxy. If we remove this proxy from the MLR model, we observe a non-significant positive trend of +2.4 ± 2.8 % decade−1. QBO effect at Izaña in Fig. 9, for total columns. The cause of the significant positive 20 trend at Wollongong is not fully explained at present. A part of it is due to a small neg- ative trend in the ELL proxy. If we remove this proxy from the MLR model, we observe a non-significant positive trend of +2.4 ± 2.8 % decade−1. aper | Discussion Paper | 4.2.3 Middle stratospheric (MidS) columns Pinatubo in 1991, and the authors find robust solar cycle signals only in the middle and upper stratosphere. In the upper stratospheric layer at Wollongong, the response to the solar et al., 2014) at about 25 km. However, the recent work of Chiodo et al. (2014) shows 5 that the apparent solar cycle signal in the tropical lower stratosphere for the period 1960–2004 is due to the two volcanic eruptions El Chichón in 1982 and Mt. Pinatubo in 1991, and the authors find robust solar cycle signals only in the middle and upper stratosphere. In the upper stratospheric layer at Wollongong, the response to the solar cycle is indeed also significant and is about 2.5 % between solar minimum and solar 10 maximum which is in agreement with previous studies (WMO, 2010). At Izaña, the so- lar contribution is found negative in the 23–32 km layer, which seems doubtful. Again, this concerned one of the shortest time series of the study (1999–2012), and could be corrected with future measurements. cycle is indeed also significant and is about 2.5 % between solar minimum and solar 10 maximum which is in agreement with previous studies (WMO, 2010). At Izaña, the so- lar contribution is found negative in the 23–32 km layer, which seems doubtful. Again, this concerned one of the shortest time series of the study (1999–2012), and could be corrected with future measurements. 4.2.3 Middle stratospheric (MidS) columns The situation for the middle stratosphere is very similar to that of the lower stratosphere: 25 all trends are found non-significant except at Wollongong where it is positive. It is in this 23–32 km layer for subtropical stations that the solar cycle shows the most important contribution (Fig. 4). This is not what has been reported in Randel and Wu (2007) 24641 Discussion Paper | Discussion Paper ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | and Tourpali et al. (2007), where the ozone response to solar cycle was maximum in the tropical lower and upper stratosphere, and minimum in the middle stratosphere. At Wollongong, the middle stratospheric ozone response is about 6 % between solar min- imum and solar maximum (see Fig. 9) while values of 1 % have been reported (Sioris and Tourpali et al. (2007), where the ozone response to solar cycle was maximum in the tropical lower and upper stratosphere, and minimum in the middle stratosphere. At Wollongong, the middle stratospheric ozone response is about 6 % between solar min- imum and solar maximum (see Fig. 9) while values of 1 % have been reported (Sioris ACPD ( g ) p ( et al., 2014) at about 25 km. However, the recent work of Chiodo et al. (2014) shows 5 that the apparent solar cycle signal in the tropical lower stratosphere for the period 1960–2004 is due to the two volcanic eruptions El Chichón in 1982 and Mt. Pinatubo in 1991, and the authors find robust solar cycle signals only in the middle and upper stratosphere. In the upper stratospheric layer at Wollongong, the response to the solar l i i d d l i ifi t d i b t 2 5 % b t l i i d l et al., 2014) at about 25 km. However, the recent work of Chiodo et al. (2014) shows 5 that the apparent solar cycle signal in the tropical lower stratosphere for the period 1960–2004 is due to the two volcanic eruptions El Chichón in 1982 and Mt. 4.2.4 Upper stratospheric (UppS) columns 15 4.2.4 Upper stratospheric (UppS) columns 15 The trends in the upper stratospheric layer are all positive in these latitudes, but significant only at Lauder (+2.8 ± 2.4 % decade−1). Our trend at Jungfraujoch station (+0.9 ± 1.0 % decade−1) corresponds well to the observed trend (+1.5 % decade−1) at OHP in Nair et al. (2013) in the 31–39 km range, although it is found significant in this 2 4.2.4 Upper stratospheric (UppS) columns 15 The trends in the upper stratospheric layer are all positive in these latitudes, but significant only at Lauder (+2.8 ± 2.4 % decade−1). Our trend at Jungfraujoch station (+0.9 ± 1.0 % decade−1) corresponds well to the observed trend (+1.5 % decade−1) at OHP in Nair et al. (2013) in the 31–39 km range, although it is found significant in this 2 g g g latter study. The MLR model explains 93 % of the variability at Jungfraujoch (R2 = 0.93), 20 namely 77 % of the variability comes from the seasonality and the remaining 16 % from the proxies, mainly the ELU and QBO (see Fig. 4). At Lauder, the trend in the 30– 40 km range from Lidar measurements is also found significantly positive for the period 2000–2012 with trend values (+2–3 % decade−1) similar to FTIR (W. Steinbrecht, per- sonal communication, 2013). 25 latter study. The MLR model explains 93 % of the variability at Jungfraujoch (R2 = 0.93), 20 namely 77 % of the variability comes from the seasonality and the remaining 16 % from the proxies, mainly the ELU and QBO (see Fig. 4). At Lauder, the trend in the 30– 40 km range from Lidar measurements is also found significantly positive for the period 2000–2012 with trend values (+2–3 % decade−1) similar to FTIR (W. Steinbrecht, per- sonal communication, 2013). 25 | Discussion Paper | 24642 24642 Discussion Paper | Discussion Paper Discussion Paper 4.2.5 Total columns ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 5 Conclusions We have exploited the time series of ozone total and partial columns (Trop, LowS, MidS, UppS) at 8 NDACC FTIR stations (Ny-Alesund, 79◦N; Thule, 77◦N; Kiruna, We have exploited the time series of ozone total and partial columns (Trop, LowS, MidS, UppS) at 8 NDACC FTIR stations (Ny-Alesund, 79◦N; Thule, 77◦N; Kiruna, 68◦N; Harestua, 60◦N; Jungfraujoch, 47◦N; Izaña, 28◦N; Wollongong, 34◦S; Lauder, 10 45◦S) to derive vertically resolved trends, using a MLR model including the main prox- ies well-known for impacting the ozone variability. 68◦N; Harestua, 60◦N; Jungfraujoch, 47◦N; Izaña, 28◦N; Wollongong, 34◦S; Lauder, 10 45◦S) to derive vertically resolved trends, using a MLR model including the main prox- ies well-known for impacting the ozone variability. 68◦N; Harestua, 60◦N; Jungfraujoch, 47◦N; Izaña, 28◦N; Wollongong, 34◦S; Lauder, 10 45◦S) to derive vertically resolved trends, using a MLR model including the main prox- ies well-known for impacting the ozone variability. 10 ) y , g g p ies well-known for impacting the ozone variability. After the seasonal variation, the TP proxy is the dominant driver of ozone variability at all stations, mainly for the troposphere, lower stratosphere and total columns, while After the seasonal variation, the TP proxy is the dominant driver of ozone variability at all stations, mainly for the troposphere, lower stratosphere and total columns, while y the EL proxy is an important contibutor to the middle and upper stratosphere, as well 15 as to the total column variabilities. At the highest latitude stations (68 to 79◦N), the EPF proxy contributes substantially to the middle stratospheric and total column vari- abilities. The VPSC proxy for polar ozone loss contributes to the lower stratosphere and total columns variabilities at the Arctic stations, but also at Jungfraujoch while is the EL proxy is an important contibutor to the middle and upper stratosphere, as well 15 as to the total column variabilities. At the highest latitude stations (68 to 79◦N), the EPF proxy contributes substantially to the middle stratospheric and total column vari- abilities. The VPSC proxy for polar ozone loss contributes to the lower stratosphere and total columns variabilities at the Arctic stations, but also at Jungfraujoch while is Paper | Discussion Paper | it non-significant at the southern hemispheric station Lauder. At the mid-latitude and 20 subtropical stations, the QBO proxy is a substantial contributor to ozone variability, es- pecially at the lowest latitude station, Izaña. 4.2.5 Total columns 24643 Discussion Paper | Discussion Paper Discussion Paper | Discussion Paper Discussion Paper ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Our non-significant trends at Jungfraujoch, Izaña and Lauder, and posi- tive trend at Wollongong are also in agreement with the recent study of Coldewey-Egbers et al. (2014), which provides trends using a similar period (1995– 2013) of merged satellite data sets. For Wollongong, since the total column positive trend is due to the ozone trends in the lower and middle stratosphere, it cannot be attributed unambiguously to the EESCs decline. ACPD 4.2.5 Total columns ACPD The total column trends are non-significant at the mid-latitude stations (−0.4 ± 1.2 % decade−1 or −1.4 ± 3.8 DU decade−1 at Jungfraujoch, −0.3 ± 1.8 % decade−1 or −1.1 ± 5.9 DU decade−1 at Lauder), non-significant at Izaña 1 1 (+0.5 ± 1.2 % decade−1 or +1.4 ± 3.6 DU decade−1), and significantly positive at Wol- 5 longong (+1.9±1.1 % decade−1 or +5.8±3.5 DU decade−1). The total column trend at Jungfraujoch is in agreement within error bars with the result of Nair et al. (2013) at OHP when they use the PWLT method (+5.5 ± 3.3 DU decade−1), but again the trend at OHP is found significantly positive. When the EESC proxy is used in their study a trend of +4.2±0.8 DU decade−1 is found. The same behaviour is seen more globally 10 in a recent study using merged satellite data from 1979 to 2012 (Chehade et al., 2014): for the latitude of Jungfraujoch, the trends are about +3–4 DU decade−1 for the 1997–2012 period, and non-significant if the PWLT method is used, while significant when the EESC proxy is used, which decreases the uncertainty on the trends. It seems p y y that at Jungfraujoch, our time series is still too short to observe this positive trend. 15 At the latitude of Izaña, the merged satellite data set shows a +3–4 DU decade−1 for the 1997–2012 period, with the more recent SBUV/SBUV-2 MOD v8.6, non-significant using the PWLT (in agreement with our study) and significant using the EESC proxy. Since our time series start at best in 1995, the EESC proxy is not really “separable” from a linear trend study at our mid-latitude and subtropical stations. When more years 20 of data will become available, the same sensitivity study (PWLT vs EESC) could be tested at least for polar stations where the turnaround point is expected around 2000. It is also interesting to note that, using the PWLT method, at the latitude of Wollon- gong, Chehade et al. (2014) found a positive significant trend of about +3 DU decade−1, 1 Discussion Paper | while at the latitude of Lauder the trend is decreased to about +1 DU decade−1 (non- 25 significant) in good agreement with what FTIR observed. When they use the EESC proxy, the trend is increasing with latitude, so that at the Lauder latitude, it reaches about 4–5 DU decade−1. 5 Conclusions The AO/AAO and ENSO proxies are sig- nificant only at Jungfraujoch and Izaña, respectively. At Wollongong, the 2.5 % ozone response to solar cycle in the upper layer is in agreement with previous studies, but the response in the middle stratosphere (∼6 %) is much larger than previously reported 25 (∼1 %). The 11 year solar cycle effect is still subject of debate (WMO, 2010; Chiodo et al., 2014), so that an additional decade of measurements would help in fixing its real 24644 Discussion Paper | Discussion Paper impact on ozone. This is particularly true for our shortest time series, Lauder, Izaña and Thule. ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD he trends at the high latitude stations are negative in the tropo The trends at the high latitude stations are negative in the troposphere, except at Kiruna where it is non significant. Except at Thule, the high latitude stations show sig nificant negative trends in the lower stratosphere. The situation is mixed in the middle 5 stratosphere, where the trend is significantly negative at Ny-Alesund, non-significant at Thule and Kiruna, and significantly positive at Harestua. The trends of the three high latitude stations with a similar time-period are all positive in the upper stratosphere, but this increase is taking place during the 1995–2003 period, while the EESC were g p g p still increasing until about 2000 in the polar region (WMO, 2010). However all four sta- 10 tions give non-significant trends in the upper stratosphere for the October 1999–2012 period, which could be the onset of the upper stratospheric ozone recovery at high latitude. The total column trends are non-significant at all high latitude stations, ex- cept at Ny-Alesund where it is negative. This is in agreement (except at Ny-Alesund) p y g g ( p y ) with model predictions that the Arctic March ozone recovery to 1980 levels will occur 15 around 2026 (WMO, 2010). 5 Conclusions However, the high year-to-year total column variability at these latitudes, driven mainly by lower stratospheric variability due to the polar tem- perature variations, does not allow yet to draw conclusions from the current trends for Arctic total ozone in the coming few years. with model predictions that the Arctic March ozone recovery to 1980 levels will occur 15 around 2026 (WMO, 2010). However, the high year-to-year total column variability at these latitudes, driven mainly by lower stratospheric variability due to the polar tem- perature variations, does not allow yet to draw conclusions from the current trends for Arctic total ozone in the coming few years. The trends for mid-latitude and subtropical stations are all non-significant, except 20 at Lauder in the troposphere and upper stratosphere, and at Wollongong for the total columns and the lower and middle stratospheric columns. Some signs of the onset of ozone mid-latitude recovery are observed only in the Southern Hemisphere, while a few more years seems to be needed to observe it at the northern stations. The trends for mid-latitude and subtropical stations are all non-significant, except 20 at Lauder in the troposphere and upper stratosphere, and at Wollongong for the total columns and the lower and middle stratospheric columns. Some signs of the onset of ozone mid-latitude recovery are observed only in the Southern Hemisphere, while a few more years seems to be needed to observe it at the northern stations. r | Discussion Paper | y To conclude, among the numerous available satellite and ground-based data sets 25 measuring vertical distributions of ozone that are useful for ozone trend evaluations (Hassler et al., 2014), the NDACC ground-based FTIR measurements have their par- ticular assets: many stations, well distributed around the globe, are now reaching al- most 20 years of measurements and will continue measuring ozone in the future. This y To conclude, among the numerous available satellite and ground-based data sets 25 measuring vertical distributions of ozone that are useful for ozone trend evaluations (Hassler et al., 2014), the NDACC ground-based FTIR measurements have their par- ticular assets: many stations, well distributed around the globe, are now reaching al- most 20 years of measurements and will continue measuring ozone in the future. This 25 24645 Discussion Paper | Discussion Pape provides long time series of ozone that are self-calibrated. 5 Conclusions Pastel (LATMOS) for use- ful discussion on equivalent latitude proxy, and R. Kivi (KMI) for discussion on ozonesondes trends in the Arctic. This study has been supported by the EU FP7 project NORS, the ESA PRODEX project A3C, as well as the AGACC-II project within the “Science for a Sustainable 20 Development” research program funded by the Belgian Science Policy Office. Appenzeller, C., Weiss, A. K., and Staehelin, J.: North Atlantic Oscillation modulates total ozone winter trends, Geophys. Res. Lett., 27, 1131–1134, 2000. 24634 Barret, B., De Mazière, M., and Demoulin, P.: Retrieval and characterization of ozone 25 profiles from solar infrared spectra at the Jungfraujoch, J. Geophys. Res., 107, 4788, doi:10.1029/2001JD001298, 2002. 24630 5 Conclusions This is also the only data set, with Umkehr measurements, that provides simultaneously total columns, tropo- spheric columns and three stratospheric columns that reach 40–45 km. This data set is suitable for an alternative determination of ozone vertical changes, as proposed in this study, but also for validation of the satellite merged data sets and detection of possible 5 drifts. provides long time series of ozone that are self-calibrated. This is also the only data set, with Umkehr measurements, that provides simultaneously total columns, tropo- spheric columns and three stratospheric columns that reach 40–45 km. This data set is suitable for an alternative determination of ozone vertical changes, as proposed in this study, but also for validation of the satellite merged data sets and detection of possible 5 drifts. ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD Acknowledgements. The National Center for Atmospheric Research is supported by the Na- tional Science Foundation. The observation program at Thule, Greenland, is supported under contract by the National Aeronautics and Space Administration and the site is also supported by the NSF Office of Polar Programs. We wish to thank the Danish Meteorological Institute for 10 support at the Thule. We would like to thank U. Raffalski and P. Völger for technical support at IRF Kiruna. The University of Liège team acknowledges the support of the F.R.S. – FNRS, the Fédération Wallonie-Bruxelles, the International Foundation High Altitude Research Stations Jungfraujoch and Gornergrat (HFSJG, Bern) and Meteoswiss. The NIWA Lauder MIR-FTIR project is core funded through New Zealand’s Ministry of Business, Innovation and Employ- 15 ment. We are grateful to the many colleagues having contributed to FTIR data acquisition at the various sites. We wish to thank S. Godin-Beekmann and M. Pastel (LATMOS) for use- ful discussion on equivalent latitude proxy, and R. Kivi (KMI) for discussion on ozonesondes trends in the Arctic. This study has been supported by the EU FP7 project NORS, the ESA PRODEX j t A3C ll th AGACC II j t ithi th “S i f S t i bl Acknowledgements. winter trends, Geophys. Res. Lett., 27, 1131 1134, 2000. 24634 Barret, B., De Mazière, M., and Demoulin, P.: Retrieval and characterization of ozone 25 profiles from solar infrared spectra at the Jungfraujoch, J. Geophys. Res., 107, 4788, doi:10.1029/2001JD001298, 2002. 24630 Appenzeller, C., Weiss, A. K., and Staehelin, J.: North Atlantic Oscillation modulates total ozone winter trends, Geophys. Res. Lett., 27, 1131–1134, 2000. 24634 B t B D M iè M d D li P R t i l d h t i ti f 5 Conclusions The National Center for Atmospheric Research is supported by the Na- tional Science Foundation. The observation program at Thule, Greenland, is supported under contract by the National Aeronautics and Space Administration and the site is also supported tional Science Foundation. The observation program at Thule, Greenland, is supported under contract by the National Aeronautics and Space Administration and the site is also supported by the NSF Office of Polar Programs. We wish to thank the Danish Meteorological Institute for 10 support at the Thule. We would like to thank U. Raffalski and P. Völger for technical support at IRF Kiruna. The University of Liège team acknowledges the support of the F.R.S. – FNRS, the Fédération Wallonie-Bruxelles, the International Foundation High Altitude Research Stations Jungfraujoch and Gornergrat (HFSJG, Bern) and Meteoswiss. The NIWA Lauder MIR-FTIR by the NSF Office of Polar Programs. We wish to thank the Danish Meteorological Institute for 10 support at the Thule. We would like to thank U. Raffalski and P. Völger for technical support at IRF Kiruna. The University of Liège team acknowledges the support of the F.R.S. – FNRS, the Fédération Wallonie-Bruxelles, the International Foundation High Altitude Research Stations Jungfraujoch and Gornergrat (HFSJG, Bern) and Meteoswiss. The NIWA Lauder MIR-FTIR g j g ( ) project is core funded through New Zealand’s Ministry of Business, Innovation and Employ- 15 ment. We are grateful to the many colleagues having contributed to FTIR data acquisition at the various sites. We wish to thank S. Godin-Beekmann and M. Pastel (LATMOS) for use- ful discussion on equivalent latitude proxy, and R. Kivi (KMI) for discussion on ozonesondes trends in the Arctic. This study has been supported by the EU FP7 project NORS, the ESA project is core funded through New Zealand’s Ministry of Business, Innovation and Employ- 15 ment. We are grateful to the many colleagues having contributed to FTIR data acquisition at the various sites. We wish to thank S. Godin-Beekmann and M. Pastel (LATMOS) for use- ful discussion on equivalent latitude proxy, and R. Kivi (KMI) for discussion on ozonesondes trends in the Arctic. This study has been supported by the EU FP7 project NORS, the ESA project is core funded through New Zealand’s Ministry of Business, Innovation and Employ- 15 ment. 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Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD Table 1. Characteristics of the FTIR stations that are contributing to the present work: location and altitude (in km a.s.l.), time-period covered by the ozone measurements used in the present trend analysis, and instrument type. References Station Latitude Longitude Altitude (km) Time-period Instrument Ny-Alesund 79◦N 12◦E 0.02 1995–2012 Bruker 120 HR Thule 77◦N 69◦W 0.22 1999–2012 Bruker 120 M Kiruna 68◦N 20◦E 0.42 1996–2007 Bruker 120 HR 2007–2012 Bruker 125 HR Harestua 60◦N 11◦E 0.60 1995–2009 Bruker 120 M 2009–2012 Bruker 125 M Jungfraujoch 47◦N 8◦E 3.58 1995–2012 Bruker 120 HR Izaña 28◦N 16◦W 2.37 1999–2005 Bruker 120 M 2005–2012 Bruker 125 HR Wollongong 34◦S 151◦E 0.03 1996–2007 Bomem DA8 2007–2012 Bruker 125 HR Lauder 45◦S 170◦E 0.37 2001–2012 Bruker 120 HR 24652 24652 ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD Table 2. Summary of the ozone retrieval parameters. All microwindow (mw) limits are given in cm−1. Ny: Ny-Alesund; Th: Thule; Ha: Harestua; Ju: Jungfraujoch. Parameters Ny-Alesund/Thule Harestua/Jungfraujoch Kiruna/Izaña Wollongong/Lauder Retrieval code SFIT2a v3.94 SFIT2av3.94 PROFFIT9b SFIT2av3.94 Microwindows 1000–1005 1000–1005 991.25–993.80 1000–1005 782.56–782.86 (Ny) 1001.47–1003.04 782.56–782.86 788.85–789.37 (Ny) 1005.0–1006.9 788.85–789.37 993.3–993.8 (Ny) 1007.347–1009.003 993.3–993.8 1011.147–1013.553 896.4–896.6 (H2O) H2O treatment – a priori profile One single profile (Ny) One single profile (Ha) Preliminary retrievals One single profile Preliminary retrievals in Preliminary retrievals in in dedicated H2O mws dedicated H2O mws (Th) dedicated H2O mws (Ju) – fit in ozone mw Scaling retrieval only Scaling retrieval only Scaling retrieval only Profile retrieval Regularization: – Sa Diagonal: 20 % (Ny) Diagonal: 5 to 11 % (Ha) Tikhonov regularization Diagonal: 10 % Diagonal: 30 % (Th) Diagonal: 10 % (Ju) L1 No inter-layer correlation No inter-layer correlation (Ha) Inter-layer correlation: Inter-layer correlation: exponential decay 4 km gaussian decay 4 km (Ju) – SNR Real SNR (depending on Constant = 100 (Ju) Depending on Constant = 150 each spectrum), exceptc Constant = 200 (Ha) each spectrum regions at: 1000.85–1001.45 1003.16–1004.5 set to SNR = 1 (Ny) Constant = 50 (Th) Instrument Fixed ideal (Ny) Fixed from LINEFIT (Ha) Fixed ideal (Kiruna) Fixed ideal Line Shape Fixed from LINEFIT 2nd order polynomial fit Fixed from LINEFIT except Bomem spectra: (Th) of EAP (Ju) (Izaña) 4th order polynomial fit of EAP a Pougatchev et al. (1995); b Hase (2000); c in order to mask strong H2O absorptions. Table 2. Summary of the ozone retrieval parameters. References All microwindow (mw) limits are given in cm−1. Ny: Ny-Alesund; Th: Thule; Ha: Harestua; Ju: Jungfraujoch. Table 2. Summary of the ozone retrieval parameters. All microwindow (mw) limits are given in cm−1. Ny: Ny-Alesund; Th: Thule; Ha: Harestua; Ju: Jungfraujoch. Discussion Paper | Discussion Pape Discussion Paper ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD Table 3. Partial column (PC) limits for the 4 altitude layers containing at least one DOFS. The random uncertainties are given for each partial column. Trop: Troposphere; LowS: Lower Stratosphere; MidS: Middle Stratosphere; UppS: Upper Stratosphere; TotC: Total Columns; Gd: Ground; Err.: Total Random Uncertainties. Layers Stations PC limits Err. Trop Izaña/Wollongong Gd-13/12 km 6 % Other stations Gd-9/10 km 5 % LowS Izaña/Wollongong 15–23 km 5 % Other stations 12–20 km 4 % MidS Izaña/Wollongong 23–32 km 5 % Other stations 20–29 km 5 % UppS Izaña/Wollongong 31–49 km 5 % Other stations 29–49 km 5 % TotC Izaña/Wollongong – 2 % Other stations – 2 % Table 3. Partial column (PC) limits for the 4 altitude layers containing at least one DOFS. The random uncertainties are given for each partial column. Trop: Troposphere; LowS: Lower Stratosphere; MidS: Middle Stratosphere; UppS: Upper Stratosphere; TotC: Total Columns; Gd: Ground; Err.: Total Random Uncertainties. Layers Stations PC limits Err. Trop Izaña/Wollongong Gd-13/12 km 6 % Other stations Gd-9/10 km 5 % LowS Izaña/Wollongong 15–23 km 5 % Other stations 12–20 km 4 % MidS Izaña/Wollongong 23–32 km 5 % Other stations 20–29 km 5 % UppS Izaña/Wollongong 31–49 km 5 % Other stations 29–49 km 5 % TotC Izaña/Wollongong – 2 % Other stations – 2 % 24654 24654 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD cussion Paper | Discussion Paper | Discussion Paper | Table 4. References Name, short description, and source of the proxies that have been tested in the stepwise regression model. Name Description Source SOLAR Solar Radio Flux at 10.7 cm ftp://ftp.ngdc.noaa.gov/STP/SOLAR_DATA/SOLAR_RADIO /FLUX/Penticton_Adjusted/monthly/MONTHLY.ADJ QBO30 zonal winds measured at Singapore at 30 hPa http://www.geo.fu-berlin.de/en/met/ag/strat/produkte/qbo/index.html QBO10 zonal winds measured at Singapore at 10 hPa http://www.geo.fu-berlin.de/en/met/ag/strat/produkte/qbo/index.html ENSO Multivariate ENSO Index (MEI) http://www.esrl.noaa.gov/psd/enso/mei/ AO/AAO Arctic Oscillation http://www.cpc.ncep.noaa.gov/products/precip/CWlink/daily_ao_index /monthly.ao.index.b50.current.ascii Antarctic Oscillation http://www.cpc.ncep.noaa.gov/products/precip/CWlink/daily_ao_index /aao/monthly.aao.index.b79.current.ascii TP Tropopause pressure http://www.esrl.noaa.gov/psd/data/gridded /data.ncep.reanalysis.tropopause.html EL(L/M/U) Equivalent latitude at three altitude levels: calculated at BIRA from ERA interim reanalysis 370, 550, and 950 K: high/mid-latitude stations 460, 700, and 1040 K: subtropical stations EPF Vertical component of the EP flux http://www.awi.de/en/research/research_divisions/climate_science /atmospheric_circulations/projects/candidoz/ep_flux_data/ VPSC Volume of Polar Stratospheric Clouds calculated at FMI Table 4. Name, short description, and source of the proxies that have been tested in the stepwise regression model. http://www.awi.de/en/research/research_divisions/climate_science /atmospheric_circulations/projects/candidoz/ep_flux_data/ calculated at FMI 24655 24655 ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD Table 5. Coefficient of determination R2 and contribution of the seasonal cycle Cseas deter- mined within the final model. See Table 3 for the limits of the layers, different for subtropical stations and mid/high latitude stations. References FTIR station Trop LowS MidS UppS Total columns Ny-Alesund R2 = 0.75 R2 = 0.92 R2 = 0.72 R2 = 0.74 R2 = 0.95 Cseas = 0.73 Cseas = 0.82 Cseas = 0.27 Cseas = 0.72 Cseas = 0.68 Thule R2 = 0.86 R2 = 0.92 R2 = 0.83 R2 = 0.81 R2 = 0.96 Cseas = 0.50 Cseas = 0.71 Cseas = 0.41 Cseas = 0.58 Cseas = 0.58 Kiruna R2 = 0.85 R2 = 0.89 R2 = 0.54 R2 = 0.78 R2 = 0.89 Cseas = 0.67 Cseas = 0.82 Cseas = 0.23 Cseas = 0.58 Cseas = 0.69 Harestua R2 = 0.77 R2 = 0.74 R2 = 0.36 R2 = 0.67 R2 = 0.75 Cseas = 0.54 Cseas = 0.51 Cseas = 0.25 Cseas = 0.45 Cseas = 0.56 Jungfraujoch R2 = 0.73 R2 = 0.83 R2 = 0.53 R2 = 0.93 R2 = 0.81 Cseas = 0.58 Cseas = 0.66 Cseas = 0.53 Cseas = 0.77 Cseas = 0.67 Izaña R2 = 0.83 R2 = 0.72 R2 = 0.80 R2 = 0.69 R2 = 0.77 Cseas = 0.87 Cseas = 0.46 Cseas = 0.45 Cseas = 0.64 Cseas = 0.56 Wollongong R2 = 0.69 R2 = 0.86 R2 = 0.42 R2 = 0.77 R2 = 0.87 Cseas = 0.69 Cseas = 0.52 Cseas = 0.09 Cseas = 0.75 Cseas = 0.63 Lauder R2 = 0.89 R2 = 0.94 R2 = 0.78 R2 = 0.89 R2 = 0.95 Cseas = 0.85 Cseas = 0.73 Cseas = 0.70 Cseas = 0.82 Cseas = 0.66 | Discussion Paper | 24656 Discussion Paper | Discussion Paper Discussion Paper | Discussio Discussion Paper ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD Table 6. Annual trend (in % decade−1) and their 95 % uncertainty ranges. Due to polar night, the measurements at Ny-Alesund, Thule and Kiruna cover only the Mid-March–September, Late-February–Mid-October, and Mid-January–Mid-November periods, respectively. All time series end in September/December 2012 for the present study. The time of start is repeated for each station. See Table 3 for the limits of the layers, different for subtropical stations and mid/high latitude stations. References Trends indicated in bold are significant. Table 6. Annual trend (in % decade−1) and their 95 % uncertainty ranges. Due to polar night, the measurements at Ny-Alesund, Thule and Kiruna cover only the Mid-March–September, Late-February–Mid-October, and Mid-January–Mid-November periods, respectively. All time series end in September/December 2012 for the present study. The time of start is repeated for each station. See Table 3 for the limits of the layers, different for subtropical stations and mid/high latitude stations. Trends indicated in bold are significant. FTIR station Trop LowS MidS UppS Total columns Ny-Alesund −5.8 ± 3.2 −4.2 ± 3.1 −5.5 ± 3.8 +6.7 ± 5.3 −3.0 ± 1.5 1995 Thule −5.3 ± 4.4 −0.4 ± 6.3 +0.2 ± 4.4 −2.3 ± 6.5 −2.1 ± 2.6 1999 (October) Kiruna −0.9 ± 2.5 −3.9 ± 2.6 +0.4 ± 2.6 +7.4 ± 3.4 −0.3 ± 1.6 1996 Harestua −3.1 ± 2.0 −5.3 ± 4.6 +4.8 ± 4.3 +7.8 ± 5.5 +1.0 ± 2.2 1995 Jungfraujoch −2.5 ± 2.7 −0.5 ± 3.3 −0.6 ± 1.2 +0.9 ± 1.0 −0.4 ± 1.2 1995 Izaña +0.7 ± 2.8 −1.7 ± 2.2 −0.1 ± 2.0 +1.6 ± 2.6 +0.5 ± 1.2 1999 Wollongong −2.2 ± 2.8 +3.1 ± 2.7 +4.0 ± 2.0 +0.2 ± 1.6 +1.9 ± 1.1 1996 Lauder +7.7 ± 3.5 −3.8 ± 4.1 −0.2 ± 3.5 +2.8 ± 2.4 −0.3 ± 1.8 2001 24657 24657 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Di ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD −1 −0.5 0 0.5 1 1.5 2 0 10 20 30 40 50 60 70 Partial columns Averaging Kernel (molec. cm−2 / molec. cm−2) Altitude (km) Ground − 10 km 12 − 20 km 20 − 29 km 29 − 49 km Sensitivity Figure 1. Partial column averaging kernels (molec. cm−2 (molec. cm−2)−1) for ozone retrievals at Jungfraujoch station. −1 −0.5 0 0.5 1 1.5 2 0 10 20 30 40 50 60 70 Partial columns Averaging Kernel (molec. cm−2 / molec. References cm−2) Altitude (km) Ground − 10 km 12 − 20 km 20 − 29 km 29 − 49 km Sensitivity 2 2 1 Partial columns Averaging Kernel (molec. cm−2 / molec. cm−2) Figure 1. Partial column averaging kernels (molec. cm−2 (molec. cm−2)−1) for ozone retrievals at Jungfraujoch station. 24658 ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 1996 1998 2000 2002 2004 2006 2008 2010 2012 0.5 1 1.5 Monthly means of ozone total columns (1E19 molec. cm−2) Ny−Alesund 1996 1998 2000 2002 2004 2006 2008 2010 2012 0.5 1 1.5 Thule 1996 1998 2000 2002 2004 2006 2008 2010 2012 0.5 1 1.5 Kiruna 1996 1998 2000 2002 2004 2006 2008 2010 2012 0.5 1 1.5 Harestua 1996 1998 2000 2002 2004 2006 2008 2010 2012 0.5 1 1.5 Jungfraujoch 1996 1998 2000 2002 2004 2006 2008 2010 2012 0.5 1 1.5 Izana 1996 1998 2000 2002 2004 2006 2008 2010 2012 0.5 1 1.5 Wollongong 1996 1998 2000 2002 2004 2006 2008 2010 2012 0.5 1 1.5 Lauder Time series of monthly means of ozone total columns at each station. ACPD Monthly means of ozone total columns (1E19 molec. cm−2) Figure 2. Time series of monthly means of ozone total columns at each station. 24659 ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Figure 3. Monthly means of ozone total and partial columns for the whole periods of mea- surements. See Table 3 for the limits of the partial columns at each station. The seasonal cycle for Southern Hemisphere stations, Wollongong and Lauder, has been shifted by 6 months for better comparison. ACPD Figure 3. Monthly means of ozone total and partial columns for the whole periods of mea- surements. See Table 3 for the limits of the partial columns at each station. References The seasonal cycle for Southern Hemisphere stations, Wollongong and Lauder, has been shifted by 6 months for better comparison. 24660 ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Trop LowS MidS UppS TotC −0.2 −0.1 0 0.1 0.2 0.3 0.4 0.5 Cfrac Ny−Alesund Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.2 −0.1 0 0.1 0.2 0.3 0.4 0.5 Cfrac Thule Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.1 0 0.1 0.2 0.3 0.4 Cfrac Kiruna Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.1 −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 Cfrac Harestua Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU 33 Discussion Paper | Discussion Paper | Discussion Paper | Discuss Trop LowS MidS UppS TotC −0.1 −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 Cfrac Jungfraujoch Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 Cfrac Lauder Trend Solar ENSO QBO AAO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.1 0 0.1 0.2 0.3 0.4 Cfrac Wollongong Trend Solar ENSO QBO AAO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Cfrac Izana Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Figure 4. Individual contributions Cfract of the proxies to the coefficient of determination R2. R2 and the dominant contribution of the seasonal cycle Cseas are given in Table 5 . ividual contributions Cfract of the proxies to the coefficient of determination R2. R2 nant contribution of the seasonal cycle Cseas are given in Table 5. References Trop LowS MidS UppS TotC −0.2 −0.1 0 0.1 0.2 0.3 0.4 0.5 Cfrac Ny−Alesund Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.2 −0.1 0 0.1 0.2 0.3 0.4 0.5 Cfrac Thule Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.1 0 0.1 0.2 0.3 0.4 Cfrac Kiruna Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.1 −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 Cfrac Harestua Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU 33 Trop LowS MidS UppS TotC −0.1 −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 Cfrac Jungfraujoch Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 Cfrac Lauder Trend Solar ENSO QBO AAO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.1 0 0.1 0.2 0.3 0.4 Cfrac Wollongong Trend Solar ENSO QBO AAO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Cfrac Izana Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU e 4. Individual contributions Cfract of the proxies to the coefficient of determination R2. R2 a ominant contribution of the seasonal cycle Cseas are given in Table 5 . References Individual contributions Cfract of the proxies to the coefficient of d and the dominant contribution of the seasonal cycle Cseas are given in Tab ACPD Trop LowS MidS UppS TotC −0.2 −0.1 0 0.1 0.2 0.3 0.4 0.5 Cfrac Thule Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.2 −0.1 0 0.1 0.2 0.3 0.4 0.5 Cfrac Ny−Alesund Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU C Trop LowS MidS UppS TotC Trop LowS MidS UppS TotC −0.1 0 0.1 0.2 0.3 0.4 Cfrac Kiruna Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU C Trop LowS MidS UppS TotC −0.1 −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 Cfrac Harestua Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC Trop LowS MidS UppS TotC 33 Trop LowS MidS UppS TotC −0.1 −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 Cfrac Jungfraujoch Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 Cfrac Lauder Trend Solar ENSO QBO AAO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.1 0 0.1 0.2 0.3 0.4 Cfrac Wollongong Trend Solar ENSO QBO AAO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Cfrac Izana Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Figure 4. Individual contributions Cfract of the proxies to the coefficient of determination R2. R2 and Figure 4. Individual contributions Cfract of the proxies to the coefficient of det 33 Trop LowS MidS UppS TotC 0.1 Trop LowS MidS UppS TotC 0.05 Trop LowS MidS UppS TotC −0.1 0 0.1 0.2 0.3 0.4 Cfrac Wollongong Trend Solar ENSO QBO AAO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Cfrac Izana Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU er | Discussion Paper | r | Disc Figure 4. Individual contributions Cfract of the proxies to the coefficient of determination R2. R2 and the dominant contribution of the seasonal cycle Cseas are given in Table 5 . Figure 4. Individual contributions Cfract of the proxies to the coefficient of determination R2. References contributions Cfract of the proxies to the coefficient of d tribution of the seasonal cycle Cseas are given in Tabl Trop LowS MidS UppS TotC −0.2 −0.1 0 0.1 0.2 0.3 0.4 0.5 Cfrac Ny−Alesund Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.2 −0.1 0 0.1 0.2 0.3 0.4 0.5 Cfrac Thule Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.1 0 0.1 0.2 0.3 0.4 Cfrac Kiruna Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.1 −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 Cfrac Harestua Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU 33 Trop LowS MidS UppS TotC −0.1 −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 Cfrac Jungfraujoch Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 Cfrac Lauder Trend Solar ENSO QBO AAO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.1 0 0.1 0.2 0.3 0.4 Cfrac Wollongong Trend Solar ENSO QBO AAO EPF VPSC TP ELL ELM ELU Trop LowS MidS UppS TotC −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Cfrac Izana Trend Solar ENSO QBO AO EPF VPSC TP ELL ELM ELU Figure 4. Individual contributions Cfract of the proxies to the coefficient of determination R2. R2 the dominant contribution of the seasonal cycle Cseas are given in Table 5 . Figure 4. References R2 and the dominant contribution of the seasonal cycle Cseas are given in Table 5. 24661 Discussion Paper | Discussion Pape ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD Figure 5. Top panels: monthly means of the tropospheric columns (Trop) at Ny-Alesund (left) and of the lower stratospheric columns (LowS) at Kiruna (right) (blue: FTIR, red: MLR model). Middle panels: same but with the seasonal signal removed. Bottom panels: the VPSC signal obtained in each case from the MLR model, for each month of the period (red line), and at each FTIR observed month (red circle). The tropopause pressure (TP) signal obtained for the LowS at Kiruna is also shown. | Discussion Paper | Figure 5. Top panels: monthly means of the tropospheric columns (Trop) at Ny-Alesund (left) and of the lower stratospheric columns (LowS) at Kiruna (right) (blue: FTIR, red: MLR model). Middle panels: same but with the seasonal signal removed. Bottom panels: the VPSC signal obtained in each case from the MLR model, for each month of the period (red line), and at each FTIR observed month (red circle). The tropopause pressure (TP) signal obtained for the LowS at Kiruna is also shown. 24662 Discussion Paper | Discussion Paper Discussion Paper | Discussion Paper | D ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. References Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD 1995 2000 2005 2010 2015 2 3 4 5 x 10 18 MidS columns at Ny−Alesund molec.cm−2 1995 2000 2005 2010 2015 2 3 4 5 x 10 18 MidS with seasonal signal removed at Ny−Alesund molec.cm−2 1995 2000 2005 2010 2015 −1 −0.5 0 0.5 1 x 10 18 EPF signal for MidS at Ny−Alesund molec.cm−2 1995 2000 2005 2010 2015 2 3 4 5 x 10 18 MidS columns at Thule molec.cm−2 1995 2000 2005 2010 2015 2 3 4 5 x 10 18 MidS with seasonal signal removed at Thule molec.cm−2 1995 2000 2005 2010 2015 −1 −0.5 0 0.5 1 x 10 18 EPF signal for MidS at Thule molec.cm−2 Figure 6. Top panels: monthly means of the middle stratospheric columns (MidS) at Ny- Alesund (left) and Thule (right) (blue: FTIR, red: MLR model). Middle panels: same but with the seasonal signal removed. Bottom panels: the EPF signal obtained in each case from the MLR model, for each month of the period (red line), and at each FTIR observed month (red circle). 1995 2000 2005 2010 2015 2 3 4 5 x 10 18 MidS columns at Thule molec.cm−2 1995 2000 2005 2010 2015 2 3 4 5 x 10 18 MidS with seasonal signal removed at Thule molec.cm−2 x 10 18 EPF signal for MidS at Thule 1995 2000 2005 2010 2015 2 3 4 5 x 10 18 MidS columns at Thule molec.cm−2 1995 2000 2005 2010 2015 2 3 4 5 x 10 18 MidS with seasonal signal removed at Thule molec.cm−2 1995 2000 2005 2010 2015 −1 −0.5 0 0.5 1 x 10 18 EPF signal for MidS at Thule molec.cm−2 1995 2000 2005 2010 2015 2 3 4 5 x 10 18 MidS columns at Thule molec.cm−2 x 10 18 MidS with seasonal signal removed at Thule Figure 6. Top panels: monthly means of the middle stratospheric columns (MidS) at Ny- Alesund (left) and Thule (right) (blue: FTIR, red: MLR model). Middle panels: same but with the seasonal signal removed. References Bottom panels: the EPF signal obtained in each case from the MLR model, for each month of the period (red line), and at each FTIR observed month (red circle). 24663 24663 ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Monthly means of the upper stratospheric columns (UppS) with the seasonal cy- ed at from top to bottom: Harestua Kiruna and Ny Alesund (blue: FTIR red: MLR 7. Monthly means of the upper stratospheric columns (UppS) with the seasonal cy- moved at, from top to bottom: Harestua, Kiruna and Ny-Alesund (blue: FTIR, red: MLR . Bottom panel: the solar cycle signal obtained at Ny-Alesund from the MLR model, the Cochrane-Orcutt transformation. ACPD nthly means of the upper stratospheric columns (UppS) t, from top to bottom: Harestua, Kiruna and Ny-Alesund ( Figure 7. Monthly means of the upper stratospheric columns (UppS) with the seasonal cy- cle removed at, from top to bottom: Harestua, Kiruna and Ny-Alesund (blue: FTIR, red: MLR model). Bottom panel: the solar cycle signal obtained at Ny-Alesund from the MLR model, before the Cochrane-Orcutt transformation. 24664 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ACPD Figure 8. Top panels: monthly means of the tropospheric columns (Trop) at Jungfraujoch (left) and Lauder (right) (blue: FTIR, red: MLR model). Middle panels: same but with the seasonal signal removed. Bottom panel: the solar cycle signal obtained at Lauder from the MLR model. Figure 8. Top panels: monthly means of the tropospheric columns (Trop) at Jungfraujoch (left) and Lauder (right) (blue: FTIR, red: MLR model). Middle panels: same but with the seasonal signal removed. Bottom panel: the solar cycle signal obtained at Lauder from the MLR model. Figure 8. Top panels: monthly means of the tropospheric columns (Trop) at Jungfraujoch (left) and Lauder (right) (blue: FTIR, red: MLR model). References Middle panels: same but with the seasonal signal removed. Bottom panel: the solar cycle signal obtained at Lauder from the MLR model. 24665 24665 Discussion Paper | Discussion Pape ACPD 14, 24623–24666, 2014 Trends of ozone total columns and vertical distribution from FTIR observations C. Vigouroux et al. Title Page Abstract Introduction Conclusions References Tables Figures ◀ ▶ ◀ ▶ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Disc Discussion Paper | Discussion Paper | D ACPD Figure 9. Top panels: monthly means of the lower stratospheric columns (LowS) at Jungfrau- joch (left), total columns (TotC) at Izaña (middle), and middle stratospheric columns (MidS) at Wollongong (right). (blue: FTIR, red: MLR model). Middle panels: same but with the sea- sonal signal removed. Bottom panels: QBO and AO signals obtained from the MLR model at Jungfraujoch (left), QBO signal at Izaña (middle), and SOLAR signal at Wollongong (right). Figure 9. Top panels: monthly means of the lower stratospheric columns (LowS) at Jungfrau- joch (left), total columns (TotC) at Izaña (middle), and middle stratospheric columns (MidS) at Wollongong (right). (blue: FTIR, red: MLR model). Middle panels: same but with the sea- sonal signal removed. Bottom panels: QBO and AO signals obtained from the MLR model at Jungfraujoch (left), QBO signal at Izaña (middle), and SOLAR signal at Wollongong (right). er | Discussion Paper | 24666 24666
https://openalex.org/W2983001284
https://www.jsdrm.ru/jour/article/download/839/750
Russian
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THE STATE OF THE WORLD ECONOMY AS A THREAT TO RUSSIA’S SECURITY: PUBLIC ESTIMATES AND INFLUENCING FACTORS
Strategičeskie rešeniâ i risk-menedžment
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cc-by
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& решения риск- cтратегические менеджмент & решения риск- cтратегические менеджмент Т. 10, № 2/2019 УДК 330.16 + 338.1 DOI: 10.17747/2618-947X-2019-2-166-173 Состояние мировой экономики как угроза безопасности России: оценки населения и влияющие факторы В. В. Каргинова-Губинова1 1 Карельский научный центр Российской академии наук Аннотация Со Состояние мировой экономики рассматривается как фактор, влияющий на национальную безопасность населения России. С помощью методов прикладной статистики и социологического подхода были проанализированы данные международного со- циологического опроса, проходившего в феврале-мае 2017 года в 38 странах. Проанализированы отчеты и статистические базы Всемирного экономического форума, Всемирного банка, Европейской экономической комиссии ООН и Центрального разведы- вательного управления США. В рамках исследования была показана множественность факторов, определяющих оценки угроз национальной безопасности с учетом персональных характеристик людей и особенностей развития стран их проживания. При- мерно одинаково характеризуют внешние и внутренние факторы угрозы своей стране жители России и Израиля. Было постро- ено уравнение множественной регрессии, которое позволяет рассчитывать текущую и прогнозную оценки населением угрозы состояния мировой экономики для безопасности страны. Полученные результаты могут быть использованы для дальнейших исследований дискурса безопасности и особенностей восприятия угроз. Также знание факторов, определяющих оценки угроз, поможет выбрать те инструменты и меры, которые создадут объективную и субъективную безопасность и ее ощущение. Ключевые слова: Ключевые слова: национальная безопасность, экономическая безопасность, риск, глобальная конкурентоспособность, экспорт товаров и услуг, социологический подход, множественная регрессия. национальная безопасность, экономическая безопасность, риск, глобальная конкурентоспос социологический подход, множественная регрессия. Для цитирования: Каргинова-Губинова В. В. Состояние мировой экономики как угроза безопасности России: оценки населения и влияющие фак- торы // Стратегические решения и риск-менеджмент. 2019. Т. 10. № 2. С. 166–173. DOI: 10.17747/2618-947X-2019-2-166-173 166 Vol. 10, № 2/2019 & decisions risk strategic management The state of the world economy as a threat to Russia's security: public estimates and influencing factors V l ti V K Valentina V. Karginova-Gubinova1 1 Institute of Economics of the Karelian Research Centre of the Russian Academy of Sciences Valentina V. Karginova-Gubinova1 1 Institute of Economics of the Karelian Research Centre of the Russian Academy of Sciences Abstract Th This article is aimed at determining the significance of the state of the world economy as a threat to national security for the public of the Russia, as well as the formalization of the factors determining this. The study is based on a sociological approach and assumes primary attention to society, not the state, and cognitive factors. Using the methods of applied statistics, the data of the international sociological survey held in February-May 2017 in 38 countries were analyzed. The reports and statistical bases of the World Economic Forum, the World Bank, the United Nations Economic Commission for Europe and the Central Intelligence Agency were also analyzed. The study showed a plurality of factors that determine the assessment of threats to national security: both the personal characteristics of people and the characteristics of the development of their countries. From all of the countries reviewed, evaluations of Russians to the greatest extent correspond to the estimates of residents of Israel. The multiple regression equation was constructed, it allows to calculate the current and forecast public assessment of the state of the world economy as a threat for the country's security. The results can be used for further studies of security discourse and threat perception. Also, knowledge of the factors that determine the threat assessment will help to choose the tools and measures that will create both objective and subjective security (its feeling). Keywords: national security, economic security, risk, global competitiveness, export of goods and services, sociological approach, multiple regression. For citation: Karginova-Gubinova V. V. The state of the world economy as a threat to Russia's security: public estimates and influencing factors. Strategic Decisions and Risk Management. 2019;10 (2): 166–173. DOI: 10.17747/2618-947X-2019-2-166-173 For citation: Karginova-Gubinova V. V. The state of the world economy as a threat to Russia's security: public estimates and influencing factors. Strategic Decisions and Risk Management. 2019;10 (2): 166–173. DOI: 10.17747/2618-947X-2019-2-166-173 1. Актуальность странах данный факт могут рассматривать как угрозу соб- ственной безопасности (Смирнова, 2010). Безопасность является одним из  базовых условий устойчивого развития территории (Эколого-экономическая безопасность, 2011; Черников, Орсоева, 2008; Ромашин, 2008; Raudeliūnienė, Tvaronavičienė, Dzemyda, 2014). Угро- зы национальной безопасности зачастую рассматриваются в качестве элемента окружающей среды (Смирнова, 2016). Они возникают в  результате столкновения интересов лю- дей, в том числе в процессе их познавательной активности (Смирнова, 2007). В силу нелинейности процессов и осо- бенностей когнитивной деятельности человека (Каргинова, 2017; Rosenzweig, 2007; Tversky A., Kahneman, 1974; Galton, 1869) абсолютно корректная рефлексия окружающей дей- ствительности невозможна (Максимова, Гончарова, Ноян- зина, 2012). Существуют как  адекватные, так и  мнимые, искусственно сформированные угрозы; их параметры оце- ниваются завышенно или заниженно (Мамытов, 2016). Так, например, государство может наращивать численность ар- мии с целью обеспечить собственную оборону, но в других В  данной работе состояние мировой экономики рас- сматривается как  угроза национальной безопасности для  населения России, предпринята попытка формали- зовать определяющие это факторы. С  этой целью пред- полагается определить общий контур угроз для  насе- ления, сопоставить российские и  общемировые данные об их значимости, выявить ключевые влияющие факторы и построить уравнение множественной регрессии оценок населения. Выбор угрозы, обусловленной состоянием мировой эко- номики, выглядит особенно актуально в условиях глобальной регионализации (Karginova, 2018). Анализ оценок населени- ем конкретных угроз национальной безопасности позволит определить, каким из них необходимо уделить приоритетное внимание, знание факторов даст возможность сформировать инструменты и принять меры, с тем чтобы минимизировать угрозы. Даже при  наличии объективных условий, обеспе- чивающих экономическую безопасность территории, без ее 167 & решения риск- cтратегические менеджмент Т. 10, № 2/2019 субъективного восприятия развития не  произойдет: будет отток капитала, населения и т.д. субъективного восприятия развития не  произойдет: будет отток капитала, населения и т.д. Следовательно, угроза может являться и онтологической, и эпистемологической характеристикой окружающей среды (Смирнова, 2007). Можно считать обоснованным примене- ние социологического подхода, который предполагает пер- востепенное внимание к обществу и когнитивным факторам (Смирнова, 2010). В частности, данный подход получил раз- витие в литературе (Buzan, Waever, Wilde, 1998; Buzan, 1983) и  будет использован в  этом исследовании для  дополнения существующих объективных представлений о безопасности субъективными. Кроме того, важно минимизировать восприятие значимо- сти угроз населением и в связи с тем, что в настоящее время люди все больше ориентируются на  оценки не  экспертов, а  таких  же простых граждан, которые практически мгно- венно распространяются с помощью репостов в социальных сетях. Рядовой читатель зачастую не относится критически к получаемой информации, не делает различия между мне- нием обывателей и  точкой зрения специалиста. 1. Актуальность А  распро- страняемая информация не  всегда соответствует действи- тельности, о чем свидетельствуют исследования (Здоровье, 2013). Соответственно, возможно повышение уровня трево- ги в обществе. 2. Теория На  примере международных угроз был показан общий механизм определения угроз. Так, восприятие угрозы – это результат осмысления возможности и масштаба негативных последствий. В  данном случае задействуются когнитивные механизмы: социальное сравнение, каузальная атрибуция, рефлексия и эмпатия. Следовательно, угрозы можно рассма- тривать в качестве социальных конструктов (Смирнова, 2016). Угрозы национальной безопасности не всегда восприни- маются рационально. Экспериментально показано, что чув- ствительность к сигналам опасности повышается, если чело- век может избежать неприятных последствий, и снижается, если такая возможность отсутствует (Brandtstädter, Voss, Rothermund, 2004). Если человек непосредственно уже стал- кивался с негативными последствиями риска, для него зна- чимость угрозы увеличивается (Koshiba, Ohtani, 2015). На  примере международных угроз был показан общий механизм определения угроз. Так, восприятие угрозы – это результат осмысления возможности и масштаба негативных последствий. В  данном случае задействуются когнитивные механизмы: социальное сравнение, каузальная атрибуция, рефлексия и эмпатия. Следовательно, угрозы можно рассма- тривать в качестве социальных конструктов (Смирнова, 2016). Угрозы национальной безопасности не всегда восприни- маются рационально. Экспериментально показано, что чув- ствительность к сигналам опасности повышается, если чело- век может избежать неприятных последствий, и снижается, если такая возможность отсутствует (Brandtstädter, Voss, Rothermund, 2004). Если человек непосредственно уже стал- кивался с негативными последствиями риска, для него зна- чимость угрозы увеличивается (Koshiba, Ohtani, 2015). Дополнительно были использованы отчеты и статисти- ческие базы Всемирного экономического форума, Всемир- ного банка, Европейской экономической комиссии ООН и Центрального разведывательного управления США. Данные литературы обработаны с  помощью методов прикладной статистики, в первую очередь посредством кор- реляционно-регрессионного анализа и  построения уравне- ния множественной регрессии. Оценка восприятия угроз безопасности проводилась с участием представителей отдельных категорий населения (Платонов, Прокопьева, 2018; Проблемы, 2015) в некоторых регионах России (Полухина, Савенко, 2014) и европейских странах. Так, социологические опросы в  Европе и  России свидетельствуют о расхождении общественно-политических рисков, оцениваемых как  доминирующие среди населения данных территорий. Это, в частности, связано с различной чувствительностью к ценностной значимости объектов и яв- лений, а также с разным уровнем политической толерантно- сти (Pankratov, 2014). 3. Методология В качестве материала для исследования взяты результаты международного опроса Pew Research Center. Респондентам предлагалось оценить, насколько та или иная угроза значи- ма для их страны (значимость каждой угрозы определялась отдельно). Исследование проходило в феврале-мае 2017 года в 38 странах по всему миру, как в развитых (в США, Вели- кобритании и др.), так и в развивающихся (например, в Тан- зании). В каждой стране опросили более 850 человек, всего почти 42 тысячи человек. В качестве показателей репрезен- тативности выборки учитывали пол, возраст, регион прожи- вания, а дополнительно в 36 странах – образование, в ряде стран – и  другие характеристики: этническую принадлеж- ность и территорию происхождения. 4. Результаты Не предлагалось оценивать: * в Турции; ** в США; *** в России. ● от конкурентоспособности экономики страны (индекс глобальной конкурентоспособности), значительные. Доминирование США сильно волнует 37%, почти в два раза больше, чем власть и влияние Китая. ● от кон глобал ● от значимости экспорта и импорта (доля экспортируе- мых и, соответственно, импортируемых товаров и ус- луг в ВВП), Сопоставление оценок угроз в России и макрорегионов мира показало наименьшее расхождение с  Северной Аме- рикой (рис. 1) и  с  Израилем, в  отношении состояния ми- ровой экономики как угрозы национальной безопасности – с Великобританией, Венгрией и США. Значимость угрозы доминирования США воспринимается как  более высокая по сравнению с аналогичной угрозой Китая, данное наблю- дение сделано не только в России, но и на Ближнем Востоке, и в Латинской Америке. В Латинской Америке, на Ближнем Востоке, в Африке и Канаде в качестве основной угрозы рас- сматривают США, в Европе и самих США – Россию, и толь- ко в Азиатско-Тихоокеанском регионе – Китай. ● от уровня экономического развития (ВВП на душу на- селения по паритету покупательной способности), ● от темпов роста экономики (годовой рост ВВП). На основании рассчитанного коэффициента корреляции Пирсона (табл. 4) показано, что между оценкой состояния мировой экономики в качестве угрозы национальной безо- пасности и  всеми рассматриваемыми показателями суще- ствует обратная связь, прежде всего с конкурентоспособно- стью экономики, на втором месте по значимости – уровень экономического развития. Доля экспорта в ВВП влияет су- щественнее, чем доля экспорта, а темп роста экономики – меньше всего. В США, России и большинстве стран (70%) Европы су- ществует зависимость между возрастом и  оценкой ИГИЛ (запрещена на территории России) в качестве угрозы наци- ональной безопасности: представители старшего поколения считают данную угрозу более существенной, чем молодежь (табл. 2). В  России расхождение наименьшее – 12 п.п.; наибольшее – в  Нидерландах, 32 п.п. (Poushter, Manevich, 2017). Далее для построения уравнения множественной регрес- сии и исключения мультиколлинеарности были рассчитаны 169 Рис. 1. Оценка угрозы в качестве значительной для страны проживания по макрорегионам, % опрошенных респондентов (по данным (Poushter, Manevich, 2017)) Угрозу от ИГИЛ (организация запрещена на территории России) не предлагалось оценивать в Турции, власть и влияние США – в США, власть и влияние России – в России Прослежена зависимость между политиче- скими взглядами респондента и его оценкой зна- чимости угроз национальной безопасности (табл. 3): правые больше обеспокоены ИГИЛ (запре- щена на территории России) и большим количе- ством беженцев, левые – глобальным изменени- ем климата. 4. Результаты Анализ материалов международного опроса Pew Research Center позволил определить доли респондентов, посчитавших угрозу значительной для своей страны, и су- ществующие расхождения в оценках жителей разных стран. Наиболее важными угрозами являются исламская группи- ровка ИГИЛ (организация запрещена на территории России) и глобальное изменение климата. Совпали мнения относи- тельно опасности кибератак из других стран и состояния ми- ровой экономики, менее значимым оказалось большое число беженцев. Большинство людей (35%) озабочены доминиро- ванием США, меньше (31%) – России и Китая (табл. 1). В исследовании вопросов международной безопасности показано, что группы людей со схожими ценностями реже воспринимают друг друга в качестве источника угрозы, если сравнивать с тем, какого мнения друг о друге придержива- ются группы с  различными ценностями (Garcia-Retamero, Muller, Rousseau, 2012). По  результатам социологического исследования в 25 странах были показаны особенности оце- нок угроз окружающей среды. Распространенность угроз в  связи с  использованием новых технологий не  приводит к восприятию этих угроз как более значительных. В то же время увеличение числа внедренных технологий повышает уровень тревоги общества. В результате наиболее серьезно угрозы окружающей среды воспринимаются в странах, где уровень использования новых технологий низкий, но стре- мительно растет (Lima, Barnett, Vala, 2005). Ситуация в  России несколько отличается от  мировой. Для  россиян угроза исламской группировки ИГИЛ значи- тельно превосходит все остальные. Наибольшее расхождение с мировыми данными наблюдается в отношении глобального изменения климата: как значительную угрозу рассматривают 35% россиян (на 26 п.п. или в 1,7 раз меньше, чем в мире). Состояние мировой экономики как угрозу национальной без- опасности россияне также воспринимаются реже, чем в сред- нем по миру, однако расхождения в данном случае не столь 168 Vol. 10, № 2/2019 & decisions risk strategic management Таблица 1 Оценка угрозы в качестве значительной для страны проживания, % опрошенных респондентов (по данным (Poushter, Manevich, 2017)) Угроза В среднем по миру Россия Расхождение, п.п. Исламская группировка ИГИЛ* 62 58 –4 Глобальное изменение климата 61 35 –26 Кибератаки из других стран 51 34 –17 Состояние мировой экономики 51 38 –13 Большое число беженцев из Ирака и Сирии 39 37 –2 Власть и влияние США** 35 37 2 Власть и влияние России*** 31 – – Власть и влияние Китая 31 19 –12 Здесь и далее для расчета средних значений по группе стран использовался показатель медианы. Не предлагалось оценивать: * в Турции; ** в США; *** в России. ц у р д р р , опрошенных респондентов (по данным (Poushter, Manevich, 2017)) Здесь и далее для расчета средних значений по группе стран использовался показатель медианы. & решения риск- cтратегические менеджмент & решения риск- cтратегические менеджмент Т. 10, № 2/2019 Территория Возраст, лет Расхождение*, п.п 18–29 30–49 50+ Среднее значение по США и странам Европы, где расхождение статистически значимо 49 67 79 30 Россия 51 55 63 12 * Между самой старшей и самой молодой возрастными группами. Таблица 2 Оценка угрозы исламской группировки ИГИЛ (запрещена на территории России) в качестве значительной для страны проживания в зависимости от возраста, % респондентов (по данным (Poushter, Manevich, 2017)) Территория Возраст, лет Расхождение*, п.п 18–29 30–49 50+ Среднее значение по США и странам Европы, где расхождение статистически значимо 49 67 79 30 Россия 51 55 63 12 * Между самой старшей и самой молодой возрастными группами. Таблица 2 Оценка угрозы исламской группировки ИГИЛ (запрещена на территории России) в качестве значительной для страны проживания в зависимости от возраста, % респондентов (по данным (Poushter, Manevich, 2017)) Угроза Левые Цен- тристы Правые Расхож- дение, п.п. Исламская группиров- ка ИГИЛ (запрещена на территории России) 63 77 79 16 Глобальное изменение климата 73 66 55 –18 Большое число бежен- цев из Ирака и Сирии 20 36 52 32 Таблица 3 Оценка угрозы в качестве значительной для страны проживания в зависимости от политических взглядов, % опрошенных респондентов (по данным (Poushter, Manevich, 2017)) Угроза Левые Цен- тристы Правые Расхож- дение, п.п. Исламская группиров- ка ИГИЛ (запрещена на территории России) 63 77 79 16 Глобальное изменение климата 73 66 55 –18 Большое число бежен- цев из Ирака и Сирии 20 36 52 32 Таблица 3 Оценка угрозы в качестве значительной для страны проживания в зависимости от политических взглядов, % опрошенных респондентов (по данным (Poushter, Manevich, 2017)) Таблица 2 курентоспособности страны; x2 – экспорт товаров и услуг, % от ВВП. коэффициенты линейной парной корреляции между рас- сматриваемыми признаками. На  основании более высокой значимости индекса глобальной конкурентоспособности по сравнению с ВВП на душу населения и экспорта товаров и услуг по сравнению с импортом, а также их высокой пар- ной корреляции показатели ВВП на душу населения и им- порт товаров и услуг исключены из дальнейшего анализа. Расчет прогнозной оценки по уравнению и ее сопостав- ление с  фактическими данными показали, что  указанные факторы с  предлагаемым уровнем значимости в  меньшей степени объясняют оценки в  Сенегале, Швеции, Польше, Германии и Индии (даны по убыванию). Для  остальных индикаторов построены различные ва- рианты уравнений множественной регрессии, рассчитана стандартная ошибка для  нескольких групп индикаторов (табл. 5). 5. Выводы Оценки угроз национальной безопасности для  страны проживания зависят как  от  персональных характеристик респондентов (в частности, возраста и политических взгля- дов), так и от особенностей развития их стран (показатели Уравнение множественной регрессии имеет вид: y = 1,0934 – 0,1170x1 – 0,0019x2, y = 1,0934 – 0,1170x1 – 0,0019x2, где y – оценка населением угрозы состояния мировой эконо- мики для безопасности страны; x1 – индекс глобальной кон- & решения риск- cтратегические менеджмент Был выбран вариант уравнения через индекс гло- бальной конкурентоспособности и экспорт товаров и услуг, так как именно этому варианту соответствует минимальная стандартная ошибка оценки (вариация фактических оце- нок относительно линии регрессии). Существенность свя- зи и  статистическая значимость уравнения регрессии под- тверждены с помощью критерия Фишера, который составил 8,378 (критическое значение – 3,245). Также были рассчитаны оценки угрозы состояния ми- ровой экономики для  национальной безопасности страны по данным за 2007–2016 годы для России (рис. 2). Средне- годовые изменения можно считать незначительными – все- го 0,66 п.п. Это обусловлено разнонаправленным влиянием глобального индекса конкурентоспособности (+9,1%) и сни- жением доли экспорта товаров и услуг в ВВП ( – 13,7%). 4. Результаты В Европе сторонники правых выше оценивают угрозу беженцев (Poushter, Manevich, 2017). В США респонденты делились на либера- лов (левых), умеренных (центристов) и консерва- торов (правых). Можно предположить, что  подобные рас- хождения по  данным России проследить труд- но из‑за  отсутствия четкого разделения партий на правых и левых. С  учетом поставленной цели основное вни- мание уделено угрозе состояния мировой эко- номики. В отношении данной угрозы указанных выше зависимостей выявлено не было (Poushter, Manevich, 2017). Исследована зависимость ее оценок: Рис. 1. Оценка угрозы в качестве значительной для страны проживания по макрорегионам, % опрошенных респондентов (по данным (Poushter, Manevich, 2017)) Угрозу от ИГИЛ (организация запрещена на территории России) не предлагалось оценивать в Турции, власть и влияние США – в США, власть и влияние России – в России 169 169 Таблица 4 Связь оценок угрозы состояния мировой экономики для безопасности страны и выбранных в рамках исследования национальных показателей (по данным (Poushter, Manevich, 2017; The Global Competitiveness, [s.a.]; The World Bank Group, [s.a.]; Статистическая база, [б.г.]; The World Factbook, [s.a.])) Связь оценок угрозы состояния мировой экономики для безопасности страны и выбранных в рамках исследования национальных показателей (по данным (Poushter, Manevich, 2017; Число индика- торов Индикатор Стандарт- ная ошибка 3 Индекс глобальной конку- рентоспособности; экспорт товаров и услуг; годовой рост ВВП 0,1427 2 Индекс глобальной конку- рентоспособности; экспорт товаров и услуг 0,1410 1 Индекс глобальной конку- рентоспособности 0,1447 Примечание: использованы данные об экспорте Япо- нии за 2016 год, Венесуэлы – за 2014 год. Таблица 5 Стандартная ошибка по уравнениям множественной регрессии (по данным (Poushter, Manevich, 2017; The Global Competitiveness; The World Bank Group, [s.a.]; Статистическая база; The World Factbook, [s.a.])) Таблица 5 Стандартная ошибка по уравнениям множественной регрессии (по данным (Poushter, Manevich, 2017; The Global Competitiveness; The World Bank Group, [s.a.]; Статистическая база; The World Factbook, [s.a.])) The Global Competitiveness, [s.a.]; The World Bank Group, [s.a.]; Статистическая база, [б.г.]; The World Factbook, [s.a.])) Показатель Коэффи- циент кор- реляции Пирсона Связь Индекс глобальной конкурен- тоспособности –0,52 Заметная обратная Экспорт товаров и услуг, % от ВВП –0,34 Умеренная обратная Импорт товаров и услуг, % от ВВП –0,24 Слабая обратная ВВП на душу населения по ППС, долл. –0,39 Умеренная обратная Годовой рост ВВП, % –0,14 Слабая обратная Примечание: использованы данные об экспорте и импорте Японии за 2016 год, Венесуэлы – за 2014 год. 170 & decisions risk strategic management Рис. 2. Изменение прогнозной оценки угрозы состояния мировой экономики для безопасности страны населением России, п.п. (по данным (Competitiveness Dataset, [s.a.]; The World Bank Group, [s.a.])) Vol. 10, № 2/2019 Vol. 10, № 2/2019 & decisions risk strategic management конкурентоспособности, экспорта, импорта и  т.д.). Суще- ствует заметная обратная связь между состоянием мировой экономики для  национальной безопасности и  конкуренто- способностью экономики страны и умеренная обратная связь с долей экспорта в ВВП и ВВП на душу населения. Таким образом, эти факторы оказывают влияние не только на реаль- ную безопасность, но и на ее восприятие. Динамика указан- ных макроэкономических характеристик воздействует на сам дискурс безопасности и воздействует как прямо, так и опо- средованно, через СМИ, общественные настроения и др. Множественность факторов, определяющих степень зна- чительности конкретной угрозы национальной безопасно- сти, является причиной существенного расхождения оценок населения разных стран. Таблица 4 По сравнению со среднемировыми данными в России более значима угроза от исламской груп- пировки ИГИЛ (запрещена в России) (она превосходит оцен- ки остальных угроз в 2–3 раза), глобальное изменение кли- мата признается значительным в 1,7 раза реже, чем в мире. Оценка россиянами угрозы состояния мировой экономики для национальной безопасности также ниже, чем в среднем по миру. Рис. 2. Изменение прогнозной оценки угрозы состояния мировой экономики для безопасности страны населением России, п.п. (по данным (Competitiveness Dataset, [s.a.]; The World Bank Group, [s.a.])) ной и социальной безопасности // Известия Алтайского государственного университета. №2-1. C. 211–215. ной и социальной безопасности // Известия Алтайского государственного университета. №2-1. C. 211–215. ной и социальной безопасности // Известия Алтайского государственного университета. №2-1. C. 211–215. 4. Мамытов Т. Б. (2016) Угрозы безопасности жиз- ненно важным интересам личности, общества и государства // Известия вузов Кыргызстана. № 10. С. 205–207. Построенное уравнение множественной регрессии по- зволяет рассчитывать текущую и прогнозную оценки угро- зы состояния мировой экономики для безопасности страны. Было показано, что с 2007 по 2017 год наблюдаются незначи- тельные флуктуации оценок населения России, что связано с разнонаправленным влиянием роста конкурентоспособ- ности национальной экономики и снижения доли экспорта товаров и услуг в ВВП. 5. Платонов А. В., Прокопьева С.А. (2018). Представле- ния студентов о национальной безопасности России // Международный научно-исследовательский журнал. № 1 (67). Ч. 4. С. 60–62. DOI: 10.23670/IRJ.2018.67.054. 6. Полухина А. В., Савенко Е. И. (2014). Исследование угроз национальной экономической безопасности Рос- сии: социологический опрос // Экономические отноше- ния. № 1. С. 21–26. DOI: 10.18334/.37332. Знание факторов, определяющих восприятие угроз на- циональной безопасности населением страны, необходимо для построения грамотной политики – выбора тех инстру- ментов и мер, которые создадут и объективную, и субъектив- ную безопасность (ее ощущение). Грамотная политика будет способствовать предпринимательской активности, предот- вратит миграцию населения, увеличит рождаемость и  т.д. Соответственно, будет обеспечено устойчивое и безопасное развитие территории. 7. Проблемы национальной безопасности: региональный уровень (2015) / В. В. Рудой [и др.]. Ростов-на-Дону: ЮРИУ РАНХиГС. 272 с. 8. Ромашин А. Ф. (2008). Продовольственная безопасность как фактор устойчивого развития России. Нижний Нов- город: Издательство Волго-Вятской академии государ- ственной службы. 127 с. Благодарность 9. Смирнова А. Г. (2007). 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Towards economic security and sustainability: key suc- cess factors of sustainable entrepreneurship in conditions of global economy // Journal of Security and Sustainability Issues. Vol. 3, № 4. P. 71–79. DOI: https://doi.org/10.9770/ jssi.2014.3.4(7). 27. Rosenzweig P. (2007). The halo effect. New York: The Free Press. 232 p. 7. Problemy natsional'noj bezopasnosti: regional'nyj urov- en' (2015) / V. V. Rudoj [i dr.]. Rostov na Donu: YURIU RANKHiGS. 272 s. [Problems of national security: regional level (2015) / V. V. Ore [et al.]. Rostov-on-Don: South-Rus- sian Institute of Management. 272 p. (In Russ.)]. 28. The Global Competitiveness Report 2016–2017 ([s.a.]). URL: http://www3.weforum.org/docs/GCR2016-2017/05FullRe- port/TheGlobalCompetitivenessReport2016-2017_FINAL. pdf. 8. Romashin A. F. (2008). Prodovol'stvennaya bezopasnost' kak faktor ustojchivogo razvitiya Rossii. Nizhnij Novgo- rod: Izdatel'stvo Volgo-Vyatskoj akademii gosudarstvennoj sluzhby. 127 s. [Romashin, A. F. (2008). 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Perception of a threat in international relations: in search of theoretical grounds. Political Expertise: POLITEX. 3(4):193–208. (In Russ.)]. 23. Lima, M. L., Barnett, J., Vala, J. (2005) Risk percep- tion and technological development at a societal level. Risk Analysis. 25(5):1229–1239. DOI: 10.1111/j.1539- 6924.2005.00664.x. 24. Pankratov, S. A. (2014). Europe and Russia: finding innova- tive resources for socio-political security. In: International Multidisciplinary Scientific Conferences on Social Sciences and Arts (SGEM 2014) (Albena, September 1–10, 2014). Sofia: STEF92 Technology Ltd. P. 135–139. DOI: 10.5593/ SGEMSOCIAL2014/B21/S4.019. 10. Smirnova A. G. (2016). Mekhanizmy konstruirovaniya polit- icheskimi liderami vneshnej ugrozy v mezhgosudarstvenny- kh otnosheniyakh. YAroslavl': YArGU. 420 s. [Smirnova, A. G. (2016). Mechanisms of construction by political leaders of an external threat in interstate relations. Yaroslavl: YarSU. 420 p. (In Russ.)]. 25. Poushter, J., Manevich, D. (2017). Globally, People Point to ISIS and Climate Change as Leading Security Threats. Pew Research Center. http://assets.pewresearch.org/wp-con- tent/uploads/sites/2/2017/07/31101043/Pew-Research- Center_2017.07.13_Global-Threats_Full-Report.pdf. 11. Smirnova A. G. (2010). Ugrozy i ikh izuchenie v sotsiologii mezhdunarodnykh otnoshenij // Sotsiologicheskij zhurnal. № 2. S. 35–49. [Smirnova, A. G. (2010). Threats and their study in the sociology of international relations. Sociologi- cal Journal. 2:35–49. (In Russ.)]. 12. Statisticheskaya baza dannykh ЕEK OON ([b.g.]) // UN- ECE. [Statistical database of the United Nations Econom- ic Commission for Europe ([s.a.]) . (In Russ.)]. UNECE. https://w3.unece.org/PXWeb/ru. 26. Raudeliūnienė, J., Tvaronavičienė, M., Dzemyda, I. (2014) Towards economic security and sustainability: key success factors of sustainable entrepreneurship in conditions of glob- al economy. Journal of Security and Sustainability Issues. 3(4):71–79. DOI: https://doi.org/10.9770/jssi.2014.3.4(7). 13. Chernikov A. P., Orsoeva M. V. (2008). Energeticheskaya bezopasnost' kak faktor ustojchivogo razvitiya regiona. Ir- kutsk: Bajkal'skij gosudarstvennyj universitet ekonomiki i prava. 173 s. [Chernikov, A. P., Orsoeva, M. V. (2008). Ener- gy security as a factor of the sustainable development of the region. Irkutsk: Baikal State University of Economics and Law. 173 p. (In Russ.)]. 27. Rosenzweig, P. (2007). The halo effect. New York: The Free Press. 232 p. 28. The Global Competitiveness Report 2016–2017 ([s.a.]). http://www3.weforum.org/docs/GCR2016-2017/05FullRe- port/TheGlobalCompetitivenessReport2016-2017_FINAL. pdf. 29. The World Bank Group ([s.a.]). В. В. Каргинова-Губинова 17. Buzan, B., Waever, O., Wilde, J. de (1998). Security: a new framework for analysis. Boulder: Lynne Rienner Pub. 239 p. Кандидат экон. наук, научный сотрудник, Институт эконо- мики – обособленное подразделение Федерального госу- дарственного бюджетного учреждения науки Федерального исследовательского центра «Карельский научный центр Рос- сийской академии наук». Область научных интересов: эконо- мическая безопасность. 18. Competitiveness Dataset ([s.a.]). World Economic Forum. http://reports.weforum.org/global-competitiveness-re- port-2015-2016/report-highlights/. 19. Galton F. (1869) Hereditary genius. An inquiry into its laws and consequences // London: Macmillan. 390 p. E-mail: vkarginowa@yandex.ru 20. Garcia-Retamero, R., Muller, S. M., Rousseau, D. L. (2012). The Impact of Value Similarity and Power on the Perception of Threat. Political Psychology. 33(2):179–193. References https://www.worldbank. org. 14. Ekologo-ekonomicheskaya bezopasnost' kak faktor ustoj- chivogo razvitiya regiona (2011) / Red. V. V. Zykov. Tyu- men': Tyumenskij gosudarstvennyj universitet. 156 s. [Eco- logical and economic security as a factor of the sustainable development of the region (2011) / Ed. V. V. Zykov. Tyu- men: Tyumen State University. 156 p. (In Russ.)]. 30. The World Factbook ([s.a.]). https://www.cia.gov/library/ publications/resources/the-world-factbook/. 31. Tversky, A., Kahneman, D. (1974) Judgment under Un- certainty: Heuristics and Biases. Science. 185:1124–1131. DOI: 10.1126/science.185.4157.1124. 15. Brandtstädter, J., Voss, A., Rothermund, K. (2004). Percep- tion of danger signals: the role of control. Experimental Psy- chology. 51(1):24–32. ИНФОРМАЦИЯ ОБ АВТОРе 16. Buzan, B. (1983) People, States & Fear: The National Se- curity Problem in International Relations. Brighton: Wheat- sheaf Books. 262 p. В. В. Каргинова-Губинова About the author 21. Karginova, V. V. (2018) Global regionalization and its im- pact on the economic security policy in the former Soviet Union. In: Practical Geography and XXI Century Chal- lenges. International Scientific and Practical Conference (Moscow, June 4–6, 2018) / Institute of Geography, Russian Academy of Sciences. Moscow. P. 645–652. Valentina V. Karginova-Gubinova PhD of Economics, Research Associate, Institute of Economics of the Karelian Research Centre of the Russian Academy of Sciences. Research Interests: Economic Security. E-mail: vkarginowa@yandex.ru 173 173
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https://zenodo.org/records/6014155/files/Vaginal%20melanoma%20(1).pdf
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Primary malignant melanoma of the vagina: a case report
Zenodo (CERN European Organization for Nuclear Research)
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Case Report A 73-year-old, gravida 2, para 2, postmenopausal Greek woman presented with abnormal vaginal bleeding of 30 days duration which had increased progressively the last ten days. Overall 5-year survival ranges from 14 to 21 %. Tumor size is the most important prognostic factor [5], but tumor thickness does not affect survival. Other potential prog- nostic factors such as age, location, FIGO stage, depth of invasion, Chung level, histology, cell type, mitotic count, vessel involvement, ulceration, p53 accumulation, type of surgery, type of radiotherapy, and chemotherapy do not seem to correlate with patients' outcome [6]. Her past surgical history was significant for anterior and pos- terior colporraphy. Her family history was unremarkable. On 'Vaginal examination there was a 5.5 x 5 x 2 cm raised, ulcerated and irregular lesion of posterior vaginal wall. There were no palpable inguinal lymph nodes, and the rest of pelvic examination was normal. Preoperative computed tomography (CT) of the chest, abdomen and pelvis, abdominal ultrasound (US), chest X-ray, colonoscopy and urethrocystoscopy were normal. No differences in survival or disease-free interval were demonstrated between patients who had radical surgical procedures compared to more conservative surgical pro- cedures [7 J. A wide local excision was performed. Pathology examination of the entire specimen demonstrated a nodular vaginal melanoma, with clear lateral and deep margins. Some remark- able histological findings were 16 mitoses/mm', Breslow depth > 15 mm, and cytoplasmic melanin pigmentation (Figure 1 ). Radiotherapy is performed as an adjuvant therapy to achieve control of hidden metastases. Radiotherapy may be of value as an alternative to surgery or an adjunct modality in patients with lesions ~ 3 cm in diameter [6]. Immunotherapy with interferon has also been shown to confer survival benefits in patients at high risk for recur- rence, but toxicity has been important [8]. Radiotherapy is performed as an adjuvant therapy to achieve control of hidden metastases. Radiotherapy may be of value as an alternative to surgery or an adjunct modality in patients with lesions ~ 3 cm in diameter [6]. The histologic diagnosis was confirmed by positive immunostaining. Tumor cells were immunopositive for S-100 protein (Figure 2), for Melan A (Figure 3) and for HMB 45 (Figure 4). Immunotherapy with interferon has also been shown to confer survival benefits in patients at high risk for recur- rence, but toxicity has been important [8]. The patient denied postoperative radiotherapy. Discussion Vaginal melanoma has a high incidence of local recur- rence and regional or distal metastasis. Fifty percent have positive lymph nodes [3] and nearly 20% of patients have distant metastases [4] at disease presentation. This may be explained by the extensive lymphatic and vascular supply to the lamina propria of the vaginal mucous membranes. Case Report She began postoperative immunotherapy with interferon-a2b three times a week. Vaginal melanoma is highly malignant and is more aggressive than nongenital and vulvar melanoma [3, 9]. Seventeen months after excision of the lesion, the patient pre- sented with abnormal vaginal bleeding again. Pelvic examina- tion revealed a vaginal mass, 5 x 3 cm, and two enlarged inguinal lymph nodes (r) 3 cm in diameter. The patient declined postoperative radiotherapy again. Summary Primary malignant melanoma of the vagina is a very rare, but very aggressive tumor. We describe a case of primary vaginal melanoma and review the literature. The patient, a 73-year-old, gravida 2, para 2 postmenopausal Greek woman, presented with abnormal vaginal bleeding for 30 days. On vaginal examination there was a 5.5 x 5 x 2 cm raised, ulcerated and irregular lesion on the posterior vaginal wall. Pathology examination of the entire specimen demonstrated a nodular vaginal melanoma. The histologic diagnosis was confirmed by positive immunostaining. The patient began postoperative immunotherapy with interferon-a2b. She died 25 months later because of cerebral metastases. In conclusion, the prognosis of vaginal melanoma is very poor, despite the treat- ment modality, because most cases are diagnosed at a late stage. Key words: Vaginal melanoma; Immunotherapy; Radiotherapy; Surgery. Two months later, she presented with a sudden onset of severe headache. CT revealed multiple cerebral metastases. At the same time invasion of urethra was seen. Introduction Primary malignant melanoma of the vagina is a very rare, but very aggressive tumor. The incidence of primary vaginal melanoma is about 0.026/100,000 women per year [ 1). The average age is the 6'h and 7th decades of life [2]. The prognosis of vaginal melanoma is very poor, despite the various treatment modalities described. She died six months later because of cerebral metastases. Primary malignant melanoma of the vagina: A case report G. Androutsopoulos1, G. Adonakis', P. Ravazoula2, G. Kourounis' 1 Department of Obstetrics and Gynecology, 'Department (if Pathology, Patras University Hospital, Rion (Greece) Revised manuscript accepted for publication May 3, 2005 Eur. J. Gynaec. Oncol. ISSN: 0392-2936 XXVI, n. 6, 2005 661 661 f71 Reid G., Schmidt R., Roberts J. et al.: " Primary melanoma of the vagina: a clinicopathological analysis". Ohstet. Cynecol. , 1989, 74, 190. [8] De Matos P., Tyler D., Seigler H.F.: "Mucosa! melanoma of the female genitalia: a clinicopathologic study of 43 cases at Duke Uni- versity Medical Center". Surgery, 19~8 , 124, 38. [9] Piura B. , Rabinovich A., Yanai-lnbar !.: "Primary malignant melanoma of the vagina: case report and review of literature". Eur. J. Cynaecol. Oncol., 2002, 23, 195. Conclusion The prognosis of vaginal melanoma is very poor, despite the treatment modality, because most cases are diagnosed at late stage. G. Androutsopoulos, G. Adonakis, P. Ravazoula, G. Kourounis 662 Fig. 1 Fig. 3 Figure I. - Nests of malignant melanocytic cells. We can see macrophages plenty of melanin. Figure 2. - Tumor cells immunopositive for_ S-100 protein. Figure 3. - Tumor cells immunopositive for Melan A. Figure 4. - Tumor cells immunopositive for HMB 45. Fig. 3 Figure I. - Nests of malignant melanocytic cells. We can see macrophages plenty of melanin. Figure 2. - Tumor cells immunopositive for_ S-100 protein. Figure 3. - Tumor cells immunopositive for Melan A. Figure 4. - Tumor cells immunopositive for HMB 45. Figure I. - Nests of malignant melanocytic cells. We can see macrophages plenty of melanin. Figure 2. - Tumor cells immunopositive for_ S-100 protein. Figure I. Nests of malignant melanocytic cells. We can see macrophages plenty of melanin. Figure 2. - Tumor cells immunopositive for_ S-100 protein. Figure 3. - Tumor cells immunopositive for Melan A. Figure 4 Tumor cells immunopositive for HMB 45 Figure 4. - Tumor cells immunopositive for HMB 45. Address reprint requests to: G.L. ADONAKIS, M.D. Aritis 7 26442 Patra (Greece) [9] Piura B. , Rabinovich A., Yanai-lnbar !.: "Primary malignant melanoma of the vagina: case report and review of literature". Eur. J. Cynaecol. Oncol., 2002, 23, 195. [8] De Matos P., Tyler D., Seigler H.F.: "Mucosa! melanoma of the female genitalia: a clinicopathologic study of 43 cases at Duke Uni- versity Medical Center". Surgery, 19~8 , 124, 38. f71 Reid G., Schmidt R., Roberts J. et al.: " Primary melanoma of the vagina: a clinicopathological analysis". Ohstet. Cynecol. , 1989, 74, 190. References [I] Weinstock M.: "Malignant melanoma of the vulva and vagina in the United states: Patterns of incidence and population-based estimates of survival". Am. J. Obstet. Cynecol., 1994, 171, 1225. l2J Ragnarsson-Olding B., Johansson H., Rutqvist L.E. et al.: "Malig- nant melanoma of the vulva and vagina". Cance1; I 993, 71, 1893. [3J Cobellis L., Calabrese E., Stefanon B., Raspagliesi F.: "Malignant melanoma of the vagina. A report of 15 cases". Eui: J. Cynaecol. Oncol. , 2000, 21, 295. [4] Ariel l.M.: "Malignant melanoma of the female genital system: a report of 48 patients and review of the literature". J. Surg. Oncol., 1981, /6,371. l5] Tjalma W.A., Monagham J.M. , de Barros Lopes A., Naik R. , Nordin A.: "Primary Vaginal melanoma and long-term survivors". Eur. J. Cynaecol. Oncol. , 2001, 22, 20. Address reprint requests to: G.L. ADONAKIS, M.D. Aritis 7 26442 Patra (Greece) Address reprint requests to: G.L. ADONAKIS, M.D. Aritis 7 26442 Patra (Greece) [61 Petru E., Nagele K., Czerwenka K., Graf A.H., Lax S., Bauer M. et al. : "Primary malignant melanoma of the vagina: long-term remis- sion following radiation therapy". Cynecol. Oneal. , 1998, 70, 23.
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https://www.zora.uzh.ch/id/eprint/223571/1/ZORA_ica_abs_5_86_2022.pdf
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Building simplification of vector maps using graph convolutional neural networks
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Zurich Open Repository and Archive Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2022 Abstract: Buildings form one of the most important object classes of topographic maps and its generalization is a very critical process to create cartographic models at multiple scales (Brassel and Weibel, 1988, Regnauld and McMaster, 2007)). In particular, the simplification operator, which reduces the number of points representing a line or polygon boundary (Stanislawski et al., 2014), serves building generalization significantly. Building simplification has experienced decades of research. The conventional approaches to automating this problem introduce a range of criteria or constraints to detect redundant vertices or edges on boundaries of building polygons. The major criteria include edge length (Regnauld et al., 1999, Sester, 2005), edge number (Haunert and Wolff, 2010), area (Buchin et al., 2016), and so on. Recent advances in deep learning have the potential to bring map generalization research to a new era. Specific to the generalization of buildings or polygons, Cheng et al. 2013 used backpropagation neural networks to detect and simplify small corners, intrusions, and extrusions in rasterized footprints of buildings. Feng et al. (2019) applied mainstream deep learning architectures for image segmentation, including U-Net, residual U-Net, and GAN, to generalize image-based building maps. While these deep learning-based approaches, which implicitly model the simplification operator, achieve promising performance for building generalization, they are prone to cause deformed boundaries in generalized buildings, owing to the purely image-based inputs. Therefore, it is reasonable to attempt to adopt vector maps as input to preserve the straight-line, often rectangular shapes of boundaries in building generalization. However, there are so far only few works on building generalization of vector maps using deep learning. Some studies have approached some pre-processing operations of generalization, e.g., structure recognition (Yan et al., 2019), building grouping (Yan et al., 2020), and shape coding (Yan et al., 2021), most of which are based on graph neural networks (GCNs). Recently, a classifier was trained using a backpropagation neural network to select the most appropriate conventional simplification algorithms for different shapes of buildings (Yang et al., 2022), but it still falls short of explicitly modeling the associated generalization operators, such as simplification, most importantly. Figure 1. Graphical illustration motivating the proposed approach Figure 1. Graphical illustration motivating the proposed approach Although in principle it has been shown that GCNs are capable of encoding vector map objects, it is still unclear how GCNs could work for end-to-end map generalization, that is, vector maps in and vector maps out. Abstracts of the International Cartographic Association, 5, 2022. European Cartographic Conference – EuroCarto 2022, 19–21 September 2022, TU Wien, Vienna, Austria. https://doi.org/10.5194/ica-abs-5-86-2022 | © Author(s) 2022. CC BY 4.0 License. ng simplification of vector maps using graph convolutional neural networks DOI: https://doi.org/10.5194/ica-abs-5-86-2022 DOI: https://doi.org/10.5194/ica-abs-5-86-2022 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-223571 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-223571 Journal Article Published Version Originally published at: Zhou, Zhiyong; Fu, Cheng; Weibel, Robert (2022). Building simplification of vector maps using graph convolu- tional neural networks. Abstracts of the ICA, 5:1-2. DOI: https://doi.org/10.5194/ica-abs-5-86-2022 Building simplification of vector maps using graph convolutional neural networks Zhiyong Zhou∗, Cheng Fu, Robert Weibel Department of Geography, University of Zurich, Zurich, Switzerland, 1st Author - zhiyong.zhou@geo.uzh.ch, 2nd Author - cheng.fu@geo.uzh.ch, 3rd Author - robert.weibel@geo.uzh.ch * Corresponding author Keywords: Building simplification, vector map, graph convolutional neural network Acknowledgements This research was supported by the Swiss National Science Foundation through project no. 200021_204081 DeepGener- alization. References Brassel, K. E. and Weibel, R., 1988. A review and conceptual framework of automated map generalization. International Journal of Geographical Information System 2(3), pp. 229–244. Brassel, K. E. and Weibel, R., 1988. A review and conceptual framework of automated map generalization. International Journal of Geographical Information System 2(3), pp. 229–244. Buchin, K., Meulemans, W., Renssen, A. V. and Speckmann, B., 2016. Area-preserving simplification and schematization of polygonal subdivisions. ACM Transactions on Spatial Algorithms and Systems (TSAS) 2(1), pp. 1–36. Buchin, K., Meulemans, W., Renssen, A. V. and Speckmann, B., 2016. Area-preserving simplification and schematization of polygonal subdivisions. ACM Transactions on Spatial Algorithms and Systems (TSAS) 2(1), pp. 1–36. Feng, Y., Thiemann, F. and Sester, M., 2019. Learning cartographic building generalization with deep convolutional neural networks. ISPRS International Journal of Geo-Information 8(6), pp. 258. Haunert, J.-H. and Wolff, A., 2010. Optimal and topologically safe simplification of building footprints. In: Proceedings of the 18th sigspatial international conference on advances in geographic information systems, pp. 192–201. Regnauld, N. and McMaster, R. B., 2007. A synoptic view of generalisation operators. In: Generalisatio information, Elsevier, pp. 37–66. d McMaster, R. B., 2007. A synoptic view of generalisation operators. In: Generalisation of geographic Elsevier, pp. 37–66. Regnauld, N., Edwardes, A. and Barrault, M., 1999. Strategies in building generalisation: modelling the sequence, constraining the choice. In: ICA Workshop on Progress in Automated Map Generalization, Ottawa, Canada, pp. 372– 378. Sester, M., 2005. Optimization approaches for generalization and data abstraction. International Journal of Geographical Information Science 19(8-9), pp. 871–897. Stanislawski, L. V., Buttenfield, B. P., Bereuter, P., Savino, S. and Brewer, C. A., 2014. Generalisatio Abstracting geographic information in a data rich world, Springer, pp. 157–195. Yan, X., Ai, T., Yang, M. and Tong, X., 2021. Graph convolutional autoencoder model for the shape coding and cognition of buildings in maps. International Journal of Geographical Information Science 35(3), pp. 490–512. g p f g p f pp Yan, X., Ai, T., Yang, M. and Yin, H., 2019. A graph convolutional neural network for classification of building patterns using spatial vector data. ISPRS journal of photogrammetry and remote sensing 150, pp. 259–273. Yan, X., Ai, T., Yang, M., Tong, X. and Liu, Q., 2020. A graph deep learning approach for urban building grouping. Geocarto International pp. 1–24. Yang, M., Yuan, T., Yan, X., Ai, T. and Jiang, C., 2022. Abstract: This study targets one of the major generalization operators involved in vector map generalization, simplification. Inspired by the excellent capability of GCNs to capture the geometric characteristics of vector map objects (Yan et al., 2019), we propose a building simplification approach using GCNs. Since the key process to simplify the polygon of a building is to decide which of its corner points should be removed, the building simplification problem is formulated as a node-level classification task on building graphs in this study (see Figure 1). Note that we assume in our solution that collinear Steiner points have been added along the segments of the building boundary to unify the input graph size (i.e., the number of points) for GCNN. In this study, the graph size is set as 64 as suggested by Yan et al. (2021) and they are interpolated equally after the regulated size (i.e., 64) subtracts the number of original corner points of a polygon. 2 of 2 Figure 2 illustrates the workflow of the proposed approach. It is composed of three parts: feature engineering to construct geometric and topological features for an individual vertex in a building graph; graph convolutional neural network (GCNN) training based on the given dataset; and post-processing to reconstruct the polygon from the graph. The features are the same as used by Yan et al. (2019), including three types for local characteristics of vertices, and four types for regional characteristics. Afterwards, the features are fed to the GCNN to train and classify if each vertex in a polygon graph should be removed or not. Finally, to create a complete vector map product at the target scale, a post-processing step, which reconstructs polygons based on those vertices that are not removed, is conducted. In the experiment, the Stuttgart dataset (Feng et al., 2019), which contains a source map at the scale of 1:5,000 (accommodating 175,756 polygons) and three target maps at the scales of 1:10,000, 1:15,000, and 1:25,000, were used to train the GCNN for building simplification. We built and trained the GCNN using PyTorch and PyTorch Geometric. Since the study is still ongoing, the results are expected to become available before the conference and they will be compared with typical machine learning approaches. Figure 2. The workflow of the proposed approach Figure 2. The workflow of the proposed approach g p European Cartographic Conference – EuroCarto 2022, 19–21 September 2022, TU Wien, Vienna, Austria. https://doi.org/10.5194/ica-abs-5-86-2022 | © Author(s) 2022. CC BY 4.0 License. Abstracts of the International Cartographic Association, 5, 2022. g p , , ropean Cartographic Conference – EuroCarto 2022, 19–21 September 2022, TU Wien, Vienna, Austria. ps://doi org/10 5194/ica-abs-5-86-2022 | © Author(s) 2022 CC BY 4 0 License Abstracts of the International Cartographic Association, 5, 2022. References A hybrid approach to building simplification with an evaluator from a backpropagation neural network. International Journal of Geographical Information Science 36(2), pp. 280– 309.
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Comparative study of shock response synthesis techniques for aerospace applications
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Content from this work may be used under the terms of the Creative Commons Attribution 3.0 license. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under license by Materials Research Forum LLC. Keywords: Shock Response Spectrum, Synthesis, Wavelets, Damped Sinusoids, Enveloped Sinusoids Abstract. The Shock Response Spectrum (SRS) is a widely used tool for analyzing and characterizing the response of mechanical systems to shock and transient events. In the aerospace industry, the SRS is used to compute the severity of the shock event on the electrical and optical equipment of a spacecraft. However, the SRS only provides magnitude information and does not retain temporal or phase information. Moving to the time domain is not a straightforward process because a time history has a unique SRS, but the converse is not true. Therefore, it is challenging to find the right time history that reproduces an SRS when simulating a given input profile using pyrotechnic devices or when computing the response to a shock input profile in the time-domain. For a given SRS an infinite combination of time pulses is possible. Synthesizing an SRS involves recovering a time-domain pulse that can accurately replicate the given SRS. There are many methods which are already widely utilized in the aerospace industry, including the use of damped sinusoids, enveloped sinusoids and wavelets. In this paper we compare different techniques, with the objective of identifying the most suitable method based on the considered frequency range and type of impulse. The case study under consideration is an SRS input profile corresponding to a real industrial case. Three artificial SRS accelerations have been generated to replicate the input, and the percentage errors of each method in comparison to the reference signal have been assessed. Further development will involve the use of optimization algorithms to generate the SRS profile with the smallest possible error. Aeronautics and Astronautics - AIDAA XXVII International Congress Materials Research Proceedings 37 (2023) 744-747 Aeronautics and Astronautics - AIDAA XXVII International Congress Materials Research Proceedings 37 (2023) 744-747 Materials Research Forum LLC Materials Research Forum LLC https://doi.org/10.21741/9781644902813-159 https://doi.org/10.21741/9781644902813-15 Shock Response Spectrum Synthesis While a unique impulse in the time domain corresponds to a specific SRS, the opposite is not true. In fact, an SRS corresponds to an infinite number of possible pulses. As a result, there are several techniques available to obtain SRS-compatible acceleration time history. In this work we will investigate the accuracy of SRS synthesis throughout the summation of damped sines, enveloped sines and wavelets. Wavelets. A wavelet is a discrete waveform of limited duration that is suited for approximating data with sharp discontinuities [4]. The original signal can be reconstructed as a summation of a set of wavelets with specified parameters. The equation of a single wavelet 𝑊𝑊𝑚𝑚(𝑡𝑡) is: 𝑡𝑡 𝑊𝑊𝑚𝑚(𝑡𝑡) = ⎩⎪⎨ ⎪⎧ 0, for 𝑡𝑡< 𝑡𝑡𝑑𝑑𝑑𝑑 𝐴𝐴𝑚𝑚sin ቂ 2𝜋𝜋𝑓𝑓𝑚𝑚 𝑁𝑁𝑚𝑚(𝑡𝑡−𝑡𝑡𝑑𝑑𝑑𝑑)ቃsin[2𝜋𝜋𝑓𝑓𝑚𝑚(𝑡𝑡−𝑡𝑡𝑑𝑑𝑑𝑑)] , for 𝑡𝑡𝑑𝑑𝑑𝑑≤𝑡𝑡≤ቂ𝑡𝑡𝑑𝑑𝑑𝑑+ 𝑁𝑁𝑚𝑚 2𝑓𝑓𝑚𝑚ቃ 0, for 𝑡𝑡> ቂ𝑡𝑡𝑑𝑑𝑑𝑑+ 𝑁𝑁𝑚𝑚 2𝑓𝑓𝑚𝑚ቃ . (1) (1) A discrete wavelet has a sinusoidal motion with a finite and odd number of half sine oscillations 𝑁𝑁𝑚𝑚 with unique parameters for frequency 𝑓𝑓𝑚𝑚, amplitude 𝐴𝐴𝑚𝑚 and time delay 𝑡𝑡𝑑𝑑𝑑𝑑. A discrete wavelet has a sinusoidal motion with a finite and odd number of half sine oscillations 𝑁𝑁𝑚𝑚 with unique parameters for frequency 𝑓𝑓𝑚𝑚, amplitude 𝐴𝐴𝑚𝑚 and time delay 𝑡𝑡𝑑𝑑𝑑𝑑. Damped sinusoids. The sinusoid approach shows a difference in the way the rise, peak and decay of the waveform is controlled, compared to the previously presented method. In this case the parameters to control are slightly different: 𝑡𝑡 𝑊𝑊𝑚𝑚(𝑡𝑡) = ൝ 0, for 𝑡𝑡< 𝑡𝑡𝑑𝑑𝑑𝑑 𝐴𝐴𝑚𝑚𝑒𝑒−𝜉𝜉𝑚𝑚2𝜋𝜋𝑓𝑓𝑚𝑚(𝑡𝑡−𝑡𝑡𝑑𝑑𝑑𝑑) sin ቂ 2𝜋𝜋𝑓𝑓𝑚𝑚 𝑁𝑁𝑚𝑚(𝑡𝑡−𝑡𝑡𝑑𝑑𝑑𝑑)ቃsin[2𝜋𝜋𝑓𝑓𝑚𝑚(𝑡𝑡−𝑡𝑡𝑑𝑑𝑑𝑑)] , for 𝑡𝑡≥𝑡𝑡𝑑𝑑𝑑𝑑. (2) 𝜉 0, for 𝑡𝑡< 𝑡𝑡𝑑𝑑𝑑𝑑𝜋𝑓𝑚𝑁𝑚 (𝑡𝑡 )ቃi [2𝜋𝑓𝑓(𝑡𝑡 )] f𝑡𝑡 . (2) (2) It can be noted an extra term 𝜉𝜉𝑚𝑚, that is the damped sinusoid damping ratio. It can be noted an extra term 𝜉𝜉𝑚𝑚, that is the damped sinusoid damping ratio. Enveloped sinusoids. The enveloped sinusoids with random phase angles approach is similar to the one of damped sinusoids. The equation for enveloped sinusoids is given by: 𝑊𝑡𝐸𝑡𝐴𝜋𝑓𝑡𝜑 𝑊𝑊𝑚𝑚(𝑡𝑡) = 𝐸𝐸(𝑡𝑡)𝐴𝐴𝑚𝑚sin(2𝜋𝜋𝑓𝑓𝑚𝑚𝑡𝑡+ 𝜑𝜑𝑚𝑚). (3) 𝜑𝑚𝑊𝑚𝑡𝐸𝑡 (3) 𝑊𝑊𝑚𝑚(𝑡𝑡) = 𝐸𝐸(𝑡𝑡)𝐴𝐴𝑚𝑚sin(2𝜋𝜋𝑓𝑓𝑚𝑚𝑡𝑡+ 𝜑𝜑𝑚𝑚). Where 𝜑𝜑𝑚𝑚 are random phase angles for each frequency n. The rise, plateau and decay of 𝑊𝑊𝑚𝑚(𝑡𝑡) is controlled by an envelope function 𝐸𝐸(𝑡𝑡) rather than damping. Where 𝜑𝜑𝑚𝑚 are random phase angles for each frequency n. The rise, plateau and decay of 𝑊𝑊𝑚𝑚(𝑡𝑡) is controlled by an envelope function 𝐸𝐸(𝑡𝑡) rather than damping. Introduction One of the most significant challenges in the space industry is the design and testing of aerospace structures and systems for reliable and safe operation in harsh environments, including the sudden and impulsive loads occurring during the launch phase. The most intense events are commonly caused by pyrotechnic devices actuating at the base of the spacecraft. The firing of these devices results in impulsive loads characterized by high peak acceleration, high-frequency content, and short duration. This poses a significant threat to the reliability and safety of electrical and optical components of the spacecraft, which are sensitive to high frequency loads. To demonstrate its compliance to shock requirements, the structure has to be tested by applying the shock load on the base interface. The accepted standard for implicit description of the pyroshock environment is the Shock Response Spectrum (SRS), which is a useful tool for estimating the damage potential of the shock pulse and for test level specification. The SRS finds its first applications in the 50’s by the seismic and aerospace community. An SRS is generated by plotting in the frequency domain the peak response of a series of Single Degree of Freedom (SDoF) oscillating systems subjected to the same transient base acceleration input. The damping is usually assumed to be 5% (Q=10), while 744 Aeronautics and Astronautics - AIDAA XXVII International Congress Materials Research Forum LLC Materials Research Proceedings 37 (2023) 744-747 https://doi.org/10.21741/9781644902813-159 Aeronautics and Astronautics - AIDAA XXVII International Congress Materials Research Proceedings 37 (2023) 744-747 Materials Research Forum LLC https://doi.org/10.21741/9781644902813-159 Materials Research Forum LLC https://doi.org/10.21741/9781644902813-159 the natural frequency of each SDoF system is chosen to be different. The primary limitation of the SRS is its inability to provide temporal or phase information, as it only gives magnitude information. As a result, when subjecting a structure to electro-dynamic shaker testing for shock qualification, the SRS cannot be directly utilized [1]. Instead, it becomes necessary to synthesize an SRS-compatible acceleration time history. A similar challenge arises when analyzing nonlinear structures, where a modal approach is not feasible, and a modal transient analysis must be conducted to account for the phase among the peak responses of individual modes. p g p p The aforementioned waveform can be obtained using a series of sinusoids [1,2] or wavelets [3], tailored to resemble an actual pyrotechnic shock pulse. Shock Response Spectrum Synthesis For all the three methods, iterations for the parameters of a set of m waveforms a time t yield a synthesized acceleration that is expressed as: 745 745 745 Aeronautics and Astronautics - AIDAA XXVII International Congress Materials Research Forum LLC Materials Research Proceedings 37 (2023) 744-747 https://doi.org/10.21741/9781644902813-159 𝑥𝑡𝑊𝑚𝑡𝑁𝑚𝑚 Aeronautics and Astronautics - AIDAA XXVII International Congress Materials Research Forum LLC Materials Research Proceedings 37 (2023) 744-747 https://doi.org/10.21741/9781644902813-159 𝑥𝑡𝑊𝑚𝑡𝑁𝑚𝑚 Aeronautics and Astronautics - AIDAA XXVII International Congress Materials Research Proceedings 37 (2023) 744-747 𝑥𝑡𝑊𝑚𝑡𝑁𝑚𝑚 Materials Research Forum LLC https://doi.org/10.21741/9781644902813-159 𝑥𝑥̈(𝑡𝑡) = ∑ 𝑊𝑊𝑚𝑚(𝑡𝑡) 𝑁𝑁𝑚𝑚 𝑚𝑚=1 . (4) 𝑥𝑥̈(𝑡𝑡) = ∑ 𝑊𝑊𝑚𝑚(𝑡𝑡) 𝑁𝑁𝑚𝑚 𝑚𝑚=1 . 𝑥𝑥̈(𝑡𝑡) = ∑ 𝑊𝑊𝑚𝑚(𝑡𝑡) 𝑁𝑁𝑚𝑚 𝑚𝑚=1 . (4) (4) An example of a synthetized time history from the SRS input in Table 1 with a duration of T= 0.06 s can be seen in Fig.1. T bl 1 Sh k l d i t An example of a synthetized time history from the SRS input in Table 1 with a duration of T= 0.06 s can be seen in Fig.1. T bl 1 Sh k l d g Table 1. Shock load input Frequency [Hz] Amplitude [g] 100 56 1000 2820 10000 2820 (a) (b) (c) Figure 1. Reconstructed time history of a shock input with (a) wavelets, (b) damped sines, (c) enveloped sines ( ) Figure 1. Reconstructed time history of a shock input with (a) wavelets, (b) damped sines, (c) enveloped sines The synthetized accelerations have been converted to SRS and compared to the reference input as shown in Figure 2. The synthetized accelerations have been converted to SRS and compared to the reference input as shown in Figure 2 The synthetized accelerations have been converted to SRS and compared to the reference input as shown in Figure 2. 746 g 2. Synthetized SRS comparison g 2 S th ti d SRS i 2. Synthetized SRS comparison Aeronautics and Astronautics - AIDAA XXVII International Congress Materials Research Forum LLC Materials Research Proceedings 37 (2023) 744-747 https://doi.org/10.21741/9781644902813-159 Aeronautics and Astronautics - AIDAA XXVII International Congress Materials Research Forum LLC Materials Research Proceedings 37 (2023) 744-747 https://doi.org/10.21741/9781644902813-159 Aeronautics and Astronautics - AIDAA XXVII International Congress Materials Research Proceedings 37 (2023) 744-747 Materials Research Forum LLC Materials Research Forum LLC https://doi.org/10.21741/9781644902813-159 https://doi.org/10.21741/9781644902813-15 Table 2. Synthesis correlation coefficient 𝐶𝐶𝐶 Table 2. Synthesis correlation coefficient Furthermore, the Synthesis Correlation Coefficient (𝐶𝐶𝐶𝐶𝐶𝐶) [5] in Table 2 has been computed in low, middle and high frequency range to compare the efficiency. Conclusions and future developments In conclusion, the investigated techniques, namely the summation of damped sines, enveloped sines, and wavelets, have shown good levels of accuracy in reproducing the desired SRS input. Further studies should be conducted by exploring different parameter settings and types of input profiles to enhance the understanding of these techniques. Additionally, the development of an optimization algorithm, such as the least square fitting method or genetic algorithm, should be pursued to combine the methods and synthesize a single SRS that minimizes the error and achieves a higher level of accuracy. 745 𝐶𝐶𝐶𝑆𝑆𝑆𝑟𝑓𝑛𝑆𝑆𝑆𝑠𝑓𝑛𝑓 Furthermore, the Synthesis Correlation Coefficient (𝐶𝐶𝐶𝐶𝐶𝐶) [5] in Table 2 has been computed in low, middle and high frequency range to compare the efficiency. 𝐶𝐶𝐶𝑆𝑆𝑆𝑟𝑓𝑛𝑆𝑆𝑆𝑠𝑓𝑛𝑓𝑓 Furthermore, the Synthesis Correlation Coefficient (𝐶𝐶𝐶𝐶𝐶𝐶) [5] in Table 2 has been computed in low, middle and high frequency range to compare the efficiency. 𝐶𝐶𝐶𝑆𝑆𝑆𝑟𝑓𝑛𝑆𝑆𝑆𝑠𝑓𝑛𝑓 𝑆𝑆𝑆𝑆𝑟𝑟(𝑓𝑓𝑛𝑛)𝑆𝑆𝑆𝑆𝑆𝑆𝑠𝑠(𝑓𝑓𝑛𝑛)𝑓𝑓|2𝑆𝑆𝑆𝑟 (𝑓𝑓𝑛𝑛)2 ∑ 𝑆𝑆𝑆𝑆𝑆𝑆𝑠𝑠(𝑓𝑓𝑛𝑛)2 𝑓𝑓2 𝑓𝑓1 . (5) 𝑆𝑆𝑟𝑆𝑆𝑆𝑠 𝐶𝐶𝐶𝐶𝐶𝐶= | ∑ 𝑆𝑆𝑆𝑆𝑆𝑆𝑟𝑟(𝑓𝑓𝑛𝑛)𝑆𝑆𝑆𝑆𝑆𝑆𝑠𝑠(𝑓𝑓𝑛𝑛) 𝑓𝑓2 𝑓𝑓1 |2 ∑ 𝑆𝑆𝑆𝑆𝑆𝑆𝑟𝑟(𝑓𝑓𝑛𝑛)2 𝑓𝑓2 𝑓𝑓1 ∑ 𝑆𝑆𝑆𝑆𝑆𝑆𝑠𝑠(𝑓𝑓𝑛𝑛)2 𝑓𝑓2 𝑓𝑓1 . 𝐶𝐶𝐶𝐶𝐶𝐶= | ∑ 𝑆𝑆𝑆𝑆𝑆𝑆𝑟𝑟(𝑓𝑓𝑛𝑛)𝑆𝑆𝑆𝑆𝑆𝑆𝑠𝑠(𝑓𝑓𝑛𝑛) 𝑓𝑓2 𝑓𝑓1 |2 ∑ 𝑆𝑆𝑆𝑆𝑆𝑆𝑟𝑟(𝑓𝑓𝑛𝑛)2 𝑓𝑓2 𝑓𝑓1 ∑ 𝑆𝑆𝑆𝑆𝑆𝑆𝑠𝑠(𝑓𝑓𝑛𝑛)2 𝑓𝑓2 𝑓𝑓1 . (5) 𝑆𝑆𝑆𝑟𝑆𝑆𝑆𝑠 (5) Where 𝑆𝑆𝑆𝑆𝑆𝑆𝑟𝑟 and 𝑆𝑆𝑆𝑆𝑆𝑆𝑠𝑠 are respectively the reference and synthetized SRS. Globally, a good level of accuracy (near the unity) has been achieved. In particular, the enveloped sines method seems to be the most effective. It can be observed that the methods are less accurate in the middle frequency range (200-1000 Hz). [1] T. Irvine, Shock Response Spectrum Synthesis Via Damped Sinusoids, Vibrationdata, 2012 [3] J. E. Alexander, A New Method to Synthesize an SRS Compatible Base Acceleration with Energy and Temporal Moments to Improve MDOF System Response, IMAC 36 Orlando, 7 Sound & Vibration, 2018. [2] T. Irvine, Shock Response Spectrum Synthesis Via Wavelets, Vibrationdata, 2000. [4] A. Grasps, An Introduction to Wavelets, IEEE Computational Science and Engineering vol. 2, num. 2, published by the IEEE Computer Society, Los Alamitos, 1995. https://doi.org/10.1109/99.388960 [5] R.G. Allemang, Vibrations: Experimental Modal Analysis, 1999. UC-SDRL-CN-20-263-662 [2] T. Irvine, Shock Response Spectrum Synthesis Via Wavelets, Vibrationdata, 2000. [4] A. Grasps, An Introduction to Wavelets, IEEE Computational Science and Engineering vol. 2, num. 2, published by the IEEE Computer Society, Los Alamitos, 1995. https://doi.org/10.1109/99.388960 [5] R.G. Allemang, Vibrations: Experimental Modal Analysis, 1999. UC-SDRL-CN-20-263-662 References [1] T. Irvine, Shock Response Spectrum Synthesis Via Damped Sinusoids, Vibrationdata, 2012 [3] J. E. Alexander, A New Method to Synthesize an SRS Compatible Base Acceleration with Energy and Temporal Moments to Improve MDOF System Response, IMAC 36 Orlando, 7 S d & Vib ti 2018 [1] T. Irvine, Shock Response Spectrum Synthesis Via Damped Sinusoids, Vibrationdata, 2012 [2] T. Irvine, Shock Response Spectrum Synthesis Via Wavelets, Vibrationdata, 2000. [4] A. Grasps, An Introduction to Wavelets, IEEE Computational Science and Engineering vol. 2, num. 2, published by the IEEE Computer Society, Los Alamitos, 1995. https://doi.org/10.1109/99.388960 [5] R.G. Allemang, Vibrations: Experimental Modal Analysis, 1999. UC-SDRL-CN-20-263-662 747
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Subjektnormen in Orientierungsrahmen: Zur (Ir)Relevanz von Authentizitätsnormen für die künstlerische Praxis
Zeitschrift für qualitative Forschung
2,019
cc-by-sa
8,383
www.ssoar.info Subjektnormen in Orientierungsrahmen: zur (Ir)Relevanz von Authentizitätsnormen für die künstlerische Praxis Geimer, Alexander Veröffentlichungsversion / Published Version Zeitschriftenartikel / journal article Zur Verfügung gestellt in Kooperation mit / provided in cooperation with: Verlag Barbara Budrich Empfohlene Zitierung / Suggested Citation: Geimer, A. (2019). Subjektnormen in Orientierungsrahmen: zur (Ir)Relevanz von Authentizitätsnormen für die künstlerische Praxis. Zeitschrift für Qualitative Forschung, 20(1), 157-174. https://doi.org/10.3224/zqf.v20i1.12 Nutzungsbedingungen: Dieser Text wird unter einer CC BY-SA Lizenz (NamensnennungWeitergabe unter gleichen Bedingungen) zur Verfügung gestellt. Nähere Auskünfte zu den CC-Lizenzen finden Sie hier: https://creativecommons.org/licenses/by-sa/4.0/deed.de Terms of use: This document is made available under a CC BY-SA Licence (Attribution-ShareAlike). For more Information see: https://creativecommons.org/licenses/by-sa/4.0 Diese Version ist zitierbar unter / This version is citable under: https://nbn-resolving.org/urn:nbn:de:0168-ssoar-62403-9 Alexander Geimer Subjektnormen in Orientierungsrahmen: Zur (Ir)Relevanz von Authentizitätsnormen für die künstlerische Praxis Identity norms within frames of orientation ‒ On the (ir)relevance of authenticity norms for the professional practice of artists Zusammenfassung Jüngst findet sich eine Vielzahl an Ansätzen, die subjekttheoretische Überlegungen in sozial-, kultur-, bildungs- und erziehungswissenschaftliche Debatten tragen; dabei jedoch zumeist von empirisch angeleiteter Theoriebildung und methodologischen Diskussionen absehen. Umgekehrt besteht eine Normvergessenheit der qualitativen Sozialforschung, so dass letztere von subjekttheoretisch informierten Ansätzen lernen kann. Der vorliegende Text wendet sich – ausgehend von einer Kritik des Bourdieu’schen Feldbegriffs – der Frage zu, wie normative Ordnungen von AkteurInnen angeeignet werden. Anhand von Interviewanalysen mittels der Dokumentarischen Methode (aus dem Kontext des DFG-Projekts Aporien der Subjektivierung) rekonstruiert der Beitrag zwei unterschiedliche Relationen zwischen Habitus der (professionellen) künstlerischen Produktion von Kunst und der Subjektnorm eines authentischen Selbstausdrucks mittels der Kunst. Demzufolge handelt es sich, im Sinne von Bohnsacks Differenzierungen des Modells des (erweiterten) Orientierungsrahmens, um eine imaginäre wie zugleich imaginative Subjektnorm, die insbesondere von jenen angeeignet wird, die sich in (v.a. biografisch und nicht erfolgsbezogen gesehen) ‚Randpositionen‘ der professionellen Kunst befinden. Zudem lässt sich eine hohe Übereinstimmung dieser Norm des authentischen Selbstausdrucks zum öffentlichen Diskurs über gelungene Kunst (wie Lebenskunst) identifiZQF 20. Jg., Heft 1/2019, S. 157‒174 Abstract There is a growing body of work in sociology, cultural and educational studies which is orientated towards subject theory. But, these discussions are rarely grounded in empirical work and methodological considerations. Conversely, accounts of qualitative methods and methodologies regarding the production of subjects by normative orders are sparsely elaborated. Against this backdrop, this paper asks – after a critique of Bourdieu’s notion of the “field” – how social actors appropriate subject norms. By means of the analysis of interviews with professional artists (which were analyzed with the documentary method in the project aporias of subjectivation), I will reconstruct two different relations between professional habitus of artistic productions and the subject norm of having to produce authentic art in an authentic way. Following Bohnsack’s methodological modifications of frames of orientations, I will specify this subject norm as imaginary and imaginated appropriated by artists especially at the ‘fringes’ of the professional field (in terms of the academic training). They share their shaping of frames of orientations by authenticity ideals with the public discourse on fine arts (and the art of living) as a tentative discourse analysis is able to demonstrate. The paper is to understood primarily as a methodological contribution to the possibilities of the reconstruction (of the appropriation) of subject norms. https://doi.org/10.3224/zqf.v20i1.12 158 ZQF Heft 1/2019, S. 157‒174 zieren, die aufschlussreiche, diskursanalytische Anschlüsse eröffnet. Der Text versteht sich v.a. als methodologischer Beitrag, der Möglichkeiten der Rekonstruktion der Aneignung von Normen eines geforderten, idealen Subjekt-Seins auslotet. Schlagworte: Subjektivierung, Subjektnormen, Authentizität, Kunst, Dokumentarische Methode, Methodologie 1 Keywords: subjectivation, subject norms, authenticity, arts, documentary method, methodology Einleitung Die qualitative Sozialforschung, insbesondere in der Tradition des interpretativen Paradigmas sowie in Anschluss an den Symbolischen Interaktionismus hat bekanntlich die Kreativität problemlösenden Handelns und die eigensinnige Aushandlung von institutionalisierten Regeln und Normen hervorgehoben (Wilson 1973[1970]; Keller 2012). Wenngleich diese Perspektive als Korrektiv zur Sozialisations- und Handlungstheorie Parsons ohne Frage wichtig und wegweisend für die Entwicklung der Methoden qualitativer Forschung war, so kann heute konstatiert werden, dass in der Differenzierung des Feldes qualitativer Methodologien normative Ordnungen eher randständig, zumindest nicht systematisch behandelt werden (Geimer 2014, 2017; Geimer/Amling 2019; beispielhaft für diese Diagnose: Hitzler 2016). Sieht man von den Anregungen Goffmans (zur Relevanz von Identitätsnormen sowie Bourdieus Relationierung von Habitus und Feld ab (s.u., sowie Bohnsack 2017), so sind es jüngst v.a. Ansätze, die sich der Kategorie des Subjekts bedienen, um den Blick auf normative Ordnungen freizulegen. In mehr oder weniger elaborierten Theorien des Subjekts kulminieren Anstrengungen (zumeist in Anschluss an Althusser, Gramsci, Foucault und/oder Butler), übersubjektive Wissensordnungen hinsichtlich ihrer normativen Appellstrukturen und hegemonialen Adressierungen begrifflich zu fassen; etwa als „Subjektideale“ (Koppetsch 2006: 667 bzw. Alkemeyer/Budde/Freist 2013, S. 14), „diskursive Subjektentwürfe“ (ebd.), „Subjektpositionierungen“ (Bührmann/Schneider 2008, S. 30) oder als „Subjektivierungsform“ (Bröckling 2007) bzw. „Subjektformierungen“ (Bührmann 2012, S. 146) oder „Subjektformen“ bzw. „Subjektrepräsentationen“ (Reckwitz 2008, S. 137) sowie „Subjektivierungsfigur[en]“ (Bröckling 2012, S. 132) oder „Subjektivierungsregime“ (ebd., S. 134), wie auch als „diskursive Subjektfiguren“ (Geimer 2012, 2013, 2014) oder „Subjektmodell[e]“ (Keller et al. 2012, S. 10; wobei letztere die Liste erweitern um: ‚Subjektkulturen‘, […] ‚Subjektcodes‘, ‚Subjektivierungsangebote‘ “, ebd.). Während die Seite der Normen eines zu bevorzugenden Subjekt-Seins – zwar im Detail unterschiedlich (vgl. Saar 2013), aber in hohem Maße programmatisch – ähnlich gefasst ist, so wurde die Frage der Aushandlung und Aneignung jener normativen Bezugspunkte bislang vergleichsweise wenig (vgl. v.a. die Ansätze einer Subjektivierungsforschung: Pfahl/Traue 2012; Bosančić 2014; Geimer et al. 2019) oder eher methodologisch-methodisch sporadisch in gegenstandsbezogenen Kontexten etwa der ‚Studies‘ (gender, disability, postcolonial, cultural, media studies, s. auch Moebius 2009, S. 162ff.) behandelt, was insbesondere bereits für die frühen (Doing) Gender Studies gilt, da interaktiv auszuhandelnde Standardisie- A. Geimer: Subjektnormen in Orientierungsrahmen 159 rungen von Geschlecht stets auch als normative Ideale gefasst wurden (vgl. West/ Zimmerman 1987). Der vorliegende Beitrag illustriert anhand einer kritischen Auseinandersetzung mit Bourdieu und mit Bezug auf neuere Entwicklungen in der Praxeologischen Wissenssoziologie /Dokumentarischen Methode – am Beispiel der Analyse der Relevanz von Authentizitätsnormen in der professionellen Kunst – Möglichkeiten einer Rekonstruktion normativer Ordnungen aus der Perspektive bzw. innerhalb des Orientierungsrahmens der AkteurInnen. Dazu wird zunächst die Relation von Feld und Habitus nach Bourdieu anhand seiner kunstsoziologischen Studien dargelegt und das Konzept des erweiterten Orientierungsrahmens als eine methodologische Alternative diskutiert (Absatz 2). Anhand von Interviews mit professionellen KünstlerInnen aus dem Projekt Aporien der Subjektivierung wird einerseits die Fruchtbarkeit dieser Weiterentwicklung für das Anliegen der Subjektivierungsforschung (Rekonstruktion von Normen des Subjekt-Seins und ihrer Aneignung/Aushandlung) illustriert (Abs. 3), sowie andererseits ein Raum für Triangulationen mit der Diskursanalyse (Abs. 4) tentativ ausgelotet. Damit wird eine empirische Perspektive eröffnet, welche auch für die gegenstandsbezogene Soziologie der Kunst insofern attraktiv ist, als sich die in diesem Bereich „nahezu onmipräsent[e]“ (Glauser 2013, S. 27) Stellung der Bourdieuschen Theorie als überarbeitungsfähig erweist. 2 Diskussion methodologischer Grundlagen zur Rekonstruktion der Aneignung und Aushandlung normativer Ordnungen 2.1 Das Feld der Kunst und die Annahme der Omnirelevanz seiner normativen Ordnung In der Analyse der „Regeln der Kunst“ konkretisiert Bourdieu (2001[1992]) sein begriffliches Repertoire hinsichtlich des Konzepts des Feldes. Damit benennt er soziale Einheiten, die im Zuge sozialer Differenzierung eigene Organisationsprinzipien sowie explizite Regeln und implizite Praktiken ausgebildet haben und entsprechend anschlussfähig an spezifische Habitus sind bzw. diese – nach ihrem Eintritt in das Feld – transformieren, so in Bezug auf das Feld der (literarischen) Kunst (aber auch dem der Wissenschaft oder etwa Ökonomie). Voraussetzung für die – wie Folge der – Teilhabe an diesen Feldern ist generell das Teilen der „Illusio“ (vgl. Bourdieu 2001[1992], S. 360ff.), also des Glaubens daran, dass in diesen Feldern etwas auf dem Spiel steht, zu gewinnen ist – in Bezug auf die Kunst: künstlerischer Wert. Dieser ist umkämpft hinsichtlich der Deutungshoheit über die Legitimität konkurrierender Werte. Das Feld der Kunst steht, so Bourdieu (ebd., S. 344ff.), im Spannungsverhältnis zweier Pole: die kommerzielle Kunst, also der Mainstream, der sich durch den hohen Marktwert definiert und die Avantgarde bzw. die l’art pour l’art von interesselosem Eigenwert, der sich durch Distinktion vom (auf Erfolg ausgerichteten) Kunstmarkt definiert. Während die kommerzielle Kunst insbesondere um Kanonisierung und dabei um den eigenen 160 ZQF Heft 1/2019, S. 157‒174 Erhalt kämpft, konkurrieren am Gegenpol Varianten der (mehr oder weniger etablierten) Avantgarden um die Vormachtstellung als legitime Counter-Culture gegenüber dem Mainstream. Gemäß ihren Habitus nehmen die AkteurInnen in diesem dynamischen Kampf unterschiedliche Positionen ein bzw. ist es die Stratifikation des Felds, die als Platzanweiser für habituelle Dispositionen funktioniert: „Alle Positionen hängen in ihrer Existenz selbst und in dem, was sie über ihre Inhaber verhängen, von ihrer aktuellen und potenziellen Situation innerhalb des Feldes […] ab.“ (ebd., S. 365, H.n.i.O.) Dies gilt besonders für die Kunst, da dieses Feld aufgrund seiner inhaltlich-gegenstandsbezogenen Gestaltbarkeit für Menschen interessant ist, die „ihre nicht ganz gesicherte, widersprüchliche soziale Identität“ einbringen können, so Bourdieu (ebd., S. 359f.) und daher für Habitus-Transformationsprozesse besonders empfänglich sind. Die Normen des Feldes werden also – ähnlich wie die umfassende Illusio und der „kollektive Glaube[n] an das Spiel“ (ebd., S. 363) – in hohem Maße theoretisch relevant gesetzt und ihre „Einverleibung“ (kritisch dazu auch Bohnsack 2017, S. 298) stets vorausgesetzt. Bourdieu etabliert so eine immer schon gesetzte Anschlussfähigkeit der Habitus im Einflussgebiet des Kunstfelds an dessen beiden Pole bzw. eine grundlegende Omnirelevanz der Normen des Felds. 2.2 Weiterentwicklungen der Dokumentarischen Methode Das Erkenntnisinteresse der Dokumentarischen Methode war bislang – zumindest ganz erheblich – auf die Rekonstruktion eines impliziten Orientierungswissens bzw. die „Interpretation vorreflexiver oder genauer: atheoretischer Wissensbestände, denen handlungsleitende Qualität zukommt“ (Bohnsack 2012, S. 147), ausgerichtet. In neueren Arbeiten1 wird allerdings diskutiert, ob dem kommunikativ-generalisierten Wissen (Identitätsnormen, Fremdzuschreibungen, institutionalisierten Erwartungen) nicht eine größere Aufmerksamkeit geschenkt werden müsse – dies manifestiert sich insbesondere in der Erweiterung des Konzepts des Orientierungsrahmens bei Bohnsack (2014, vgl. auch die Studien von KhanZvornicanin 2016, von Sichart 2016), in dessen Zusammenhang die Relevanz einer weiteren Differenzierung des kommunikativen Wissens herausgestellt wird (dazu detailliert: Bohnsack 2017, S. 151ff.). Gemäß dieser Modifikationen erscheinen die Orientierungsrahmen von AkteurInnen in einer umfassenderen Komplexität, insofern die „theoretischen (Selbst-) Reflexionen“ der AkteurInnen und die von ihnen „als exterior erfahrenen Normen“ und das „gesellschaftliche Identifiziertwerden im Sinne der Fremdzuschreibung einer sozialen Identität“ (Bohnsack 2014, S. 35) nicht nur einem Habitus (und „Orientierungsrahmen im engeren Sinne“) gegenübergestellt, sondern in das Modell noch ausdrücklicher integriert werden, wobei der Fokus auf das Spannungsverhältnis gerichtet wird, in dem diese Wissensformen stehen und das es aufzudecken gelte: „Das Spannungsverhältnis von Habitus und Norm, welches ich, wenn es um die normativen Erwartungen an die Selbstpräsentation der AkteurInnen geht, auch als Spannungsverhältnis von Habitus und Identität bezeichne, stellt den Regel-, nicht den Ausnahmefall der alltäglichen Praxis dar.“ (Bohnsack 2017, S. 49; H.i.O.) Bohnsack greift zur Präzisierung der Ebene der „Identität“ (die als Teil des Orientierungsrahmens im erweiterten Sinne der Ebene des kommunikativgeneralisierten Wissen entspricht) auch auf den Begriff der „Identitätsnormen“ A. Geimer: Subjektnormen in Orientierungsrahmen 161 von Goffman (1967[1963], S. 130) zurück (Bohnsack 2014, S. 39), die sich „im Sinne der Diskursanalyse (u. a. Keller/Schneider/Viehöfer 2008) als ,Subjektcode(s)’ oder ,Subjektposition(en)’ verstehen“ (ebd.) lassen. Der Kurzschluss von der theoretischen Identifikation von Normen auf die Notwendigkeit ihrer Verinnerlichung, den Bourdieu, wie oben skizziert, bei der Analyse des künstlerischen Felds vornimmt, lässt sich so vermeiden bzw.: Die Kategorie des Orientierungsrahmens im weiteren Sinne ermöglicht „die Struktur der Handlungspraxis […] in ihrer Relation zur Dimension der Normen und Identitätserwartungen […] systematisch zum Gegenstand der empirischen Analyse zu machen. Die Relationierung mit den Kategorien der institutionalisierten Normen und der Identitätsnormen lässt sich als […] Äquivalent zur gesellschaftstheoretisch angeleiteten Bourdieu’schen Kategorie des Feldes verstehen.“ (Bohnsack 2017, S. 26, vgl. 302) Die (im Vergleich zur ‚klassischen‘ Version der Dokumentarischen Methode verstärkte) Berücksichtigung der (mehr oder weniger) exterior erfahrenen Identitätszuschreibungen und -erwartungen lässt sich also als ein Ansatzpunkt für die Rekonstruktion von Bezugnahmen auf diskursiv-hegemoniale Ordnungen verstehen und kann damit entscheidend helfen, die oben aufgeworfenen Fragen zur Relation zwischen bereichs- bzw. feldspezifischen normativen Ordnungen und der habituellen Logik der (hier: künstlerischen) Praxis zu beantworten. Diese Modifikation der Dokumentarischen Methode scheint damit in hohem Maße geeignet, subjektivierungsanalytische Fragen nach der handlungsleitenden Relevanz von Subjektnormen2 (s.o.) zu bearbeiten. Vor diesem Hintergrund ist insbesondere – neben der Fassung des Orientierungsrahmens als spannungsvoller Einheit von Identität und Habitus – die ebenfalls von Bohnsack eingeführte Differenzierung von Formen eines impliziten kommunikativ-generalisierten Wissens von Bedeutung. Denn die Kategorien eines imaginativen kommunikativen Wissens und eines imaginären kommunikativen Wissens können nicht nur dazu dienen, die Schnittstellen zwischen Habitus und Subjektnormen weiter zu konkretisieren, sie lassen auch den potenziell handlungsleitenden Charakter von diskursivhegemonialen Appellstrukturen genauer bestimmen: „Während also die AkteurInnen an der Performanz und Habitualisierung der imaginativen sozialen Identitäten orientiert sind und somit deren Bezug zur Praxis gegeben ist, gehen die AkteurInnen im Falle der imaginären sozialen Identitäten selbst nicht davon aus, diese virtualen Entwürfe zur Performanz zu bringen, das heißt eine Beziehung zur Performanz im Sinne einer möglichen Enaktierung, einer Neuorientierung wird von ihnen selbst ausgeschlossen beziehungsweise nicht mit dargestellt“ (Bohnsack 2017, S. 138). Diese beiden Kategorien werden von Bohnsack anhand von empirischem Material folgendermaßen illustriert: Zum einen erscheint am Beispiel einer Gruppendiskussion mit Jugendlichen ein alternatives Lebensmodell als „Traum“, der sich vor dem Hintergrund der „Unentrinnbarkeit der eigenen Alltagspraxis“ (ebd., S. 145) als imaginär erweist. Zum anderen scheint an einem (von Bohnsack aus dem Kontext des Projekts Aporien der Subjektivierung herangezogenen) Beispiel von Interviews mit PolitikerInnen eine „zunehmende Passung von Identitätsnorm und Handlungspraxis, von Norm und Habitus“ (Bohnsack 2017, S. 152) auf, die impliziert, dass es sich hier um imaginative soziale Identitäten handelt, also solche, die handlungsleitend sein können. Die Fruchtbarkeit dieser Unterscheidung möchte ich im Weiteren mittels der Ergebnisse des genannten DFG-Projekts im Bereich der Kunst detailliert herausarbeiten und also zeigen, wie diskursiv-normative Ordnungen in der Handlungspraxis von KünstlerInnen (auch nicht) wirksam werden. 162 ZQF Heft 1/2019, S. 157‒174 Dabei lassen sich verschiedene Formen der Relevantsetzung der identifizierten Authentizitätsnorm für einen Orientierungsrahmen (im erweiterten Sinn) der Produktion von Kunst herausarbeiten; zugleich lässt sich die Irrelevanz und Zurückweisung des Imperativs, authentisch zu sein, vor dem Hintergrund von anderen Habitus (Orientierungsrahmen im engeren Sinn) herausstellen. 2.3 Untersuchungsdesign im Rahmen des Projekts Aporien der 3 Subjektivierung Auf gegenstandsbezogene Vorarbeiten kann aufgrund des spezifischen, methodologischen Rahmens kaum zurückgegriffen werden. Die wenigen neueren empirischen Studien zum Feld der Kunst können kaum den Ansprüchen qualitativrekonstruktiver Forschung standhalten – weswegen dieser Beitrag kein Kapitel zum Forschungsstand umfasst. Hinsichtlich der freien (nicht industrie/designbezogenen) Kunst sind jenseits von historischen oder diskurstheoretischen Schriften über Künstlerbiografien (vgl. z.B. Mader 2009, Pelizäus-Hoffmeister 2006) kaum einschlägige Arbeiten zu finden. Thurn (1985) sowie Ingrisch (2012) nutzten zwar Interviews mit KünstlerInnen; das aber, erstens, nicht rekonstruktionslogisch im Sinne qualitativer Forschung und zweitens ohne einen systematischen Bezug auf Normen in der professionellen Kunst.4 Im Rahmen des Projekts Aporien der Subjektivierung wurden insgesamt 24 Interviews im Bereich der professionellen Kunst erhoben, von denen elf detailliert mittels der Dokumentarischen Methode ausgewertet wurden. Diese Interviews zielten auf die detaillierte Rekonstruktion der Aneignung und Aushandlung normativer Erwartungen, denen sich KünstlerInnen gegenübersehen. Es ging also sowohl um die von den AkteurInnen wahrgenommenen, normativen Anforderungen, als auch um die Analyse des impliziten Orientierungswissens, das die berufliche Handlungspraxis der Befragten im Sinne eines modus operandi strukturiert. Die Interviews waren in verschiedene thematische Abschnitte gegliedert (Biografie, zunächst allgemein, dann berufliche Karriere/n; Berufsalltag; professionelles Selbstverständnis). Die Abschnitte wurden jeweils durch einen allgemeinen Erzählstimulus eingeleitet, der auf die Erfahrungen der Befragten abzielte; dadurch wurde den KünstlerInnen die Gelegenheit gegeben, ihre eigenen Schwerpunkte in Bezug auf den (beruflichen) Werdegang und den Arbeitsalltag zu setzen. Nach dieser offenen, narrativen Phase und einer zweiten Phase der immanenten Nachfragen wurden dann auch Selbst-Positionierungen bzw. Argumentationen erfragt. Die Auswahl der zu befragenden KünstlerInnen folgte der Strategie der Suche nach (potenziell) starken Kontrasten (vgl. Glaser/Strauss 1967): Es wurden zum einen KünstlerInnen in einer frühen Karrierephase und etablierte KünstlerInnen adressiert; denn nicht zuletzt mit Blick auf die Expertiseforschung (Gruber 1999) ist anzunehmen, dass die unterschiedliche Dauer der Auseinandersetzung mit (beruflichen) Anforderungen und damit verbundenen normativen Erwartungen zu unterschiedlichen Formen des Umgangs mit diesen Anforderungen und Erwartungen führen könnte. Zum anderen war das Sampling auf die sogenannten freien Künste und hier auf VertreterInnen aus dem Bereich Zeichnung/Malerei (‚alte Medien‘) sowie solche aus dem Bereich Foto/Video (‚neue Medien‘) beschränkt, die sich in Bezug auf Arbeitspraktiken (Techniken, Materialien) stark unterscheiden (vgl. Tabelle 1). 163 A. Geimer: Subjektnormen in Orientierungsrahmen Tabelle 1: Interviews mit KünstlerInnen5 Frühe Karrierephase Erfahrene Professionelle m Einstein (10+M) Daniel (10+M) Dimitrij (10+M) Heinrich (10+M) Herr K. (30+M) Luca (20+) Otto (30+M) W Marlene (10+Z) Britta (10+M) Mascha (5+M) m Mirko (5+F) Igor (10+V) W Kathrin (6+V) Olga (8V) Irina (10+V) Alte Medien Neue Medien Steffi (20+) Laura (20+M) Anna (40+M) Amelie (15+Z) Lew (20+F) Hans (20+F) Ulrich (20+F) Boris (20+V) Sergej (20+F) - Die Auswertung der Interviews folgt zwei wesentlichen Prinzipien: Der Unterscheidung verschiedener Textsorten (im Wesentlichen nach Schütze 1987) und dem Prinzip der komparativen Sequenzanalyse (Nohl 2013; Bohnsack 2013). Die Unterscheidung der Textsorten diente dazu, die Passagen zu identifizieren, in denen Erzählungen und Beschreibungen eigener Handlungspraxis dominieren. Diese gelten in der Dokumentarischen Methode als primäre Grundlage der Rekonstruktion des impliziten Orientierungswissens der AkteurInnen (vgl. z.B. Nohl 2012, S. 20ff.). Da das Vorhaben aber auch auf die Analyse des expliziten Deutungswisssens der Befragten abzielte, rückten ebenso Passagen, in denen reflexive Positionierungen, Selbstentwürfe usw. dominierten, in den Fokus der Analyse. Diese wurden mit Blick auf die sich darin äußernden normativen Erwartungen, Fremdidentifizierungen usw. analysiert, und dann in Relation zu jenen Passagen gesetzt, in denen Erzählungen oder Beschreibungen eigener Handlungspraxis vorlagen. Komparativ war die Auswertung in mehrfacher Weise angelegt: Erstens wurden innerhalb eines Falles nach Homologien in Bezug auf das implizite Orientierungswissen gesucht, das sich, so die zentrale Annahme der Dokumentarischen Methode, in mehreren Passagen mit Erzählungen und Beschreibungen in gleichartiger Weise dokumentiert. Zweitens wurden die Relationen zwischen diesen impliziten und den kommunikativ-generalisierten oder expliziten Wissensbeständen der Befragten rekonstruiert, wobei letztere anhand der argumentativen Textteile herausgearbeitet wurden. Drittens wurden jeweils Homologien und Heterologien in den Blick genommen, die auf die Anerkennung (wie Zurückweisung) normativer Anforderungsprofile verweisen. 3 Ergebnisse der Interviewanalysen: Relevanz und Irrelevanz von authentizitätsbezogenen Subjektnormen Anhand der komparativen Analyse der Interviews lassen sich insbesondere zwei Aspekte herausarbeiten, über die sich die Fälle zu vier deutlich abgrenzbaren Ty- 164 ZQF Heft 1/2019, S. 157‒174 pen ordnen lassen. Zum einen ist das die Form, in der die künstlerische Praxis jeweils beschrieben wird, die auf die habituelle Logik künstlerischer Praxis oder den Orientierungsrahmen im engeren Sinne verweist (im Folgenden als Produktionshabitus bezeichnet); zum anderen ist das die Form, in der Subjektnormen aufgegriffen und/oder bearbeitet werden, die – relationiert mit der habituellen Logik künstlerischer Praxis oder eben dem impliziten Orientierungswissen, das diese Praxis strukturiert – auf den Orientierungsrahmen im weiteren Sinne verweist. Es ist diesbezüglich festzustellen, dass sich überhaupt nur in Bezug auf einen Produktionshabitus ein handlungsrelevanter Bezug auf eine in hohem Maße ähnliche (da jeweils authentitizitätsbezogene) Subjektnorm nachzeichnen lässt. Dieser Produktionshabitus bzw. die Formen der Relationierung von Produktionshabitus und Relevantsetzung von Authentizitätsnormen werden im Folgenden ausführlicher dargestellt (Abs. 3.1). Es ist insbesondere diese Relationierung, die dem Anliegen der Subjektivierungsanalyse zur Identifikation hegemonialer, normativer Anrufungen entsprechend (vgl. Pfahl/Traue 2012; Bosančić 2014; Geimer 2014) eine Rekonstruktion derselben aus Perspektive der AkteurInnen ermöglicht. Aus Platzgründen kann ich die Produktionshabitus (Orientierungsrahmen im engeren Sinne), für die sich in den empirischen Analysen kein handlungsrelevanter Bezug zu Authentizitätsnormen (bzw. entsprechende Distanzierungen) nachzeichnen lassen, nur in einer kurzen Zusammenfassung darstellen (Abs. 3.2). 3.1 Orientierungsrahmen im weiteren Sinne: Kunst als Medium des Selbstausdrucks und die Relevanz von Authentizitätsforderungen Im Weiteren stelle ich mit Bezug auf das empirische Material anhand von drei Fällen (Luca, Mirko, Daniel), die sich über die komparative Analyse einem Typ zuordnen lassen, einerseits die implizite Logik ihrer künstlerischen Praxis vor, andererseits aber auch deren Spannungsverhältnis zu authentizitätsbezogenen Subjektnormen. In allen drei Fällen wird die künstlerische Praxis eng an die persönliche Erfahrung gekoppelt: Kunst erscheint in den Beschreibungen eigener Produktionspraxis als erfahrungsgeleiteter Selbstausdruck bzw. als Ausdruck der „Seele“, wie das ein Interviewter (Daniel, s.u.) bezeichnet. Es sollen besonders schlimme oder auch positive, biografische Erfahrungen in der Kunst be- und verarbeitet werden und die Güte der Kunst bemisst sich daran, ob das gelingt. Für die Logik des künstlerischen Schaffensprozesses in den Fällen dieses Typs ist wesentlich, dass dieser Prozess ‚von innen‘ angeregt wird; es sind zwar mitunter eine Rahmung durch externe Vorgaben nötig oder Initialzündungen, die dazu führen, dass man seinen Stil und seine Medien für sich entdeckt; wichtig ist aber, dass diese zur Veräußerlichung eines inneren Selbst und der dieses prägenden Erfahrungen beitragen. Im Sinne der oben skizzierten Differenzierungen Bohnsacks zwischen einem imaginären und imaginativen Wissen (Abs. 2.2) lassen sich in den Fällen dieses ersten Typs zwei Varianten unterscheiden. Obschon es nämlich einem Unter-Typ (so bei Mirko) gelingt, unter bestimmten Bedingungen (etwa mittels „Kiffen“ in einem abgeschiedenen Dachzimmer bei lauter Opernmusik) das ganz Eigene, Persönliche im Kunstschaffen zu realisieren, konturiert sich das freie Einbringen der Persönlichkeit ins Werk hier als positiver, aber doch unerreichbarer Horizont – das Wissen um die Authentizitätsnorm lässt A. Geimer: Subjektnormen in Orientierungsrahmen 165 sich daher als ein imaginäres kommunikatives Wissen fassen, das sich kaum habituell umsetzen lässt und in einem ganz erheblichen Spannungsverhältnis zur Logik der eigenen Praxis der Produktion steht. In der folgenden Passage bearbeitet Mirko diese Ambivalenz der Norm des authentischen Selbstausdrucks: und ich bin irgendwie froh dass ich keine Ahnung dass ich kein Kindheitstrauma hab was ich irgendwie durch Kunst abarbeiten muss. //Hmm.// Jedenfalls nicht in der starken Form; ich hab natürlich auch irgendwie mein //Hmm.// Päckchen zu tragen aber (.) irgendwie is es (2) alles in gemäßigten Bahnen bei mir. //Hmm.// Aber das is im Endeffekt auch grade das Problem. //Hmm.// Dass ich das Gefühl habe, als Künst@ler@ (.) muss man quasi //Hmm.// (.) brauch man son Lebensthema. //Hmm.// Um sich (2) un man brauch auch diese Portion: Wahnsinn oder so um (.) um wirklich was Großes zu schaffen. Deswegen seh ich mich auch irgendwie eher als (2) Gebrauchs ((atmet laut aus)) künstler, weil ich irgendwie ne- also eher wie n Handwerker weißt du also n //Hmm.// n Tischler (2) hat n hat ne ne Fertigkeit Sachen zu bauen //Hmm.// aus Holz weil er n Geschick hat, n //Hmm.// und n Talent hat, mit Holz umzugehen, und ich hab irgendwie n Talent (.) zu zeichnen oder hatte es früher heute zeichne ich kaum noch aber ich //Hmm.// hab n Talent mit ((atmet laut ein)) ich=ab diese Technik verstanden und ich hab n Talent (.) mit Trickfilm umzugehen und daraus was zu schaffen, das halt //Hmm.// das macht mich noch nicht zum Künstler in mein Augen; //Hmm.// (2) das (.) macht mich zum //Hmm.// (.) künstlerischen Handwerker oder wie immer man das nennen will, //Hmm.// ch=seh mich da tatsächlich dem Handwerk näher. Auch wenn ich gerne (.)//Hmm.// rein vom Schaffen her (.) n Künstler wäre vielleicht aber. //Hmm.// Das Thema, dass Mirko ein „Lebensthema“ fehlt, das ihn umtreibt, wiederholt sich in mehreren Passagen und aus unterschiedlichen Perspektiven der Annäherung und Abgrenzung von der Authentizitätsnorm, die zur Findung und Bearbeitung dieses Lebensthemas anreizt. Hier verdeutlicht Mirko, dass er gewissermaßen das (Un-)Glück, hat, kein Künstler sein zu müssen. Er sieht bei seinen KollegInnen (wie er auch zuvor schildert), dass deren Künstler-Sein in der Bearbeitung von traumatischen Erfahrungen gründet. Dass sein „Päckchen“, das er zu tragen hat, also zu klein ist, ist im „Endeffekt“ das „Problem“ bzw. dass er keine grundlegenden, psychischen Probleme hat, ist sein Problem. Eine – ihm allerdings nicht als dauerhaft tragfähig erscheinende Lösung – ist, den „Wahnsinn“ künstlich mittels Drogen zu induzieren, wie er an anderer Stelle ausführlich ausführt. Anstatt dies allerdings zu forcieren, versucht sich Mirko damit abzufinden, kein Künstler zu sein und sich selbst als „künstlerischen Handwerker“ zu verstehen, der eine Begabung für Techniken und Medien hat. Zugleich „rein vom Schaffen“ her, also vom Prozesscharakter des Entstehens der Werke, wäre er gerne ein Künstler, der sich selbst in seinen Arbeiten ausdrückt. Mirko orientiert sich ganz offensichtlich an einem Subjekt-Ideal, welches sich auf ein spezifisches Modell (der Genese) des Künstlers bezieht, das er nicht erreichen kann und versteht dies zugleich als Glücksfall (er ist nicht traumatisiert genug), den er andererseits bedauert (er ist kein vollwertiger Künstler). Auch an anderer Stelle oszilliert die Verortung seiner künstlerischen Praxis zwischen „Künstler“, „Handwerker“ und „Agenturtyp“. Aufschlussreich ist diesbezüglich sein „Traum“, einen Film zu produzieren, von dem er „selbst überzeugt“ sagen kann: „Das bin ich, das ist ein Teil von mir“. Allerdings lässt sich dieser Traum der reinen Kunst und des Selbstausdrucks – der zugleich auch keine „öffentliche Therapie“ sein sollte – nicht realisieren. Dennoch versucht Mirko in seinen Auftragsarbeiten „seine eigene eigene Note, oder sich sich sel- seinen eigenen Anteil so zu finden“. In anderen Fällen desselben Typus hingegen besteht eine stärkere Passung zwischen jener Subjektnorm, sich authentisch auszudrücken und der Logik der 166 ZQF Heft 1/2019, S. 157‒174 künstlerischen Praxis: Der positive Horizont des Selbstausdrucks wird dann in der eigenen Arbeit mit und an den eigenen Erfahrungen in der Kunst möglichst enaktiert; entsprechend möchte Daniel in seinen Werken etwas von sich „preisgeben“, will, dass man in seiner Kunst „enorm viel über“ ihn „erfährt“ („So wie ich so ticke °oder° was fürn (.) was für ne Seele ich sozusagen bin“), was ihm aus seiner Perspektive und auch aus der Perspektive signifikanter Anderer auch gelingt. Dies wird deutlich, wenn er manche Reaktionen des Publikums auf seine Arbeiten beschreibt und betont, dass es „viele aus den romanischen Ländern“ – „vor allem französische Frauen“ – seien, die seine „besondere Sensibilität“ in seinen Arbeiten „gespürt“ hätten (vgl. dazu auch Geimer 2014 über Authentizitätsideale in der Popmusik). Der persönliche Ausdruck führt zum gewünschten Eindruck mancher Rezipientinnen, die Daniel so erkennen, wie er sich in den Werken preisgibt und selbst versteht: „Und die ham sofort diese Metaebene gespürt, un=und auch dieses Brüchige was ich habe. Gab ja mehrere Brüche so auch=in meim Leben, und äh (.) un- das fließt aber positiv in die Kunst hinein.“ Vor diesem Hintergrund lässt sich argumentieren, dass in diesem Unter-Typ des Habitus Kunst als Medium des Selbstausdrucks ein imaginatives kommunikatives Wissen sichtbar wird, das die habituelle Logik der Produktion anleiten kann. Ein weiterer Fall eben dieses Unter-Typs liegt mit Luca vor. In der folgenden Passage beschreibt er, dass (und vorher: wie) er zu seinem eigenen Stil gefunden hat und was das für ihn bedeutet: Und jetz weiß ich ich hab meinen Stil jetz, also meine Bilder sind unverwechselbar und es=is nachweislich das kann man nirgendswo so=was sehen; //Hmm.// das is, es freut mich dass ich meine Wege gefunden habe=weil das soll der Sinn des sein das wenn man was macht, //Hmm.// das man seine eigene Wegen geht. //Hmm.// Un mit diese Serie Kreise des Lebens und Spuren von Leben=das=is so=was von intim das=is wirklich ähm kein Mensch nachmachen kann; weder nachmachen was ich bis jetz gemacht habe oder ich von jemand nachmachen; weil das sind, hier es geht wirklich um Erinnerung von meine venezolanische Zeit beziehungsweise um ein neues (.) ja Wesen in sein=seine Kreise aufzunehmen un=das=is äh ich bin diese Serie Kreise des Lebens //Hmm.// hab ich äh meine Tochter inspiriert als sie //Hmm.// geboren wurde und da sind wirklich so (im unten) ich glaube vielleicht //Hmm.// könnte man so meinen=ich glaub Kunst geht darum dass man wirklich an eigene Erfahrungen selbst ähm verarbeitet und //Hmm.// daraus entstehen Sachen die andern Mensch auch interessiert weil man sich auch identifiziert; darum geht das. //Hmm.// Luca stellt heraus, dass es vor allem die „eigenen Wege“ sind, die es zu finden gilt, was ihm gelungen sei. Diese Authentizität der eigenen Arbeiten steht für ihn in engem Zusammenhang mit dem Distinktionswert, denn sie garantiert eine Unverwechselbarkeit: Man kann das nirgendwo anders sehen und die Arbeiten sind so „intim“, dass sie weder (von anderen) nachgemacht werden, noch (von ihm) nachgemacht sein können. Sie sind authentisch als Selbstausdruck, der für sich und die Manifestation seiner Erfahrung steht und zertifizieren damit die Echtheit der Werke – womit der Ausdruck des Selbst auch marktrelevant wird. Das nicht zuletzt deshalb, weil die Verarbeitung eigener Erfahrungen die Möglichkeit für kommunikativ erfolgreiche Anschlüsse erhöht: Andere Menschen können sich „interessier[en]“ und „identifizier[en]“. Insofern ist festzuhalten, dass sich der Anspruch, authentisch zu sein, bei Luca gerade dadurch enaktieren lässt, dass die Norm so angeeignet wird, dass sie zwar befolgt wird, aber zugleich ihres moralischen Charakters der Zweckfreiheit entkleidet wird. Seine Kunst ‚ist‘ also authentisch; dies aber auch und ganz wesentlich, um sie verkaufen zu können. A. Geimer: Subjektnormen in Orientierungsrahmen 167 3.2 Orientierungsrahmen im engeren Sinne: Habitus des Schaffens von Kunst und die Irrelevanz von Authentizitätsforderungen Ein zweiter Produktionshabitus kann nun (aus Platzgründen kurz) im Kontrast zu dem Habitus des Selbstausdrucks darüber charakterisiert werden, dass in den Beschreibungen und Erzählungen der Befragten (Fälle Einstein, Britta, Marlene) sich ihre eigene künstlerische Praxis als ein forschendes Experimentieren konturiert. Dies kann eher auf ästhetisch-theoretische oder haptisch-materielle Aspekte des Materials, mit dem gearbeitet wird, bezogen sein. In Bezug auf den Umgang mit authentizitätsbezogenen Subjektnormen wird sehr deutlich, dass die Erwartung, authentisch zu sein, zudem in allen drei Fällen (auf exmanente Nachfrage) zwar ge- und erkannt, aber nicht anerkannt wird und in keinem Bezug zur eigenen künstlerischen Praxis steht. Auch in den Interviews, anhand derer sich ein dritter Habitus der künstlerischen Produktion (Fälle Otto, Kathrin und Olga) dokumentiert, werden weder Erfahrungen des Schaffens, die auf Authentizitätsnormen verweisen, geschildert, noch kann auf die explizite Nachfrage nach der Relevanz von Authentizitätsforderungen ein persönlicher Bezug zu diesen hergestellt werden. Den Fällen dieses Typs ist gemeinsam, dass sich ihr Zugang zur künstlerischen Praxis als ein theoretisch-konzeptioneller verstehen lässt, der sozialkritisch fundiert ist: Die Werke werden so gestaltet, dass sie ein hohes Maß an Möglichkeit zur Irritation aufweisen und zum Nachdenken über soziale Konventionen anregen. Im Unterschied zu den genannten Typen wird das Schaffen in den Fällen von Karl und Steffi kaum als aktive Produktion beschrieben, sondern vielmehr als Erfahrung, der man sich überlassen muss oder als ein Zustand, in den sich der/die KünstlerIn begibt und der für die Dauer des Schaffens zu erhalten ist. Die Rolle des/der KünstlerIn besteht für diese KünstlerInnen insofern darin, sich im Sinne eines Mediums, in dem sich die Kunst selbst ausdrückt, verfügbar zu halten und sich den objektiv neuen Möglichkeiten, welche die Welt der Kunst bietet, zu öffnen. Deren Realisierung lässt sich mit einem Bildtitel von Sigmar Polke zusammenfassen, welchen Steffi zitiert: „Höhere Wesen befahlen: Rechte obere Ecke schwarz malen!“. Und Karl formuliert genau dieses Prinzip, gemäß dem sich die Kunst selbst mitteilt, mit eigenen Worten: „Man sacht also ich weiß gar nicht warum aber der (.) grüne Fleck soll jetzt rot werden; (.) glaub des wird besser @(.)@ (.).“ Beide zeigen sich in ihrer künstlerischen Praxis auch nicht von Authentizitätsnormen angeleitet; Karl hinterfragt diese vielmehr vehement anhand eines Beispiels (Mozarts Biographie). 4 Diskussion der Ergebnisse. Ausblick auf diskursanalytische Anschlüsse Hinsichtlich der Frage welche Gemeinsamkeiten bestehen könnten, die dazu führen, dass Mirko, Daniel und Luca eine geteilte, starke Orientierung an der Norm eines authentischen Selbstausdrucks aufweisen, lassen sich aus dem Material und im Sinne einer soziogenetischen Interpretation keine Hinweise finden; den- 168 ZQF Heft 1/2019, S. 157‒174 noch allerdings zeichnen sich Aspekte einer soziogenetischen Typenbildung ab. Alle Fälle des Typus Kunst als Medium des Selbstausdrucks befinden sich im Feld der Kunst in einer gewissen ‚Randposition‘. Dies weniger hinsichtlich der Positionierung auf dem Kunstmarkt oder des finanziellen Erfolgs, aber alle anderen Fälle haben ein Kunststudium hinter sich und sind (mehr oder weniger etablierte) ‚freie‘ KünstlerInnen schon von ihrer Ausbildung her.6 Wirft man nun einen schlaglichtartigen – hier nur tentativ vorzunehmenden, aber aufschlussreichen – Blick auf öffentliche Diskurse über die Kunst findet sich eine hohe Übereinstimmung hinsichtlich der Relevanz von Authentizitätsnormen, was sich u.a. anhand des Ratgebers „Der Weg des Künstlers“ (Cameron 1996 [1992]) darlegen lässt. In dieser Anleitung zur Findung und Pflege künstlerischer Aktivität wird „Kreativität [als] unsere wahre Natur“ (ebd., S. 16) zu verstehen gegeben, als eine innere, zutiefst persönliche Qualität des Seins, die es für KünstlerInnen zunächst zu finden und dann zu erhalten gilt. Die Norm, authentisch zu sein, funktioniert also in diesem Dokument eines öffentlichen Diskurses als eine potenziell „subjektivierende Macht, welche die Menschen zur Suche nach ihrer Wahrheit“ anreizt (Bröckling 2017, S. 21). Die Entdeckung dieser ‚Eigentlichkeit‘ verspricht die Möglichkeit, diese auch mitteilbar und zum Ausgangspunkt und Gegenstand der eigenen Arbeit zu machen: „Wir begegnen unserer Wahrheit und dadurch uns selbst; wir begegnen uns selbst und dadurch unserem Selbstausdruck. Wir werden authentisch, weil wir zu einer Quelle geworden sind, aus der die Arbeit fließt.“ (Cameron 1996, S. 152) Die (Fokussierungs)Metapher der „Quelle“ veranschaulicht weniger, dass die künstlerische Produktion nur läuft, fließt oder sprudelt, sondern v.a. dass dies wie ‚von selbst‘, tief aus dem eigenen Selbst heraus geschieht; ohne die Mühen reflexiver Distanzierung oder kritischer Befragung, sondern in schlafwandlerischer Sicherheit der Selbsterleuchtung derer, die sich wahrhaftig kennen. Dieser Diskurs überschneidet sich in hohem Maße auch mit den Authentizitätsidealen zu einer generell gelungenen Lebensführung im Sinne der ‚Kunst des Lebens‘ – entsprechend ergehen Hinweise zur „Kunst du selbst zu sein“ (Sierck 2016) oder zu der „Kunst, ehrlich, authentisch und einfach du selbst zu sein“ (Jones 2015). Kunst und Authentizität bilden in der Ratgeber-Literatur also einen – nicht nur adressiert an KünstlerInnen sondern auch LebenskünstlerInnen – engen Verweishorizont. Mit Ferrara lässt sich dieser als zirkuläre Relation zwischen Urteilen über die Wohlgeformtheit von Kunstwerken und von Identitäten fassen: „our judgements on the well-formedness of works of art share many features in common with our judgements on the […] authenticity of an individual identity“ (Ferrara 1998, S. 142); etwa hinsichtlich der Einzigartigkeit und Unersetzbarkeit (ebd., S. 143), die authentische Werke und authentische Personen aufzuweisen haben. Entsprechend gilt es nicht nur, aber besonders für KünstlerInnen, „inneren Kontakt“ (Cameron 1996, S. 41) zu sich herzustellen und „die eigene Identität zu entdecken“ (ebd., vgl. auch zum Zusammenhang von Kreativität und Authentizität: Bröckling 2017, S. 415). Denn: Erst wer sich entdeckt hat, kann auch von anderen entdeckt werden, sich selbst auch sichtbar und wahrnehmbar machen und in öffentliche Kontexte (authentisch) einbringen – das heißt, dass wir „um einen Selbstausdruck zu haben, zuerst ein Selbst haben müssen, das wir ausdrücken können“ (Cameron 1996, S. 149). Ganz in diesem Sinne wird auch in dem YouTube-Ratgeber „Sei du selbst – 5 Tipps, um authentisch zu sein!“7 als erster Tipp von einer Diplom-Psychologin genannt: „Ganz klar; wer authentisch leben möchte; der muss erst mal wissen wer er überhaupt ist; was er für ein Mensch ist, der muss seine eigenen Charakterei- A. Geimer: Subjektnormen in Orientierungsrahmen 169 genschaften kennen […].“ Vor diesem Hintergrund ist also festzustellen, dass öffentliche Diskurse zur (Lebens)Kunst eine starke Homologie zum rekonstruierten Habitus des Selbstausdrucks in der Akzentuierung des normativen Leitbilds des Authentisch-Seins aufweisen. Bedenkt man, dass die Vertreter dieses Habitus eine Fallgruppe darstellen, die ausnahmslos von ‚Randpositionen‘ in der Kunst besetzt ist (s.o.), lässt sich argumentieren, dass öffentlich-diskursive Authentizitätsnormen in die künstlerische Praxis dann imaginativ hineinragen, oder dieser imaginär gegenüberstehen (und ein ambivalentes Selbstverständnis produzieren), wenn die Akteure längere Zeit nicht in der freien Kunst beheimatet waren bzw. eine andere (wenngleich verwandte) berufliche Sozialisation kennen. Mit anderen Worten: Unter diesen Voraussetzungen können Common Sense-Normen des Selbstausdrucks eine größere Relevanz für die künstlerische Praxis entfalten. Umgekehrt erweisen sich im professionellen Kerngebiet der Kunst (das heißt: an Kunsthochschulen) sozialisierte AkteurInnen kaum beeindruckt von der Common Sense-Forderung nach der Authentizität der Kunst und ihres künstlerischen Schaffens. Diese Überlegungen legen an dieser Stelle also eine Weiterführung und Ausweitung der vorliegenden Studien nahe; etwa um diskursanalytische Arbeiten, um über diese auch soziogenetische Analysen über die Relevanz von imaginativen und imaginären Subjektnormen voranzutreiben (und um die sich deutlich abzeichnenden Homologien zu validieren und ggf. weiter zu differenzieren8). 5 Fazit Der theoretisch-methodologische Teil des Beitrags problematisierte die HabitusFeld-Relation Bourdieus, der zufolge Normen des Felds stets (wenngleich ggf. durch Brüche, etwa infolge Milieuwechsel) inkorporiert und zu Komponenten des Habitus werden. Indem feldspezifische Normen und Habitus a priori theoretisch zusammengezogen werden, sind sie empirisch nicht ausreichend zu unterscheiden bzw. diktiert das Feld das Subjekt, wie auch Schirato und Webb (2010, S. 260) pointieren: „If the field ‘is’ the subject to a large extent, then any […] relation to the doxa and illusio of the field must be a constitutive part of that field.” Im Rahmen neuerer Konzepte der Praxeologischen Wissenssoziologie (Bohnsack 2017) und im Sinne einer dokumentarisch inspirierten Subjektivierungsanalyse (vgl. bspw. Geimer/Amling 2017; 2019), ließ sich hier die Relation von Norm und Habitus weitergehend präzisieren, indem ein Orientierungsrahmen im weiteren Sinne und drei Orientierungsrahmen im engeren Sinne (eines Habitus) rekonstruiert wurden. Die letzteren (drei) Habitus der Produktion von Kunst weisen keinerlei praxisrelevanten Bezüge zu Authentizitätsnormen auf und lassen sich folgendermaßen unterscheiden: Kunst als Medium ästhetischer Forschung, Kunst als Medium politischer Kommunikation bzw. ästhetischer Intervention, Kunst als Medium der Realisierung neuer Möglichkeiten der Kunst. Ich habe kurz die Eigenheiten dieser Habitus des Machens von Kunst differenziert; allerdings bestehen auch Gemeinsamkeiten zwischen diesen Typen, insbesondere dahingehend, dass sich in allen Fällen zeigt, dass die freie Kunst vom anwendungsbezogenen Design zu unterscheiden ist: Fast keiner der Befragten berichtet von (eigenen) Auftragsarbeiten. Lediglich jener Habitus, demgemäß Kunst ein Medium des Selbstausdrucks darstellt, weist in dieser Hinsicht einen abweichenden Fall auf. 170 ZQF Heft 1/2019, S. 157‒174 Mirko arbeitet auch als Auftragskünstler (etwa mit Videoinstallationen im Theater) und für seine künstlerische Praxis erweist sich die Subjektnorm des Schaffens aus eigenen Erfahrungen insofern als relevant, als es sich um ein nicht einholbares Ideal handelt, an dem sich Mirko misst und das er zugleich verwirft. Künstlerische Authentizität bleibt für Mirko imaginär und unerreichbar und doch ein höchst relevanter Bezugspunkt, vor dessen Hintergrund er sich selbst (‚glücklicherweise‘ als defizitär, s.o.) versteht. Anders bei den Fällen Daniel und Luca, die auf unterschiedliche Weise die Norm der Authentizität in die Logik ihrer künstlerischen Praxis imaginativ übernehmen: Daniel legt seine Werke so an, dass grundlegende Persönlichkeitsstrukturen („Seele“, „Brüche“) darin gefunden werden können und berichtet, dass v.a. Frauen aus Südeuropa diese in seinen Arbeiten erkennen. Luca bezieht sich ebenfalls auf eigene Erfahrungen (z.B. Geburt der Tochter, Lebensgefühl und Farben seiner Heimat Venezuela), um authentische Anschlüsse herzustellen und Interesse und Identifikationen auszulösen; allerdings wird hier die Zweckfreiheit des Selbstausdrucks (der Daniel und Mirko folgen) aufgegeben: sich selbst authentisch auszudrücken, bedeutet für Luca auch: nicht kopiert zu werden, die Konkurrenz am Markt zu gewinnen, also so unverwechselbar originell zu sein, dass jedes Bild ein offensichtliches Original darstellt. Diese Analysen weisen also darauf hin, dass imaginativ angeeignete Subjektnormen in einem geringeren Spannungsverhältnis zum Habitus stehen als imaginär angeeignete Normen, indem sie sowohl eher enaktiert wie entsprechend affirmativer wahrgenommen werden. Weitere Relationen zwischen Habitus und (anderen) Subjektnormen (etwa in der professionellen Politik, vgl. Geimer 2017; Geimer/Amling 2019), die auch gar nicht im Rahmen eines Spannungsverhältnisses behandelt werden und vor allem auf habituelle Entsprechungen zu Subjektnormen sowie Aneignungsprozesse derselben verweisen, können in diesem Beitrag nicht diskutiert werden. Die hier vorgenommenen Analysen sind in zwei Aspekten in besonderem Maße relevant: Erstens hinsichtlich potenzieller Verschränkungen mit diskursanalytischen Untersuchungen, welche erlauben, die öffentliche Repräsentation von kommunikativ-generalisierten Authentizitätsnormen weitergehend in den Blick zu nehmen. Vor diesem Hintergrund ergeben sich auch für die Kunstsoziologie spannende, empirische Anschlussfragen. Denn die Kunst stellt, so Danko und Glauser (2012, S. 4) in einem Überblick zu klassischen wie neueren Perspektiven der (speziellen) Kunstsoziologie, für die (allgemeine) Soziologie eine „schwierige Herausforderung [dar], insofern Kunstwerke weithin als Ausdruck des Individuellen fungieren und Kunst maßgeblich entlang von Kategorien wie Originalität, Authentizität […] codiert wird“. Wie ich hier, wenngleich schlaglichtartig, aber deutlich zeigen konnte, ist eine generalisierende Einschätzung gerade der Relevanz von Authentizitätsnormen erheblich zu differenzieren, da eben nicht alle, sondern lediglich spezifische Habitus der Produktion von Kunst an Authentizität(snormen) ausgerichtet sind und diese Ausrichtung zugleich als diskursiv vortypisiert verstanden werden kann (vgl. Abs. 4), was für andere Habitus der Produktion der Kunst aber kaum eine Prägekraft entfaltet. Insofern können aus den dargestellten Ergebnissen und ihrer Diskussion auch methodologische Hinweise zu Triangulationen gewonnen werden: Diskursanalytische Arbeiten und die Rekonstruktion von (imaginären und imaginativen) Subjektnormen im Rahmen der Praxeologischen Wissenssoziologie können sich gegenseitig validieren. Um diese Perspektive weiter auszuloten, wären nun potenzielle Anschlüsse an Varianten der Diskursanalyse (u.a. im Rahmen der Dokumen- A. Geimer: Subjektnormen in Orientierungsrahmen 171 tarischen Methode, zuletzt etwa Nohl 2016) zu prüfen, die ich hier aus Platzgründen nicht diskutieren kann. Zudem ermöglicht, zweitens, die Rekonstruktion der Relation normativer Ordnungen und handlungsleitender Orientierungen (im Konzept des erweiterten Orientierungsrahmens) der jüngst – vor allem subjekttheorisch und poststrukturalistisch informiert – geführten Diskussion um den Stellenwert von hegemonialen Normen eines geforderten Subjekt-Seins eine empirische Fundierung und kann einer spezifisch rekonstruktiven Subjektivierungsforschung eine systematische Gestalt verleihen. Allerdings ist auch diese Perspektive mit Einschränkungen verbunden. Dass keine normativen, gemeinsamen Bezugspunkte in allen Habitus der künstlerischen Produktion und drei Orientierungsrahmen ‚nur‘ im engeren Sinne zu identifizieren waren, bedeutet freilich nicht, dass keine entsprechend handlungsleitenden Normen für jene drei Habitus bestehen. Vielmehr waren andere Subjektnormen – in dem gewählten Untersuchungsdesign bzw. mittels der vorgestellten Interviewanalyse9 – nicht hinsichtlich ihrer handlungsleitenden Relevanz zu beobachten. Subjektivierung als empirisch nachweisbare Ausrichtung eines Habitus an Normen eines zu bevorzugenden Subjekt-Seins (hier des Authentisch-Seins als KünstlerIn) kann also, einerseits, nicht nur nie die AkteurInnen in ihrer Ganzheit umfassen, sondern, andererseits, auch empirisch lediglich beobachtbar für bestimmte Habitus-Typen sein. Von daher würde eine solche Interpretation des vorliegenden Materials auch fehlgehen, welche von Authentizitätsnormen unbeeindruckte Habitus (und die drei Orientierungsrahmen im engeren Sinne) als ‚autonomere‘, ‚freiere‘ (oder gar ‚authentischere‘) Subjekte‘ begreifen würde. Abschließend ist daher der Beitrag der Dokumentarischen Methode zur Analyse der Prägekraft normativer Ordnungen zwar hervorzuheben, aber auch insofern einzuschränken, als dass die Rekonstruktion von Orientierungsrahmen im erweiterten Sinne (der Relation von Habitus und Subjektnormen) einen Zugriff auf die Totalität eines Habitus ebenso wenig impliziert wie eine objektive Identifikation normativer Ordnungen. Anmerkungen 1 2 3 4 Vgl. die Überlegungen zu einer dokumentarischen Subjektivierungsforschung (Geimer 2014, 2017; Geimer/Amling 2017), zur Organisationsforschung (Amling/Vogd 2017) oder Diskursanalyse (Nohl 2016) sowie bereits der Evaluationsforschung (Bohnsack/Nentwig-Gesemann 2010). Ich spreche im Weiteren nicht von Identitäts-, sondern Subjektnormen, um den normativen Appellcharakter derselben in Anschluss an subjekttheoretische Diskurse zu betonen (vgl. Absatz 1, sowie Geimer 2017; Geimer/Amling 2019). Ein Subjekt in diesem Sinne ist immer eines das werden soll und dadurch Handlungsspielräume gewinnen kann: „There are two meanings of the word ,subject': subject to someone else by control and dependence, and tied to his own identity by a conscience or self-knowledge.” (Foucault 1982: 781) Es handelt sich um das DFG-Projekt Aporien der Subjektivierung. Zur Aneignung und Aushandlung hegemonialer Subjektfiguren mittels einer Weiterentwicklung der Dokumentarischen Methode am Beispiel der beruflichen Sozialisation in der professionellen Politik und Kunst (April 2015 - August 2017). Während Thurn schon angesichts des umfassenden Samples von 110 KünstlerInnen auf quantitative Analysetechniken zurückgreifen muss, so zieht Ingrisch (2012) ihr Material heran, um zuvor postulierte Orientierungsdilemma der Spätmoderne und dann deren 172 5 6 7 8 9 ZQF Heft 1/2019, S. 157‒174 Lösung exemplarisch zu erörtern – und um auszuloten, wie „Kreativität und Improvisationskunst, und also das, was KünstlerInnen uns an Werten und Gestaltungslust vermitteln, über theoretische Annahmen hinweg Inspiration für gesellschaftliche Problematiken bieten“ (ebd.: 12). Die Namen der Befragten sind anonymisiert; die Zahlen hinter den Namen stehen für die Dauer der Berufserfahrung; die Buchstaben für die Medien, mit denen die KünstlerInnen jeweils arbeiten (M: Malerei; Z: Zeichnung; V: Video; F: Foto); die fett gedruckten Fälle wurden detailliert ausgewertet. Mirko arbeitet auch und vor allem an (gut finanzierten) Auftragsarbeiten und zudem (fest angestellt) als Werkstattleiter an einer Kunsthochschule (was er als einen „goldenen Käfig“ bezeichnet). Luca und Daniel arbeiten zwar im Bereich der klassischen, ‚freien Kunst‘, sind allerdings beide Quereinsteiger ohne Kunststudium, die zunächst andere (verwandte) Berufe lange ausgeübt haben: Luca war bis zum Alter von etwa 40 Jahren nur Kunsthändler (und ist das noch immer, wobei er auch sich selbst vermarktet), Daniel bis etwa zu diesem Alter Restaurateur (und hat diesen Beruf aufgegeben). https://www.youtube.com/watch?v=ouMpWifE6uA Eine stärkere Differenzierung der Ergebnisse lässt sich zudem mittels der vergleichenden Analyse von Gruppendiskussionen mit (teilweise gleichfalls interviewten) professionellen KünstlerInnen vornehmen, vgl. dazu Geimer/Amling 2018. 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Extended cleavage specificity of human neutrophil cathepsin G: A low activity protease with dual chymase and tryptase-type specificities
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RESEARCH ARTICLE Editor: Albert Jeltsch, Universita¨t Stuttgart, GERMANY Received: April 10, 2017 Accepted: February 22, 2018 Published: April 13, 2018 Copyright: © 2018 Thorpe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2018 Thorpe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This study was funded by the Swedish Research Council (621-2011-5007). GDL Pharmaceutical Consulting and Contracting provided support in the form of salary for Lawrence de Garavilla and the provision of reagents. The specific roles of this author are articulated in the ‘author contributions’ section. The funders had no role in study design, data collection Extended cleavage specificity of human neutrophil cathepsin G: A low activity protease with dual chymase and tryptase- type specificities Michael Thorpe1, Zhirong Fu1, Gurdeep Chahal1, Srinivas Akula1, Jukka Kervinen2, Lawrence de Garavilla3, Lars Hellman1* 1 Department of Cell and Molecular Biology, Uppsala University, Uppsala, The Biomedical Center, Uppsala, Sweden, 2 Fraunhofer USA Center for Molecular Biotechnology, Newark, United States of America, 3 GDL Pharmaceutical Consulting and Contracting, Downingtown, Pennsylvania, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * Lars.Hellman@icm.uu.se * Lars.Hellman@icm.uu.se * Lars.Hellman@icm.uu.se Abstract Human neutrophils express at least four active serine proteases, cathepsin G, N-elastase, proteinase 3 and neutrophil serine protease 4 (NSP4). They have all been extensively stud- ied due to their importance in neutrophil biology and immunity. However, their extended cleavage specificities have never been determined in detail. Here we present a detailed cleavage specificity analysis of human cathepsin G (hCG). The specificity was determined by phage display analysis and the importance of individual amino acids in and around the cleavage site was then validated using novel recombinant substrates. To provide a broader context to this serine protease, a comparison was made to the related mast cell protease, human chymase (HC). hCG showed similar characteristics to HC including both the primary and extended specificities. As expected, Phe, Tyr, Trp and Leu were preferred in the P1 position. In addition, both proteases showed a preference for negatively charged amino acids in the P2´ position of substrates and a preference for aliphatic amino acids both upstream and downstream of the cleavage site. However, overall the catalytic activity of hCG was ~10-fold lower than HC. hCG has previously been reported to have a dual specific- ity consisting of chymase and tryptase-type activities. In our analysis, tryptase activity against substrates with Lys in P1 cleavage position was indeed only 2-fold less efficient as compared to optimal chymase substrates supporting strong dual-type specificity. We hope the information presented here on extended cleavage specificities of hCG and HC will assist in the search for novel in vivo substrates for these proteases as well as aid in the efforts to better understand the role of hCG in immunity and bacterial defence. OPEN ACCESS Citation: Thorpe M, Fu Z, Chahal G, Akula S, Kervinen J, de Garavilla L, et al. (2018) Extended cleavage specificity of human neutrophil cathepsin G: A low activity protease with dual chymase and tryptase-type specificities. PLoS ONE 13(4): e0195077. https://doi.org/10.1371/journal. pone.0195077 Editor: Albert Jeltsch, Universita¨t Stuttgart, GERMANY Extended cleavage specificity of human cathepsin G non-oxygen dependent mechanisms as well as neutrophil extracellular traps (NETs), which are web-like structures consisting of nuclear DNA, histones and granule components, to kill bacteria [3–5]. In addition to secretory vesicles, three types of cytoplasmic granules are found within neutrophils, the specific granules, the azurophilic granules, and the gelatinase granules [6, 7]. These granules contain a number of different components and the majority of them are involved in bacterial defence. Several abundant antibacterial peptides including cathelicidins and defensins, are found in these granules together with other antibacterial substances such as bacterial permeability increasing protein (BPI), lysozyme, lactoferrin and several serine prote- ases [6–8]. Four such active proteases have been identified: cathepsin G (CG), N-elastase (NE), proteinase 3 (PR3) and neutrophil serine protease 4 (NSP4) [9–12]. Human neutrophils also express a close homologue to these proteases, azurocidin, which is a potent antibacterial pro- tein that lacks proteolytic activity [13]. This loss of proteolytic activity is caused by mutations in two of the three amino acids in the catalytic triad of the active site [13]. These four proteases and protease homologs (azurocidin) are primarily considered neutrophil specific proteases, however CG is also expressed in other cells, particularly macrophages, upon infection [14]. Human CG but not its mouse and rat counterparts, is also expressed in mast cells and low lev- els of these proteases are also found in a subpopulation of monocytes due to their expression during early stages of myelo-monocyte development. and analysis, decision to publish, or preparation of the manuscript. and analysis, decision to publish, or preparation of the manuscript. Competing interests: Lawrence de Garavilla is an employee of GDL Pharmaceutical Consulting and Contracting. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Abbreviations: aa, amino acid(s); Ang, angiotensin; HC, human chymase; hCG, human cathepsin G; Ni-NTA, nickel-nitrilotriacetic acid. Abbreviations: aa, amino acid(s); Ang, angiotensin; HC, human chymase; hCG, human cathepsin G; Ni-NTA, nickel-nitrilotriacetic acid. The four active neutrophil proteases have a number of different functions and also an array of potential substrates. Knock out experiments show that several of these enzymes are impor- tant for bacterial and fungal defence [15–21]. Two potential bacterial targets have been identi- fied as flagellin of Pseudomonas aeruginosa and the outer membrane protein A of E. coli [22, 23]. Introduction Neutrophils constitute 50 to 75% of all leukocytes in human blood and are thereby the most abundant human white blood cell [1, 2]. They are short-lived cells, which use both oxygen and 1 / 28 PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 Human cathepsin G can also activate coagulation factor VIII, which may contribute to the enhanced coagulation in areas of infection where neutro- phils accumulate [29]. Finally, in support of the role of these proteases in coagulation, both the single (CG) and double knock out mice (NE+CG) show a bleeding time that is twice as long as the wild-type mice [21]. The role of mammalian neutrophils in coagulation links this aspect to innate immunity, which is similar to what is observed in many invertebrate species where coagulation has a central role in antimicrobial immunity [30]. Of the four active neutrophil serine proteases hCG, is one of the more abundant enzymes, and is probably the most extensively studied protease of these four [31]. A relatively detailed analysis of hCG has previously been performed using peptide libraries where hCG was com- pared with its mouse counterpart mCG [32]. Human cathepsin G but not mCG displayed a dual specificity as both a chymase and a tryptase, where the later activity favoured lysine over arginine [31–33]. The lower activity for human but not mouse CG, compared to human chy- mase (HC), has been attributed towards the widening of its active site, which may also explain the broader specificity seen with hCG [32]. Although hCG and the other neutrophil proteases are relatively well characterized there are a number of important unanswered questions con- cerning them. Their extended specificities have never been determined in detail and almost no quantitative information concerning the importance of various positions in and around the cleavage site has been presented. Such information can be used to increase the resolution dur- ing screenings of the human genome and genomes of pathogens sensitive to these enzymes in order to identify novel in vivo substrates. This would serve as a tool for understanding the gen- eral roles of them in immunity with a particular focus in bacterial defence. In order to close this gap in our understanding of these enzymes, here we present a detailed analysis of the extended cleavage specificity of hCG. Substrate phage display was used in order to obtain an estimate of the most favoured (up to 9) amino acids in a region surrounding the cleavage site. These sequences were then verified and/or modified in order to pinpoint specific amino acid involvement in this cleavage. Patients with PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 2 / 28 Extended cleavage specificity of human cathepsin G Papillon-Lefèvre syndrome show a particular severe form of periodontitis due to bacterial infection with Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. These patients lack cathepsin C, which is an enzyme involved in the activation of all the neutrophil proteases. The role of the neutrophil protease here is related to neutralizing bacterial toxins and also activating one of the neutrophil antibacterial peptides, hCAP-18 [27]. N-elastase and hCG may also have indirect antimicrobial effects by their recently identified effect on blood coagulation. These two neutrophil proteases can enhance coagulation by the cleavage of an inhibitor of the tissue factor pathway (TFPI), an effect that is strongly potentiated by histones and DNA released by the NETs [28]. The bacteria are subsequently trapped by the coagulation in small blood vessels and are thereby inhibited from entering tissues, which results in a decrease in bacterial numbers [28]. Human cathepsin G can also activate coagulation factor VIII, which may contribute to the enhanced coagulation in areas of infection where neutro- phils accumulate [29]. Finally, in support of the role of these proteases in coagulation, both the single (CG) and double knock out mice (NE+CG) show a bleeding time that is twice as long as the wild-type mice [21]. The role of mammalian neutrophils in coagulation links this aspect to innate immunity, which is similar to what is observed in many invertebrate species where coagulation has a central role in antimicrobial immunity [30]. Papillon-Lefèvre syndrome show a particular severe form of periodontitis due to bacterial infection with Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. These patients lack cathepsin C, which is an enzyme involved in the activation of all the neutrophil proteases. The role of the neutrophil protease here is related to neutralizing bacterial toxins and also activating one of the neutrophil antibacterial peptides, hCAP-18 [27]. N-elastase and hCG may also have indirect antimicrobial effects by their recently identified effect on blood coagulation. These two neutrophil proteases can enhance coagulation by the cleavage of an inhibitor of the tissue factor pathway (TFPI), an effect that is strongly potentiated by histones and DNA released by the NETs [28]. The bacteria are subsequently trapped by the coagulation in small blood vessels and are thereby inhibited from entering tissues, which results in a decrease in bacterial numbers [28]. Furthermore, knocking out both CG and NE impairs the elimination of Mycobacterium bovis lung infections in mice [20]. In the same study, therapeutic administration of liposomes loaded with NE and CG also resulted in significant enhancement of mycobacterial protection [20]. Experimental infections using another mycobacterial species, the human pathogen Myco- bacterium tuberculosis, also show reduced survival rates of both single (CG) and double knock out mice (NE+CG) without having any direct effect on bacterial replication themselves. How- ever, there are increased levels of several inflammatory cytokines including TNF-α, IL-6 and MIP2, in the knock out mice during early infection indicating dys-regulated cytokine expres- sion [21]. Modulation of the expression of these same cytokines was also observed with another pathogen Pseudomonas aeruginosa. However, a strong down-regulation in the knock out animals was seen, highlighting a complex role of these proteases in cytokine regulation [24]. Interestingly, CG and NE are directly involved in Fc receptor dependent activation of neutrophils by immune complexes [25]. The activation results in an integrin dependent clus- tering of neutrophils, and in the absence of NE and CG the knock out mice show a severe defect in MIP-2 secretion and oxygen radical production [25]. In line with the role of these enzymes in cell adhesion and clustering, CG is also involved in arterial myeloid cell recruit- ment by strengthening adhesion of the cells during high shear flow [26]. The killing ability of neutrophils within different gram positive bacteria as well as between gram positive and negative bacteria differs markedly. For example Staphylococcus aureus is killed primarily by oxygen dependent mechanisms involving reactive oxygen species (ROS) whereas the killing of another gram positive bacteria Streptococcus pneumonia is independent of ROS but dependent on phagocytosis and complement mediated opsonisation [18]. This par- ticular study also showed that killing of the Streptococcus was dependent on active serine prote- ases. Here the effect seemed to be a combined protease response as individual inhibition of specific proteases did not lead to a loss in killing activity (NSP-4 was not analysed) but inhibit- ing all three reduced killing to baseline [18]. Both NE and CG appear to have non-redundant roles in lung protective immunity against Streptococcus pneumonia [19]. PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 Chromogenic substrate assay A large panel of different chromogenic substrates was used to determine the primary specifici- ties of hCG and HC. In order for complete specificity coverage the panel included different chymase, elastase, tryptase and asp-ase substrates. In Fig 2 hCG cleaved the classical chymase substrates with Phe and Tyr efficiently as well as the substrate with a Leu in the P1 position. However, all other substrates remained uncleaved including the two tryptase substrates (Z-GPR-pNA, Suc-VLGR-pNA) with an Arg in the P1 position (Fig 2 first column). The HC also cleaved the Phe and Tyr substrates efficiently but not the P1 Leu containing substrate highlighting an apparent difference between the two enzymes under these conditions (Fig 2 second column). As further controls for this assay, two additional serine proteases, thrombin and granzyme B, with tryptase and asp-ase specificities, respectively were tested (Fig 2 panels 3 and 4). Importantly, approximately 10 times more enzyme was needed for hCG indicating a much lower activity of this enzyme compared to the HC (100 ng HC compared to 1000 ng for hCG). Our results showed that hCG is approximately 10 times less active than the HC at a similar molar ratio. As expected, hCG preferred Tyr and Phe in its P1 position but also cleaved efficiently after Trp and Leu. Interestingly hCG also showed a preference for negatively charged amino acids in the P2´position, which is similar to HC as well as the functional homologue of HC in mice, mouse mast cell protease 4 (mMCP-4) [34]. We also verified the activity of hCG on Lys containing substrates in the P1 position. Our results showed that hCG displayed only 2 times lower activity on Lys compared to Tyr and Phe, which clearly shows that it contains tryptase-type cleavage specificity after positively charged Lys. However, it showed little activity after Arg. Finally, we also showed that HC has a low tryptase-type activity on Arg, which was approximately 20 times lower than its favourable chymase activity. Recently an alternative method to study cleavage specificity has been developed using a peptide library and subsequent analysis of cleavage products by mass spectroscopy (MS) PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 3 / 28 Extended cleavage specificity of human cathepsin G [35, 36]. In order to study the advantages and disadvantages of this method we have analyzed the cleavage of the identified consensus cleavage sites using our novel thioredoxin-based cleav- age assay, with four different human neutrophil proteases: hCG, N-elastase, proteinase 3 and NSP4. Here, we saw that for proteases with a relatively broad specificity, such as N-elastase and proteinase 3, the peptide library/MS method provided similar results to our analyses. However, for more specific proteases such as hCG, the phage display results were much more reliable as they originate from peptide libraries several orders of magnitude larger in complex- ity compared to the proteomics method used. Human cathepsin G and human mast cell chymase Human cathepsin G used for both the phage display analysis and subsequent verification by the recombinant substrates was a commercially available preparation from BioCentrum (Kra- kow, Poland) that had been purified by several steps from human peripheral blood neutrophils (Fig 1A). Recombinant HC was also used for a comparative analysis of activity and extended specificity (Fig 1B). This enzyme was produced via baculovirus infected insect cells as previ- ously described [37]. Human N-elastase and proteinase 3 were also included for comparison (Fig 1A) and both were also commercially available from peripheral blood neutrophils. In Fig 1A both hCG and proteinase 3 are relatively stable after purification whereas N-elastase showed unavoidable self cleavage at a low rate even when stored at +4˚C. The preparations of human thrombin (Th) and human granzyme B (GB) were also included (Fig 1B) as they were used as reference proteases in the chromogenic and recombinant substrate assays. Determination of the extended cleavage specificity by substrate phage display To obtain a more complete view of the extended specificity of hCG we performed unbiased screening for the most favoured targets by substrate phage display. The phage library used to determine the extended cleavage specificity of hCG contains approximately 5x107 phage clones. Each phage clone expresses a unique sequence of 9 random amino acids (nonamer) on their surface, followed by a His6-tag in the C-terminus of capsid protein 10. The phages are subsequently immobilized on Ni-NTA agarose beads via interactions with the His6-tag. The purified hCG was used to screen the phage library for peptides susceptible to cleavage. After PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 4 / 28 Extended cleavage specificity of human cathepsin G Fig 1. Analysis of the human cathepsin G preparation used in the determination of the extended cleavage specificity. The enzyme had previously been purified from blood neutrophils and the purity of the preparation wa determined by separation on SDS-PAGE and visualized with Coomassie Brilliant Blue staining. In panel A comme preparations of two additional neutrophil proteases N-elastase (NE) and proteinase 3 (P3) were also included in th figure for comparison. Proteinase 3 and hCG were intact after short term storage at +4˚C. However, N-elastase sho several degradation products indicating self-cleaving activity. In panel B three additional enzymes, the human mas chymase (HC), human thrombin (Th) and human granzyme B (GB) were included as they were used as reference proteases in the chromogenic and recombinant substrate assays. https://doi org/10 1371/journal pone 0195077 g001 Fig 1. Analysis of the human cathepsin G preparation used in the determination of the extended cleavage Fig 1. Analysis of the human cathepsin G preparation used in the determination of the extended cleavage specificity. The enzyme had previously been purified from blood neutrophils and the purity of the preparation was determined by separation on SDS-PAGE and visualized with Coomassie Brilliant Blue staining. In panel A commercial preparations of two additional neutrophil proteases N-elastase (NE) and proteinase 3 (P3) were also included in the figure for comparison. Proteinase 3 and hCG were intact after short term storage at +4˚C. However, N-elastase showed several degradation products indicating self-cleaving activity. In panel B three additional enzymes, the human mast cell chymase (HC), human thrombin (Th) and human granzyme B (GB) were included as they were used as reference proteases in the chromogenic and recombinant substrate assays. Fig 1. Determination of the extended cleavage specificity by substrate phage display Analysis of the human cathepsin G preparation used in the determination of the extended cleavage specificity. The enzyme had previously been purified from blood neutrophils and the purity of the preparation was determined by separation on SDS-PAGE and visualized with Coomassie Brilliant Blue staining. In panel A commercial preparations of two additional neutrophil proteases N-elastase (NE) and proteinase 3 (P3) were also included in the figure for comparison. Proteinase 3 and hCG were intact after short term storage at +4˚C. However, N-elastase showed several degradation products indicating self-cleaving activity. In panel B three additional enzymes, the human mast cell chymase (HC), human thrombin (Th) and human granzyme B (GB) were included as they were used as reference proteases in the chromogenic and recombinant substrate assays. https://doi.org/10.1371/journal.pone.0195077.g001 https://doi.org/10.1371/journal.pone.0195077.g001 the first selection step (biopanning), the released phages, which are cleaved in their unique random region, were amplified in E. coli and subjected to additional biopannings. Selections of phages susceptible to cleavage by the protease, were performed over 5 biopannings, after which hCG induced the release of 71 times more phages compared to a PBS control (data not shown). After the last biopanning, 120 individual phage clones were isolated and the sequences encoding the randomly synthesized nona-peptides were determined for 96 of them (one 96 well plate). The nucleotide sequences were then translated into nona-peptides, which were aligned based on the primary cleavage specificity observed from the chromogenic substrate assay (Fig 2). The alignment of the phage display sequences is performed by hand starting with the substrates having only one aromatic amino acid or one Leu. Based on this alignment we PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 5 / 28 Extended cleavage specificity of human cathepsin G Fig 2. Chromogenic substrate assay. A panel of different chromogenic substrates was used to determine th specificity of hCG and HC. The panel included different chymase, elastase, tryptase and aspase substrates. T acid sequences of the substrates are listed at the left side of the figure. Human thrombin and human granzym included as reference enzymes for tryptase and asp-ase activities, respectively. https://doi.org/10.1371/journal.pone.0195077.g002 Fig 2. Chromogenic substrate assay. A panel of different chromogenic substrates was used to determine the primary specificity of hCG and HC. The panel included different chymase, elastase, tryptase and aspase substrates. The amino acid sequences of the substrates are listed at the left side of the figure. PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 Verifying the consensus sequence by the use of a new type of recombinant protein substrate In order to verify the results from the phage display analysis, a new type of recombinant sub- strates that has been validated in a number of previous studies was used [39–45]. The consen- sus sequence obtained from the phage display analysis was inserted into a linker region between two E.coli thioredoxin (Trx) molecules by ligating a double stranded oligonucleotide encoding the cleavable sequence into a BamHI and a SalI site of the vector construct (Fig 5A). For purification purposes a His6-tag was added to the C-terminal of the second Trx protein (Fig 5A). A number of related and unrelated substrate sequences were also produced with this system, by ligating the corresponding oligonuclotides into the BamHI/SalI sites of the vector. All of these substrates were expressed as soluble proteins in E.coli and purified on IMAC col- umns to obtain a protein with a purity of 90–95%. These recombinant proteins were subse- quently used to study the preference of hCG and HC for these different sequences (Figs 5–10). Previously we have used several such 2xTrx clones encoding the consensus sequences obtained from phage display analyses of the HC, a HC double mutant (Arg143Gln + Lys192Met) as well as for the dog and opossum mast cell chymases [38–40, 46]. This set of substrates was used for an initial reference and comparison between HC and hCG. In Fig 5 the preference for nega- tively charged amino acids in the P2´position for both proteases as seen from the phage display analysis was confirmed. We could also see that hCG was more tolerant to Trp in the P1 posi- tion in comparison to the HC. However, these four substrates differ in a few additional posi- tions surrounding the cleavage site, which may influence the rate of cleavage. Based on both chromogenic substrate analyses for hCG (Fig 2) and the phage display (Fig 3), it was apparent that hCG also efficiently cleaves after Leu in the P1 position. As all four of the consensus substrates analysed in the 2xTrx system (Fig 5) contained both aromatic amino acids and several Leu residues it was difficult to obtain an accurate estimate of the preference for the four potential preferred P1 residues, Phe, Tyr, Trp and Leu. We therefore decided to make a new set of 2xTrx substrates with only single aromatic amino acids containing, Phe, Tyr or Trp in the cleavable sequence (Figs 6, 7 and 8). Determination of the extended cleavage specificity by substrate phage display Human thrombin and human granzyme B were included as reference enzymes for tryptase and asp-ase activities, respectively. Fig 2. Chromogenic substrate assay. A panel of different chromogenic substrates was used to determine the primary specificity of hCG and HC. The panel included different chymase, elastase, tryptase and aspase substrates. The amino acid sequences of the substrates are listed at the left side of the figure. Human thrombin and human granzyme B were included as reference enzymes for tryptase and asp-ase activities, respectively. https://doi org/10 1371/journal pone 0195077 g002 https://doi.org/10.1371/journal.pone.0195077.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 6 / 28 Extended cleavage specificity of human cathepsin G then continue using the same basic pattern and align the sequences containing several poten- tial cleavage sites. What we can conclude from this alignment is that in addition to the similar preference of Phe/Tyr/Trp/Leu in the P1 position hCG, like HC [38], has a clear specificity for negatively charged amino acids in the P2’ position. A strong preference for aliphatic amino acids both N and C terminal of the cleavage site was also observed (Figs 3 and 4). Compared to HC a marked difference in the number of Leu in the P1 position was observed (Figs 3 and 4). However we could not observe a high frequency of Arg or Lys containing phages that could indicate tryptase activity. The phage display method primarily selects the most favourable sub- strates and secondary specificities can thereby easily remain undetected, a fact that needs to be taken into consideration when analysing complex enzymes with several specificities. The phage display technology does not allow for the identification of the exact cleavage site. How- ever, by combining the information from chromogenic substrates and the verification by the recombinant substrates described in the next section we obtain a very accurate picture of the cleavage preference. PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 Verifying the consensus sequence by the use of a new type of recombinant protein substrate Here it was important to ensure that none of the surrounding amino acids in the 2xTrx substrate could serve as a cleavable P1 site. A sub- strate lacking Phe, Tyr, Trp and Leu was therefore constructed and analysed using both HC and hCG (Fig 6A and 6B). This substrate has Val in the positions where aromatic amino acids or Leu was originally located. In Fig 6A and 6B both proteases did not cleave this control P1 Val substrate whereas the substrate with Phe in the same position was efficiently cut, which PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 7 / 28 Extended cleavage specificity of human cathepsin G Fig 3. Phage displayed nonamers susceptible to cleavage by hCG after five biopannings. After the last selection step, phages released by proteolytic cleavage were isolated and the sequences encoding the nonamers were determined. The general sequence of the T7 phage capsid proteins are PGG(X)9HHHHHH, where (X)9 indicates the randomized nonamers. The protein sequences were aligned into a P5-P4´ consensus, where cleavage occurs between positions P1 and P1´. The amino acids are colour coded according to the side chain properties as shown in a separate panel in the Fig 3. Phage displayed nonamers susceptible to cleavage by hCG after five biopannings. After the last selection step, phages released by proteolytic cleavage were isolated and the sequences encoding the nonamers were determined. The general sequence of the T7 phage capsid proteins are PGG(X)9HHHHHH, where (X)9 indicates the randomized nonamers. The protein sequences were aligned into a P5-P4´ consensus, where cleavage occurs between positions P1 and P1´. The amino acids are colour coded according to the side chain properties as shown in a separate panel in the PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 8 / 28 Extended cleavage specificity of human cathepsin G bottom of the figure. The sequences with one aromatic amino acid (potential cleavage site) are aligned first, followed by sequences containing two, three or four aromatic amino acids. It is not a perfect alignment but so far the best that has been obtained by any method. bottom of the figure. The sequences with one aromatic amino acid (potential cleavage site) are aligned first, followed by sequences containing two, three or four aromatic amino acids. It is not a perfect alignment but so far the best that has been obtained by any method. Verifying the consensus sequence by the use of a new type of recombinant protein substrate https://doi.org/10.1371/journal.pone.0195077.g003 https://doi.org/10.1371/journal.pone.0195077.g003 once again confirmed the P1 specificity of the enzyme. In panel D, hCG preferred substrates with Phe over Tyr or Leu in the P1 position by a factor of less than 2 and over Trp with a factor of 2–3 (Fig 6D). In contrast the HC cleaved all three substrates with aromatic amino acids almost to the same extent (Fig 6C). The cleavage rate for the Leu substrate was only slightly lower (2–3 times) than for the three aromatic amino acids (Fig 6C). Based on above results we concluded that the amino acids surrounding the P1 site were of major importance for the efficiency of cleavage by both enzymes. We therefore decided to study the influence of different amino acids in the P1´and the P2´positions (Fig 7A and 7B). Here the P2´amino acid was changed from Asp to an Ala. Only a very minor effect on the cleavage activity was observed for both enzymes indicating that Ala rather than Gly can replace the negative charge for efficient cleavage. Changing Ser in the P1´position to Leu or Arg resulted in a 3-5-fold drop in cleavage activity for both enzymes, thus also highlighting the importance of the amino acids in this position (Fig 7A and 7B). Interestingly, here the HC showed very similar kinetics for these three substrates although phage display selected few or no sequences with Trp and Leu and no activity on chromogenic substrates containing a Leu in the P1 position (Figs 2 and 3). The lack of cleavage of the chromogenic Leu substrate indicates that the HC is more dependent on downstream residues when cleaving Leu containing sub- strates compared to substrates with aromatic amino acids in the P1 position. In addition to having chymase activity, hCG has also been attributed to display tryptase activity with preference for Lys over Arg [31–33]. In order to address this aspect we produced substrates with Arg, Lys and Asp in the P1 position (Fig 8). There were indications of cleavage in the Arg containing substrate although some 10x times less than towards chymase substrates (Fig 8B). However, hCG cleaved the Lys substrate very efficiently almost as equally well as the Tyr substrate. This shows that, in contrast to HC, hCG also is a potent tryptase, but primarily for Lys containing substrates. PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 Verifying the consensus sequence by the use of a new type of recombinant protein substrate Interestingly the HC showed some (although very low) activity towards the Arg substrate suggesting that it has some tryptase activity. Importantly, the HC showed no cleavage of the Lys containing substrate revealing a major difference between these two enzymes. A few additional substrates were tested to obtain a detailed view of the importance of resi- dues surrounding the cleavage site. Using modified versions of the HC consensus in Fig 5 (VVLFSEVL, VVLLSEVL, VVLRSEVL) we saw in this setting that Leu showed 2-3-fold lower cleavage rate than Phe in the P1 position (Fig 9A). The substrate with P1 Arg following a Leu showed a 3–5 fold reduction in cleavage for both enzymes (Fig 9A and 9B). Exchanging this Arg for a Ser (VVLRSEVL to VVLSSEVL) resulted in no effect for hCG but an 3–5 fold enhancement for HC (Fig 9 panels C and D). Exchanging the Arg for an Asp (VVLRSEVL to VVLDSEVL) resulted in a dramatic drop in activity for hCG, so severe that the enzyme cleaved at the Leu in the end of the sequence (Fig 9 panels C and D). The cleavage at the end of the linker region can be deduced from the change in size of the cleavage products (Fig 9 panels C and D). In contrast, the Asp enhanced the cleavage efficiency over Arg for the HC by a factor of 2 (Fig 9 panels C and D). We also made three additional substrates where the Val-Leu-Phe- Ser of the HC consensus has been changed to Val-Leu-Val-Leu, Leu-Leu-Val-Ser or Val-Ser- Val-Ser (VVLVLEVL, VLLVSEVL, VVSVSEVL) (Fig 9E and 9F). Unsurprisingly, very low cleavage was seen with the first two substrates whereas a remarkably high activity for hCG was observed for the third substrate VVSVSEVL, resulting in increased cleavage at the Leu residue PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 9 / 28 Extended cleavage specificity of human cathepsin G Fig 4. Distribution of amino acids in positions P4 to P4´ in phage displayed nonamers cleaved by hCG after five biopannings. Based on the alignment (Fig 3) the percentage of each amino acid present in each position P4 to P4´ as calculated The amino acids are Fig 4. Distribution of amino acids in positions P4 to P4´ in phage displayed nonamers cleaved by hCG after five biopannings. Verifying the consensus sequence by the use of a new type of recombinant protein substrate Based on the alignment (Fig 3) the percentage of each amino acid present in each position P4 to P4´ as calculated. The amino acids are ordered from left to right: aromatic, aliphatic, hydrophilic, basic (positively charged) and acidic (negatively charged). https://doi org/10 1371/journal pone 0195077 g004 Fig 4. Distribution of amino acids in positions P4 to P4´ in phage displayed nonamers cleaved by hCG after five biopannings. Based on the alignment (Fig 3) the percentage of each amino acid present in each position P4 to P4´ as calculated. The amino acids are Fig 4. Distribution of amino acids in positions P4 to P4´ in phage displayed nonamers cleaved by hCG after five biopannings. Based on the alignment (Fig 3) the percentage of each amino acid present in each position P4 to P4´ as calculated. The amino acids are ordered from left to right: aromatic, aliphatic, hydrophilic, basic (positively charged) and acidic (negatively charged). https://doi.org/10.1371/journal.pone.0195077.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 10 / 28 Extended cleavage specificity of human cathepsin G Fig 5. Analysis of the cleavage specificity by the use of recombinant protein substrates. Panel A shows the overall structure of the recombinant protein substrates used for analysis of the efficiency in cleavage by the hCG and HC. In these substrates two thioredoxin molecules are positioned in tandem and the proteins have a His6-tag positioned in their C termini. The different cleavable sequences are inserted in the linker region between the two thioredoxin molecules by the use of two unique restriction sites, one Bam HI and one SalI site, which are indicated in the bottom of panel A. In panel B a hypothetical cleavage is shown to highlight possible cleavage patterns. Panels C and D shows the cleavage of 4 different substrates by hCG and HC, the HC consensus obtained from phage display, the consensus of a HC .org/10.1371/journal.pone.0195077 April 13, 2018 11 / 2 Fig 5. Analysis of the cleavage specificity by the use of recombinant protein substrates. Panel A shows the overall structure of the recombinant protein substrates used for analysis of the efficiency in cleavage by the hCG and HC. In these substrates two thioredoxin molecules are positioned in tandem and the proteins have a His6-tag positioned in their C termini. Verifying the consensus sequence by the use of a new type of recombinant protein substrate The different cleavable sequences are inserted in the linker region between the two thioredoxin molecules by the use of two unique restriction sites, one Bam HI and one SalI site, which are indicated in the bottom of panel A. In panel B a hypothetical cleavage is shown to highlight possible cleavage patterns. Panels C and D shows the cleavage of 4 different substrates by hCG and HC, the HC consensus obtained from phage display, the consensus of a HC Fig 5. Analysis of the cleavage specificity by the use of recombinant protein substrates. Panel A shows the overall structure of the recombinant protein substrates used for analysis of the efficiency in cleavage by the hCG and HC. In these substrates two thioredoxin molecules are positioned in tandem and the proteins have a His6-tag positioned in their C termini. The different cleavable sequences are inserted in the linker region between the two thioredoxin molecules by the use of two unique restriction sites, one Bam HI and one SalI site, which are indicated in the bottom of panel A. In panel B a hypothetical cleavage is shown to highlight possible cleavage patterns. Panels C and D shows the cleavage of 4 different substrates by hCG and HC, the HC consensus obtained from phage display, the consensus of a HC PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 11 / 28 Extended cleavage specificity of human cathepsin G double mutant, the dog chymase consensus and the opossum chymase consensus [34, 46, 52]. The name and sequence of the different substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated above the corresponding lanes of the different gels. The uncleaved substrates have a molecular weight of approximately 25 kDa and the cleaved substrates appear as two closely located bands with a size of 12–13 kDa. In panel E we show the results from scanning of the gels where on e clearly can see the effect of P2´negative charge for both HC and hCG. The gel pictures in panels C and D were densitometrically scanned and the result was presented as two separate diagrams in panel E, one for HC and one for hCG. https://doi.org/10.1371/journal.pone.0195077.g005 https://doi.org/10.1371/journal.pone.0195077.g005 at the C terminal end of the sequence, indicating that a Ser in the now position P6 is of major importance for efficient cleavage for hCG (Fig 9E). Analysing the effect of human lactoferrin on the cleavage activity of hCG In a recent study by Eipper et al they show that human lactoferrin can enhance the activity of hCG by a factor of 5–7 [47]. In their study they used an active site binding assay to assess the activity of hCG. In order to confirm and possibly get a better understanding of this enhancing effect we used the recombinant 2xTrx substrates to assay the potential activating effect by lac- toferrin on hCG. By adding lactoferrin into the cleavage reaction at 25 μg/ml, 250 μg/ml or 2500 μg/ml, we could show in our assay system that there was a 3–5 fold enhancement in cleav- age activity on hCG by lactoferrin but not of an unrelated protein such as bovine serum albu- min (BSA) (Fig 11A and 11B). However, the enhancement was only observed at the two highest concentrations of lactoferrin, 250 and 2500 μg/ml (Fig 11A). Here the molar ratio between lactoferrin to hCG was approximately 37 and 370. The enhancing effect was also only 3–5 fold even at the highest lactoferrin concentration and no enhancement was seen at the low concentration of lactoferrin despite having an excess of lactoferrin to hCG of 3.6 times. Verifying the consensus sequence by the use of a new type of recombinant protein substrate This position does not seem to have the same effect on the HC, which also cleaved at the Leu C-terminal end but at a much lower rate (Fig 9F). Here, the second substrate VLLVSEVL was a relatively good target for the HC in con- trast to hCG (Fig 9E and 9F). In order to verify the importance of a Ser in the P6 position for hCG we produced two additional substrates where the Ser in the P6 position was exchanged for a Val or an Asp (VVSVSEVL to VVVVSEVL, VVDVSEVL). A marked drop in activity was observed for both of these two new substrates with hCG (Fig 10). A Ser in position P6 of a sub- strate appears to be of major importance for the cleavage by hCG indicating that residues rela- tively far from the P1 cleavage site can have major impact on cleavage efficiency of this enzyme (Fig 10). The importance of a Ser in position P6 was not possible to extract from the phage display data as the variable region of the phage only is 9 amino acids long and cleavage often occur in the middle of this region, making predictions of specificity only possibly up to posi- tion P4. Analysing the difference in efficiency and reliability of combinatorial peptide library with mass spectrometry to determine the extended cleavage specificity In two recent studies by O’Donoghue et al., a novel method to study the extended specificities of some pure pathogen and immune related proteases and proteases in mixtures has been developed [35, 36]. This new method is based on a library of 14 amino acid long peptides where the products after cleavage by the analysed proteases are then subject to mass spectro- scopic techniques (MS). This is a relatively rapid analysis, which may become an interesting alternative to phage display. As we now have a good consensus substrate for hCG and have also recently studied two other human neutrophil proteases (hNE and hPR3) by phage display (unpublished results), we can now compare the two methods. The consensus sequences from both methods were PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 12 / 28 Extended cleavage specificity of human cathepsin G Fig 6. Analysis of the cleavage specificity by the use of recombinant protein substrates. Panels A and B show the cleavage of two substrates, one containing Phe in the P1 position and one where this residue has been exchanged for a Val residue, thereby the construct does not contain any aromatic amino acids or a Leu serving as a negative control. Panels C and D show the analysis of the chymotrypsinogen-like P1 preference for the two enzymes with substrates having different aromatic amino acids or Leu in the P1 position. Panels E and F show an analysis of the P1´and Fig 6. Analysis of the cleavage specificity by the use of recombinant protein substrates. Panels A and B show the cleavage of two substrates, one containing Phe in the P1 position and one where this residue has been exchanged for a Val residue, thereby the construct does not contain any aromatic amino acids or a Leu serving as a negative control. Panels C and D show the analysis of the chymotrypsinogen-like P1 preference for the two enzymes with substrates having different aromatic amino acids or Leu in the P1 position. Panels E and F show an analysis of the P1´and Fig 6. Analysis of the cleavage specificity by the use of recombinant protein substrates. Panels A and B show the cleavage of two substrates, one containing Phe in the P1 position and one where this residue has been exchanged for a Val residue, thereby the construct does not contain any aromatic amino acids or a Leu serving as a negative control. Analysing the difference in efficiency and reliability of combinatorial peptide library with mass spectrometry to determine the extended cleavage specificity Panels C and D show the analysis of the chymotrypsinogen-like P1 preference for the two enzymes with substrates having different aromatic amino acids or Leu in the P1 position. Panels E and F show an analysis of the P1´and PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 13 / 28 Extended cleavage specificity of human cathepsin G P2´specificities. The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated above the corresponding lanes of the different gels. The gel pictures in panels A, B, C and D were densitometrically scanned and the result was presented as two separate diagrams in panels E and F. https://doi.org/10.1371/journal.pone.0195077.g006 https://doi.org/10.1371/journal.pone.0195077.g006 produced in the 2xTrx system and used to study the efficacy in cleavage by the enzymes. In the MS-study non-natural amino acids were used such as nor-leucine, which complicates the comparison. The most similar natural amino acids are methionine or leucine. We therefore inserted Met in the positions of the MS consensus sequences where they had used nor-leu- cines. As seen from Fig 12, the consensus sequences for both methods worked fine with the rel- atively unspecific enzymes hNE and hPR3. However for the more specific protease hCG the phage display sequence was clearly superior. Only minor cleavage was observed with the con- sensus sequence obtained by the proteomics method. This indicates that the proteomics method can be a method of choice for proteases with a broad specificity but not for more spe- cific proteases where the number of cleavable peptides in the starting pool needs to be high. The substrate identified for NSP-4 by the MS technology was also a very poor substrate as we even after using a relatively large amount of enzyme only observed minor cleavage, which indi- cates that also NSP-4 is a relatively specific protease. In the proteomic studies only 124 origi- nating peptides were used whereas the phage display library contains approximately 50 million different nine amino acid long sequences, indicating that for more specific proteases a larger starting complexity is required. With both technologies the cleavage can occur at multiple positions which results in 50 million + and 124 + potential cleave sites. However, one very pos- itive characteristic of the proteomics method is the identification of substrates that are not Fig 7. Analysis of the cleavage specificity by the use of recombinant protein substrates. Analysing the difference in efficiency and reliability of combinatorial peptide library with mass spectrometry to determine the extended cleavage specificity Panels A and B show an analysis of the P1´and P2´specificities. The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated above the corresponding lanes of the different gels. https://doi.org/10.1371/journal.pone.0195077.g007 Fig 7. Analysis of the cleavage specificity by the use of recombinant protein substrates. Panels A and B show an analysis of the P1´and P2´specificities. The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated above the corresponding lanes of the different gels. https://doi.org/10.1371/journal.pone.0195077.g007 PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 14 / 28 Extended cleavage specificity of human cathepsin G Fig 8. Analysis of the potential tryptase activity by HC and hCG using recombinant protein substrates. Using the Phe substrate (Fig 6) as a reference to determine the cleavage rate by HC and hCG on three substrates having Arg, Lys or Asp in the P1 position. The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated above the corresponding lanes of the different gels. The gel pictures in panels A and B were densitometrically scanned and the result was presented as two separate diagrams in panel C, one for HC and one for hCG. Fig 8. Analysis of the potential tryptase activity by HC and hCG using recombinant protein substrates. Using the Phe substrate (Fig 6) as a reference to determine the cleavage rate by HC and hCG on three substrates having Arg, Lys or Asp in the P1 position. The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated above the corresponding lanes of the different gels. The gel pictures in panels A and B were densitometrically scanned and the result was presented as two separate diagrams in panel C, one for HC and one for hCG. https://doi org/10 1371/journal pone 0195077 g008 Fig 8. Analysis of the potential tryptase activity by HC and hCG using recombinant protein substrates. Using the Phe substrate (Fig 6) as a reference to determine the cleavage rate by HC and hCG on three substrates having Arg, Lys or Asp in the P1 position. The sequences of the different substrates are indicated above the pictures of the gels. Analysing the difference in efficiency and reliability of combinatorial peptide library with mass spectrometry to determine the extended cleavage specificity The time of cleavage in min is also indicated above the corresponding lanes of the different gels. The gel pictures in panels A and B were densitometrically scanned and the result was presented as two separate diagrams in panel C, one for HC and one for hCG. optimal but still cleaved relatively efficiently, such as the cleavage of threonine containing sub- strates by hPR3 and lysine containing substrates by hCG, which had not been observed during the phage display screening as this method primarily identifies the most optimal substrates. PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 https://doi.org/10.1371/journal.pone.0195077.g009 Discussion The granule associated serine proteases of the neutrophils have been studied quite extensively for many years, however, their extended cleavage specificities have never been studied in detail [9–12, 32]. The fine specificity of the enzymes and their selectivity for the different potential in vivo substrates they may encounter is to a large extent determined by their extended specificity, PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 15 / 28 Extended cleavage specificity of human cathepsin G Fig 9. Analysis of the cleavage specificity by the use of recombinant protein substrates. An analysis changing amino acids in and around the cleavage site on the cleavage by HC and hCG. The sequences o substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated abov corresponding lanes of the different gels. https://doi.org/10.1371/journal.pone.0195077.g009 Fig 9. Analysis of the cleavage specificity by the use of recombinant protein substrates. An an changing amino acids in and around the cleavage site on the cleavage by HC and hCG. The seque substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicate corresponding lanes of the different gels. https://doi.org/10.1371/journal.pone.0195077.g009 Fig 9. Analysis of the cleavage specificity by the use of recombinant protein substrates. An analysis of the effect of changing amino acids in and around the cleavage site on the cleavage by HC and hCG. The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated above the corresponding lanes of the different gels. https://doi.org/10.1371/journal.pone.0195077.g009 https://doi.org/10.1371/journal.pone.0195077.g009 PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 16 / 28 Extended cleavage specificity of human cathepsin G Fig 10. Analysis of the effect of particular amino acids in the P6 position for the cleavage by hCG. The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated above the corresponding lanes of the different gels. Fig 10. Analysis of the effect of particular amino acids in the P6 position for the cleavage by hCG. The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated above the corresponding lanes of the different gels. Fig 10. Analysis of the effect of particular amino acids in the P6 position for the cleavage by hCG. PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 Discussion The effect on the activity by hCG on the HC consensus substrate (VVLFSEVL) was analysed in a cleavage reaction as before (Figs 5–9). In panel A three different concentrations of lactoferrin were used: 25, 250 and 2500μg/ml. A 3–5 fold enhancement was observed at the highest lactoferrin concentration (2500μg/ml) and a 2–3 fold enhancement at the middle concentration (250μg/ml) but no enhancement at the low (250μg/ml) lactoferrin concentration. In panel B, bovine serum albumin (BSA) was used as a control to ensure that the enhancing effect was not simply an effect of higher protein concentration and thereby a stabilizing effect on hCG, but a specific lactoferrin dependent effect. As seen in panel B, no enhancement but instead a slight reduction in the cleavage of the recombinant substrate was observed by the addition of BSA. https://doi org/10 1371/journal pone 0195077 g011 Fig 11. Analysis of the effect of human lactoferrin on the activity of hCG. The effect on the activity by hCG on the HC consensus substrate (VVLFSEVL) was analysed in a cleavage reaction as before (Figs 5–9). In panel A three different concentrations of lactoferrin were used: 25, 250 and 2500μg/ml. A 3–5 fold enhancement was observed at the highest lactoferrin concentration (2500μg/ml) and a 2–3 fold enhancement at the middle concentration (250μg/ml) but no enhancement at the low (250μg/ml) lactoferrin concentration. In panel B, bovine serum albumin (BSA) was used as a control to ensure that the enhancing effect was not simply an effect of higher protein concentration and thereby a stabilizing effect on hCG, but a specific lactoferrin dependent effect. As seen in panel B, no enhancement but instead a slight reduction in the cleavage of the recombinant substrate was observed by the addition of BSA. https://doi.org/10.1371/journal.pone.0195077.g011 https://doi.org/10.1371/journal.pone.0195077.g011 more specific proteases. However, for relatively unspecific and complex mixtures of proteases it may be a very interesting alternative to phage display. Interestingly also the proteomics method identified Thr as a good P1 residue for hPR3, which has not been observed with phage display. If the proteomics study increased the number of peptides by a factor of 10 and did not use non-natural amino acids as for example nor-Leucine the technique may become a valuable addition to the methods used to determine the specificity of also more specific proteases in a near future. Discussion The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated above the corresponding lanes of the different gels. https://doi.org/10.1371/journal.pone.0195077.g010 https://doi.org/10.1371/journal.pone.0195077.g010 which is why such information can be of major importance for the biological functions of these enzymes. A detailed map of their extended specificity can also aid our search for their most important in vivo substrates. Different methods can be used to study the extended specificity of an enzyme. The classical chromogenic substrates usually only cover the N terminal side of the cleavage sites whereas FRET peptides, peptide libraries and phage display can cover larger regions of the site includ- ing both N and C terminal sequences thereby providing a more complete view of the full extended specificity. Recently a novel proteomics method has also been developed to study protease specificity. This technique has been used to study the involvement of the different neutrophil proteases in the formation of NETS. Here, the authors concluded that N-elastase shows the most enzyme activity in the NETS [36]. Based on our analysis of the consensus cleavage sites identified by the proteomics and phage display studies we can now conclude that both techniques gave similar and accurate consensus sites for the relatively unspecific prote- ases such as N-elastase and proteinase 3. However, for the more specific proteases the com- plexity of the peptide library used for the proteomics study was most likely too limited to give a reliable result. The consensus sites for hCG by the proteomics method were suboptimal and only minor cleavage was seen with these substrates in the 2xTrx analysis (Fig 12). This result also indicates that the protease activity of the NETS may need to be re-evaluated. The peptide substrates for the latter enzyme in the proteomics study were not optimal, which may explain why its activity in the NETS was most likely underestimated. The analysis of the consensus sequences for the two techniques also indicates that the low complexity of the peptide library in the proteomics study with only 124 different peptides may limit its use for the analysis of PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 17 / 28 Extended cleavage specificity of human cathepsin G Fig 11. Analysis of the effect of human lactoferrin on the activity of hCG. Discussion The use of natural amino acids only would also make the results more easy to con- firm and validate with other non-MS based methods. PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 18 / 28 Extended cleavage specificity of human cathepsin G Fig 12. Analysis of the cleavage of a panel of consensus substrates for four human neutrophil proteases. The cleavage of a number of consensus cleavage sites for four different human neutrophil proteases (N-elastase, hCG, hPR3 and NSP4) were studied with the 2x-Trx system. The ‘A’ consensus sites come from phage display analyses performed in our lab and the substrates B and C comes from a proteomics study by O’Donoghue et al [36]. The substrates originating from the two different methods for N-elastase and proteinase 3 both show very good cleavage, whereas for hCG the consensus sites obtained by the proteomics method shows only minor cleavage, indicating a relatively poor site. No phage display has yet been performed on hNSP4, therefore only a proteomics site was studied where minor cleavage after using a relatively high concentration of the enzyme was seen. https://doi.org/10.1371/journal.pone.0195077.g012 Fig 12. Analysis of the cleavage of a panel of consensus substrates for four human neutrophil proteases. The cleavage of a number of consensus cleavage sites for four different human neutrophil proteases (N-elastase, hCG, hPR3 and NSP4) were studied with the 2x-Trx system. The ‘A’ consensus sites come from phage display analyses performed in our lab and the substrates B and C comes from a proteomics study by O’Donoghue et al [36]. The substrates originating from the two different methods for N-elastase and proteinase 3 both show very good cleavage, whereas for hCG the consensus sites obtained by the proteomics method shows only minor cleavage, indicating a relatively poor site. No phage display has yet been performed on hNSP4, therefore only a proteomics site was studied where minor l ft i l ti l hi h t ti f th z Fig 12. Analysis of the cleavage of a panel of consensus substrates for four human neutrophil proteases. The cleavage of a number of consensus cleavage sites for four different human neutrophil proteases (N-elastase, hCG, hPR3 and NSP4) were studied with the 2x-Trx system. The ‘A’ consensus sites come from phage display analyses performed in our lab and the substrates B and C comes from a proteomics study by O’Donoghue et al [36]. PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 Discussion The substrates originating from the two different methods for N-elastase and proteinase 3 both show very good cleavage, whereas for hCG the consensus sites obtained by the proteomics method shows only minor cleavage, indicating a relatively poor site. No phage display has yet been performed on hNSP4, therefore only a proteomics site was studied where minor cleavage after using a relatively high concentration of the enzyme was seen. https://doi.org/10.1371/journal.pone.0195077.g012 https://doi.org/10.1371/journal.pone.0195077.g012 The analysis of the hCG presented in this communication now shows that this enzyme has an extended specificity that is very similar in many aspects to the HC. These two enzymes are encoded from the same chromosomal locus, the chymase locus, and likely originate from the same original gene via gene duplication [48–51]. Both lack the cysteine bridge Cys191/Cys220 (chymotrypsinogen numbering) that is present in most non hematopoetic serine proteases, which is thought to open up the catalytic site and possibly facilitate a broader specificity of these enzymes [32]. Our analysis of hCG now shows that hCG, like the HC, has a preference for negatively charged amino acids in the P2´position, a preference for aliphatic amino acids both N and C terminal of the cleavage site, and also efficiently cleaves sites with a Leu in the P1 position. The previously identified dual specificity, being both a chymase and a tryptase-type, has also been verified here and we can now show that the chymase activity is only 2 fold higher for the most optimal substrates, containing a Phe in the P1 position, compared to the Lys con- taining tryptase substrates. However, the activity on an Arg containing substrate was approxi- mately 10 times lower than the chymase activity indicating a strong preference for Lys for tryptase activity, as previously indicated [31–33]. Interestingly, the activity on Trp containing substrates was highly dependent on positions surrounding the cleavage site. Very low activity PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 19 / 28 Extended cleavage specificity of human cathepsin G was observed for the HC on the opossum chymase consensus substrate, which contains a Trp in the P1 position, whereas the substrate with multiple Val residues in P2, P3 and P4 positions showed no difference between Phe, Tyr and Trp containing substrates (Figs 5 and 6). Here a clear difference was observed between the two enzymes. Discussion With the VVV substrates, hCG showed a preference for Phe over Tyr by a factor 2 and over and Trp by a factor 3–5. In con- trast, the activity on Leu containing substrates was very similar for the two enzymes, approxi- mately only 2 times lower than the optimal Phe substrate (Fig 6C and 6D). It was noteworthy that these proteases showed a marked difference between their overall activity. The difference was approximately 10-fold on a molar basis. This lower activity of hCG has previously also been observed and proposed to be dependent on the widening of the active site, which results in a broader specificity, being both chymase and tryptase-type, but at the same time lowering the affinity to the substrates and thereby lowering cleavage rate [32]. We have previously shown that the preference for negatively charged amino acids in the P2´position for the HC is governed by Arg143 and Lys192 [40]. They both contribute to this preference to approximately the same extent [40]. We now observe that hCG also shows a sim- ilar preference for negatively charged amino acids in the P2´position (Figs 3 and 5). Analysis of its primary amino acid sequence shows that hCG also has positively charged amino acids in the same positions of the enzyme indicating that the Arg143 and Lys192 could be responsible for this preference in target sequences (data not shown). Interestingly, the preference was more obvious for both enzymes when using substrates with a Gly in the P2´position compared to Ala where only a minor difference to Asp could be observed (Fig 7A and 7B). A preference for acidic residues in P2´ position has also been observed for the opossum α-chymase, rMCP- 5 and mMCP-4 [34, 46, 52]. All of these chymases contain Arg143 and Lys192 residues. Other chymases that also have Arg143 and Lys192 are the α-chymases from the crab eating macaque and baboon, the sheep mast cell chymase, mMCP-5, and hamster chymase-2. Furthermore, the β-chymases rMCP-3, hamster chymase-1, the guinea pig α-chymase and gerbil chymase-1 also contain Arg143 and Lys192 [53]. The preference for acidic P2´ residues seems to be highly preserved among the α-chymases. However, there are exceptions. The dog α-chymase holds Lys143 and Lys192 residues, and rMCP-1 holds Gln143 and Lys192 and both of these enzymes have no or only very weak preference for acidic residues in P2´ [34, 39]. PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 Discussion Apparently, a minor change from the positively charged Arg to a slightly smaller but still positively charged side chain of a Lys residue in position 143 is enough to partially affect the preference for acidic P2´ residues. The gerbil chymase 2 also holds Lys143 and Lys 192 and may therefore also have a lower preference for acidic aa in the P2’ position [54]. The strong activity of HC on P1 Leu containing recombinant substrates in Figs 6C and 9B was unexpected based on the results from the chromogenic substrate assay and the phage dis- play (Figs 2 and 3). This finding indicated a marked difference in the activity on short trun- cated substrates as represented by the chromogenic substrates, which lack amino acids C- terminal of the cleavage site. The smaller amino acid Leu compared to the aromatic Phe and Tyr may therefore need additional support by residues both upstream and downstream of the cleavage site for proper positioning and binding to the active site of the enzyme. The very large difference on the cleavage rate of a substrate by relatively similar amino acids surrounding the cleavage site was also somewhat unexpected. This was typified in the activity of two Trp con- taining substrates where the opossum consensus (VGLWLDRV) was a poor substrate for HC (Fig 5C) whereas the VVVWSEVV substrate was as good as the Phe consensus substrate for this enzyme (Fig 6C). In contrast hCG was less active on both of these Trp containing substrates (Figs 5 and 6). The notable effect by Ser in position P6 was also unexpected and differed between the two enzymes where only hCG showed this marked preference (Fig 10). This latter finding closely resembles the situation seen for thrombin, another member of the PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 20 / 28 Extended cleavage specificity of human cathepsin G chymotrypsin gene family [43]. Thrombin is for some substrates highly dependent in its cleav- age activity of sites located either close or further away from the active site. The fibrinogen alpha chain use a region located just upstream of the cleavage site to enhance cleavage of a site that is relatively poor in the absence of this region. This enhancing site is located only 4–8 amino acids upstream of the P1 residue [43]. Discussion In contrast, cleavage sites for thrombin within coagulation factors V and VIII such enhancing regions extend more than 100 amino acids upstream of the cleavage site and these regions interact with positively charged regions on the enzyme, so called exosites [43]. Here hCG showed a tendency of this more local interaction when the cleavage was dependent on a site located 6 amino acids upstream of the actual cleav- age site. Recently the activity of hCG has been shown to be modulated by other granule stored pro- teins. Lactoferrin is an iron binding protein stored in the neutrophil granules, which can enhance activation of hCG [47]. The activity of the enzyme can be increased by a factor of 5–7 with the addition of lactoferrin [47]. Similarly to the cooperation seen with lactoferrin and hCG, human thrombin has interactions with other proteins when cleaving protein C. Throm- bin is a potent activator of protein C by cleaving an activation site, which is highly dependent on the accessory molecule thombomodulin. In order for this to efficiently occur, thrombomo- dulin needs to be bound to the complex. Despite this, the exact mechanism of protein C activa- tion is not known and in this regard the situation for hCG and lactoferrin is also, to our knowledge, not fully elucidated. However, both of these examples highlight the very complex nature sometimes involved in substrate interactions and cleavage. Here we could confirm the enhancing activity of lactoferrin on hCG by incubating lactoferrin and recombinant 2x Trx substrates with hCG (Fig 11A). However, the effect was only seen with an excess of lactoferrin to hCG with the highest concentrations corresponding to 37 and 370 times more lactoferrin to hCG, indicating that the effect is not necessarily direct specific binding, which is generally an equimolar interaction, but more dependent on other unknown factors. Other factors such as the accessibility of the potential cleavage site can become more rele- vant than the sequence itself. A relatively poor site can be cleaved relatively efficiently when fully exposed, whereas an ideal consensus site, when being well hidden in the structure, might not be cleaved at all even in the presence of the enzyme at a high concentration. In that con- text, initial cleavages might also facilitate access to otherwise non-exposed sites. PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 Discussion Taken together, in order to fully address protease’s accessibility to a potential cleavage site or sites, any proposed substrate needs to be tested experimentally. This is highlighted in a recent study where the cleavage sensitivity of 50 different human cytokines and chemokines for cleavage by the HC and hCG was tested. The general view suggested that the HC and hCG would cleave a broad range of targets yet, remarkable, in this study only a few cytokines were cleaved despite most of them contained what appeared to be close to optimal sites for HC or hCG [55]. Only 4 out of 50 cytokines tested were cleaved by the HC, with a few additional by hCG but only under equimolar substrate to enzyme ratios [55]. This highlights factors such as accessibility of the site as being of major importance for actual cleavage. Among the targets identified in above study included several alarmins, such as IL-18 and IL-33, suggesting a role for these two enzymes in limiting excessive inflammation [55]. A num- ber of additional potential targets have also been identified for both of these enzymes. Angio- tensin (Ang) is a well studied target where Ang I is cleaved efficiently by both HC and hCG resulting in a product Ang II, an active peptide involved in blood pressure regulation [56–58]. Interestingly hCG is relatively resistant to inactivation by plasma protease inhibitors (serpins) when bound to the neutrophil cell surface [59]. This is in contrast to when it is free in plasma and can possibly explain why surface bound hCG can act as a potent Ang II converter even in the presence of inhibitors [59]. For many of the potential targets the difference between HC PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 21 / 28 Extended cleavage specificity of human cathepsin G and hCG activities is most likely due to the broader specificity of hCG compared to the HC. The relatively strong tryptase activity of hCG most likely results in the capacity of this enzyme to cleave a broader range of substrates. However, the lower overall enzymatic activity of hCG also needs to be taken into account when analysing its in vivo function. Here there is the possi- bly enhancing activity in the presence of lactoferrin, which could have an important role in its regulation. Discussion Despite this the potentiating activity was only 3–5 fold higher in our assay even at a very high molar excess of lactoferrin, indicating only a modest enhancement under physiologi- cal conditions. In summary, we demonstrate here that hCG shows some enzyme characteristics that are closely comparable to HC. Both enzymes are chymase-type proteases with a preference for Phe and Tyr over Trp in the P1 position of substrates as well as they show potent activity toward Leu containing substrates. However, they differ in aspects relating to their tryptase activity where hCG readily cleaves Lys containing substrates in the P1 position whereas HC only shows a very weak activity against Arg and no activity towards Lys. Somewhat surprisingly, the phage display analysis of hCG did not select any tryptase-like targets, which suggests that the technique can primarily be used to identify the most optimal targets. Targets that are cleaved only 2–3 times less efficiently than the optimal ones are apparently primarily selected against during the panning. This needs to be taken into consideration when analysing the specificity of enzymes so that significant secondary specificities would not be overlooked. hCG also has an overall much lower enzymatic activity, approximately 10-fold, as compared to HC. These two enzymes also differ in the preference for the amino acids in the P1 position where nega- tively charged residues are strongly non-preferred by hCG but are favoured for HC. Interest- ingly there is also a strong preference for Ser in the P6 position for hCG but not for HC. This is an indication that for positions relatively far from the active site can be of major importance for the cleavage, not only for thrombin exosite interactions but also for the granule stored ser- ine proteases of hematopoietic cells. We hope that the extended specificities presented here for both hCG and HC can now be used as a refined tool to identify and validate old and potentially new in vivo targets for these two important immune proteases. As we increase our understanding of the function of these proteases and identify new targets, both of endogenous and exogenous (viral and bacterial) origin, this knowledge hopefully will facilitate the identification of important immunomodula- tory or antimicrobial targets for treatment. PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 Determination of cleavage specificity by phage-displayed nonapeptide library A library of 5x107 unique phage-displayed nonameric peptides was used to determine the cleavage specificity of the hCG as previously described [34, 52, 61]. In these T7 phages, the C-terminus of the capsid protein 10 were manipulated to contain a nine aa long random pep- tide followed by a His6-tag [61]. An aliquot of the amplified phages (~109 pfu) were bound to 100 μl Ni-NTA beads by their His6-tags for 1 h at 4˚C under gentle agitation. Unbound phages were removed by washing ten times in 1.5 ml 1 M NaCl, 0.1% Tween-20 in PBS, pH 7.2, and two subsequent washes with 1.5 ml PBS. The beads were finally resuspended in 1 ml PBS. Activated and heparin-Sepharose purified HC mutant (~0.1 μg) was added to the resus- pended beads and left to digest susceptible phage nonapeptides under gentle agitation at room temperature over night. PBS without protease was used as control. Phages with a ran- dom peptide that was susceptible to protease cleavage were released from the Ni-NTA matrix, and the supernatant containing these phages was recovered. To ensure that all of the released phages were recovered the beads were resuspended in 100 μl PBS (pH 7.2) and the supernatant, after mixing and centrifugation, was added to the first supernatant. To ensure that the His6-tags had been hydrolyzed on all phages recovered after protease digestion, 15 μl fresh Ni-NTA agarose beads were added to the combined phage supernatant and the mixture agitated for 15 min followed by centrifugation. A control elution of the phages still bound to the beads, using 100 μl 100 mM imidazole showed that at least 1 x 108 phages were attached to the matrix during each selection. Ten μl of the supernatant containing the released phages was used to determine the amount of phages detached in each round of selection. Dilutions of the supernatant were plated in 2.5 ml of 0.6% top agarose containing 300 μl of E. coli (BLT5615), 100 μl diluted supernatant and 100 μl 100mM IPTG. The remaining volume of the supernatant was added to a 10 ml culture of BLT5615 (OD ~0.6). The bacteria had 30 min prior to phage addition been induced to produce the T7 phage capsid protein by the addition of 100 μl 100 mM IPTG to the culture. The bacteria lysed approximately 75 minutes after phage addition. Production and purification of proteases The hCG used in this study had been purified from peripheral blood neutrophils and was pur- chases from BioCentrum (Krakow, Poland). Human proteinase 3 and N-elastase were also purified from peripheral blood neutrophils and purchased from Lee Biosolutions (St. Louis, Missouri, USA) and Athens Research & Technology (Athens, GA, USA), respectively. The constructs and procedure for the production of recombinant HC from bacculovirus infected insect cells has previously been described in detail [37]. Human activated plasma thrombin, purchased from Sigma Aldrich (Sigma T-6884), and human granzyme B produced in-house in the human cell line HEK-293 EBNA using the vector pCEP-Pu2 [40] were used in the chromo- genic substrate assay. Protein purity and concentration was estimated by separation on 12.5% SDS-PAGE gels. Protein samples were mixed with sample buffer, and β-mercapto-ethanol was added to a final concentration of 5%. To visualize the protein bands, the gel was stained with colloidal Coo- massie Brilliant Blue [60]. PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 22 / 28 Extended cleavage specificity of human cathepsin G Analysis of primary specificity by cleavage of chromogenic substrates Enzymatic activity was measured towards a panel of chromogenic substrates. These sub- strates were purchased from Bachem (Bubendorf, Switzerland) and Chromogenix (Mo¨lndal, Sweden). Measurements were performed in 96-well microtiter plates with a substrate con- centration of 0.18 mM in 200 μl PBS. Hydrolysis was monitored spectrophotometrically at 405 nm for up to 10 hrs in a Versamax microplate reader (Molecular Devices, Sunnyvale, CA). Generation of recombinant substrates for the analysis of the cleavage specificity A new type of substrate was developed to verify the results obtained from the phage display analysis. Two copies of the E. coli thioredoxin gene were inserted in tandem into the pET21 vector for bacterial expression (Fig 5A). In the C-terminal end a His6- tag was inserted for purification on Ni2+ IMAC columns. In the linker region, between the two thioredoxin mole- cules, the different substrate sequences were inserted by ligating double stranded oligonucleo- tides into two unique restriction sites, one BamHI and one SalI site (Fig 5A). The sequences of the individual clones were verified after cloning by sequencing of both DNA strains. The plas- mids were then transformed into the E.coli Rosetta gami strain for protein expression (Nova- gen, Merck, Darmstadt, Germany). A 10 ml overnight culture of the bacteria harbouring the plasmid was diluted 10 times in LB + Amp and grown at 37 ˚C for 1–2 hours until the OD (600 nm) reached 0.5. IPTG was then added to a final concentration of 1 mM. The culture was then grown at 37˚C for an additional 3 h under vigorous shaking, after which the bacteria were pelleted by centrifugation at 3500 rpm for 12 minutes. The pellet was washed once with 25 ml PBS + 0.05% Tween 20. The pellet was then dissolved in 2 ml PBS and sonicated 6 x 30 seconds to open the cells. The lysate was centrifuged at 13000 rpm for 10 minutes and the supernatant was transferred to a new tube. Five hundred μl of Ni-NTA slurry (50:50) (Qiagen, Hilden, Ger- many) was added and the sample was slowly rotated for 45 min at RT. The sample was then transferred to a 2 ml column and the supernatant was allowed to slowly pass through the filter leaving the Ni-NTA beads with the bound protein in the column. The column was then washed four times with 1 ml of washing buffer (PBS + 0.05% Tween + 10 mM Imidazole + 1 M NaCl). Elution of the protein was performed by adding 150 μl elution buffer followed by five 300 μl fractions of elution buffer (PBS + 0.05% Tween 20 + 100 mM Imidazole). Each frac- tion was collected individually. Ten μl from each of the eluted fractions was then mixed with 1 volume of 2 x sample buffer and 1 μl β-mercapto-ethanol and then heated for 3 min at 80˚C. Determination of cleavage specificity by phage-displayed nonapeptide library The lysate was centrifuged to remove cell debris and 500 μl of the phage sub-library was added to 100 μl fresh Ni-NTA beads, to start the next round of selection. After binding the sub-library for 1 h at 4˚C under gentle agitation, the Ni-NTA beads were washed 15 times in 1.5 ml 1 M NaCl, 0.1% Tween-20 in PBS, pH 7.2, followed by two subse- quent washes with 1.5 ml PBS. Following five rounds of selection, 100 plaques were isolated from LB plates after plating in top agarose. Each phage plaque, corresponding to a phage clone, was dissolved in phage extraction buffer (100 mM NaCl and 6 mM MgSO4 in 20 mM Tris-HCl pH 8.0) and vigorously shaken for 30 min in order to extract the phages from the agarose. The phage DNA was then amplified by PCR, using primers flanking the variable region of the gene encoding the modi- fied T7 phage capsid-protein. After amplification, 96 PCR fragments were sent unpurified to GATC Biotech Germany for sequencing. 23 / 28 PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 Extended cleavage specificity of human cathepsin G Generation of a consensus sequence from sequenced phage inserts Phage insert sequences were aligned by hand assuming a preference for aromatic aa in position P1. Sequences with only one aromatic aa were aligned first and sequences with more than one possible cleavage site were then aligned to fit this pattern. Amino acids with similar character- istics were grouped together as follows: aromatic (Phe, Tyr, Trp); negatively charged (Asp, Glu); positively charged (Lys, Arg); small aliphatic (Gly, Ala); larger aliphatic (Val, Leu, Ile, Pro); hydrophilic (Ser, Thr, His, Asn, Gln, Cys, Met). The nomenclature by Schechter and Ber- ger [62] was adopted to designate the aa in the substrate cleavage region, where P1-P1’ corre- sponds to the scissile bond. PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 Generation of recombinant substrates for the analysis of the cleavage specificity The samples were analysed on a SDS bis tris 4–12% PAGE gel and the second and third frac- tions that contained the most protein were pooled. The protein concentration of the combined fractions was determined by Bio-Rad DC Protein assay (Bio-Rad Laboratories Hercules, CA USA). Approximately 60 μg of recombinant protein was added to each 120 μl cleavage reaction (in PBS). Twenty μl from this tube was removed before adding the enzyme, the 0 minute time point. The active enzyme was then added (approximately 35 ng of the HC or 350 ng of hCG) and the reaction was kept at room temperature during the entire experiment. Samples (20 μl) were removed at the indicated time points (15 min, 45 min and 150 min) and stopped by addi- tion of one volume of 2 x sample buffer. β-mercaptoethanol (1 μl) was then added to each sam- ple followed by heating for 3 min at 80 ˚C. Samples (20 μl each) were then analysed on 4–12% pre-cast SDS-PAGE gels (Invitrogen, Carlsbad, CA, USA). The gels were stained overnight in colloidal Coomassie staining solution and de-stained for several hours according to previously PLOS ONE | https://doi.org/10.1371/journal.pone.0195077 April 13, 2018 24 / 28 Extended cleavage specificity of human cathepsin G described procedures [60]. Some of the most important protein gels were analyzed using the UN-SCAN-IT Gel Analysis Software from Silk Scientific Inc. (Orem, Utah USA). The gel pic- tures had first to be turned into grey scale pictures before the densitometric analysis. The scan- ning results are presented as separate panels in Figs 5, 6 and 8. Writing – original draft: Lars Hellman. Writing – original draft: Lars Hellman. Writing – review & editing: Michael Thorpe, Zhirong Fu, Gurdeep Chahal, Srinivas Akula, Jukka Kervinen, Lars Hellman. Conceptualization: Michael Thorpe, Lars Hellman. Conceptualization: Michael Thorpe, Lars Hellman. Formal analysis: Michael Thorpe, Zhirong Fu, Srinivas Akula, Lars Hellman. 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